CN103585105A - Sphingolipid activator protein C for transmembrane medicine delivering system as well as associated protein and peptide-promoted fusion protein - Google Patents

Abstract

The present invention includes methods of using fusogenic proteins to deliver pharmaceutical agents and/or imaging agents into and/or through the membranes of the skin, mucosa and other cells, and across the blood-brain barrier. Fusogenic proteins associate with phospholipid membranes such as liposomes. Liposomes may include dioleoylphosphatidylserine, a negatively charged long-chain lipid. Alternatively, the liposomes comprise negatively charged long-chain lipids, neutral long-chain lipids and neutral short-chain lipids. Preferred fusogenic proteins include saposin C and other proteins, polypeptides and peptide analogs derived from saposin C. Active agents contained in liposomes may include biomolecules and/or organic molecules. This technology can be used in cosmetic and medical applications where the goal is to deliver active agents into and/or under biofilms or across the blood-brain barrier and neuronal membranes.

Description

Saposin C and associated protein and the short Fusogenic properties of peptide for cross-film drug delivery system

The application is to be dividing an application of November 7, application number in 2008 application of the same name that is 200880132489.0 the applying date.

The fund No.ROl DK57690-01 bottom that You of the present invention NIH authorizes is divided and is obtained government-funded.Government can have some right in the present invention.

Cross reference related application

The application is the PCT patent application series No.PCT/US2007/010357 of submission on April 27th, 2007 and the U.S. Provisional Patent Application series No.60/745 of submission on April 28th, 2006,969 part continues and requires its interests, for all objects are by the complete by reference this paper that is incorporated to of described application.

Invention field

The application relates to compositions and the method to object cell or tissue by developer and/or therapeutic agent targeted delivery, wherein by described pack containing or otherwise be incorporated in protein/lipid nano-capsule bubble (nanovesicle).More specifically, the present invention relates to compositions and method to object cell or tissue by developer and/or therapeutic agent targeted delivery, wherein described pack is contained in protein/lipid nano-capsule bubble or otherwise integrate with it, described protein/lipid nano-capsule bubble comprises can targeted cells film and send Saposin C (saposin C) protein fragments of this type of reagent.

Background of invention

The therapeutic efficiency of pharmaceutical agent or therapeutic agent depends on pharmaceutical agent the sending to site of action of sufficient dosage.Developed many delivery modality, comprised for example through intestinal (oral), parenteral (intramuscular, intravenous, subcutaneous) and local application.In most of the cases, select administration system with carry out reliably and easily dosage send.

Similar to the problem that the transdermal delivery of pharmaceutical preparation is intrinsic, blood brain barrier is the obstacle of CNS drug delivery.In fact, blood brain barrier is considered to " bottleneck " of brain drug development, and may be the unique most important restriction to the development in future of neurotherapy.(Pardridge, W.M., The Blood-Brain Barrier:Bottleneck in Brain Drug Development; The Journal of the American Society for Experimental NeuroTherapeutics, the 2nd volume, 3-14; Jan, 2005; Pardridge, W.M.Brain drug targeting:the future of brain drug development.Cambridge, UK:Cambridge University Press, 2001).BBB is formed by brain capillary endothelium, and it stops nearly 100% macromole (for example monoclonal antibody, recombinant protein, antisense or gene therapeutic agents) and more than 98% all small-molecule drugs to the transportation in brain.Although the mean molecule quantity of CNS active medicine is 357 dalton, even less, 100 dalton molecules are histamine for example, when being infused into mice and make it scatter more than 30 minutes, can not pass through BBB.In fact, comprehensive pharmaceutical chemistry data base's assessment is shown: in more than 7000 kinds of small-molecule drugs, only have 5% treatment CNS, and this 5% Cure of depression, schizophrenia and insomnia.

Therefore, most drug can not be passed BBB.Unfortunately, many obstacles of central nervous system (CNS) can be benefited from the pharmacotherapy of the improvement of pointing to CNS.Although the known reagent through BBB has been carried out to relatively few research, has had the feature that can predict the probability of sending to the success in CNS.This category feature is: the 1) molecular weight below 400-500 dalton threshold value, and 2) fat-solubility.At present, only have 4 class CNS obstacles to respond this quasi-molecule, comprise affective disorder, chronic pain and epilepsy.Migraine can be considered to CNS obstacle, also can be included in this category.On the contrary, suffer from Ji Bingliruaercihaimoshi disease, parkinson disease, Huntington Chorea, A.L.S., multiple sclerosis, neuro-AIDS, the brain cancer, apoplexy, brain or spinal cord injuries receptor, infantile autism, lysosomal storage disease, fragile X mental retardation, hereditary ataxia and blind patient extremely limited in drug treatment choice.(L-DOPA treatment has obtained some successes in disturbances in patients with Parkinson disease, and multiple sclerosis can be used as treating for the immune cytokine of periphery) (conventionally referring to, Partridge, the same).

In many listed obstacles above, through sending of BBB, be the speed limit problem of gene therapy or algucerase.Many these type of obstacles can be with the medicine of having found, enzyme or gene therapy.Yet these medicines can not pass BBB, and because this is former thereby fail to be considered for therapeutic use.Due to the impervioursness of BBB, must use the additive method that drug delivery is entered to CNS.These class methods comprise micromolecule, the use destroying through cranium brain (trans-cranial brain) drug delivery and BBB.Yet, these class methods none provide can be in a large amount of patients the actual solution for BBB problem realizing.(Pardridge,W.M.,'The Blood-Brain Barrier")。

Sphingolipid activator protein (family of little (approximately 80 aminoacid) thermally-stabilised glycoprotein) is that in the body of several lysosomal enzymes in the catabolic pathway of glycosyl sphingolipid, hydrolysing activity is necessary (referring to Grabowski; G.A.; Gatt; S. and Horowitz; M. (1990) Crit.Rev.Biochem.Mol.Biol.25,385-414; Furst, W. and Sandhoff, K., (1992) Biochim.Biophys.Acta1126,1-16; Kishimoto, Y., Kiraiwa, M. and O'Brien, J S. (1992) J.Lipid.Res.33,1255-1267).4 member A, B, C and D of sphingolipid activator protein family from single precursor protein-Prosaposin (prosaposin) Proteolytic enzyme (referring to Fujibayashi; S., Kao, F.T.; Hones; C, Morse, H.; Law; M. and Wenger, D.A. (1985) AmJ.Hum.Genet.37,741-748; O'Brien, J.S., Kretz, K.A., Dewji, N., Wenger, D.A., Esch, F. and Fluharty, AL. (1988) Science241,1098-1101; Rorman, E.G. and Grabowski, GA. (1989) Genomics 5,486-492; Nakano, T., Sandhoff, K., Stumper, J., Christomanou, H and Suzuki, K. (1989) J.Biochem. (Tokyo) 105,152-154; Reiner, O., Dagan, O. and Horowitz, M. (1989) J.Mol.Neurosci.1,225-233).Sphingolipid activator protein A, B, C and the complete amino acid sequence of D and the genomic organization of Prosaposin and cDNA sequence have been reported (referring to Fujibayashi; S., Kao, F.T.; Jones; C, Morse, H; Law; M. and Wenger, D.A. (1985) Am.J.Hum.Genet.37,741-748; O'Brien, J.S., Kretz, K A., Dewji, N., Wenger, D.A, Esch, F. and Fluharty, A.L. (1988) Science241,1098-1101; Rorman, E.G and Grabowski, G A. (1989) Genomics 5,486-492).The lacking completely of the Prosaposin causing because of the sudden change of start codon cause to combination lysosomal hydrolase lack the storing up of similar multiple glycosyl sphingolipid substrate (referring to Schnabel, D., Schroder, M., Furst, W., Klien, A., Hurwitz, R., Zenk, T., Weber, J., Harzer, K, Paton, B.C., Poulos, A., Suzuki, K and Sandhoff, K (1992) J.Biol.Chem.267,3312-3315).

Sphingolipid activator protein (Saposin) is defined as sphingolipid activator protein (sphingolipid activator proteins) or coenzyme.Structurally, sphingolipid activator protein A, B, C and D have the similarity of about 50-60%, comprise that 6 strict conservative cysteine residues are (referring to Furst, W. and Sandhoff, K, (1992) Biochim.Biophys.Acta1126, 1-16), described cysteine residues forms the interior disulphide bridges of 3 identical domains of its position (placement) (referring to Vaccaro, A.M., Salvioli, R., Barca, A., Tatti, M., Ciaffoni, F., Maras, B., Siciliano, R., Zappacosta, F., Amoresano, A. and Pucci, P. (1995) J.Biol.Chem.270, 9953-9960).All sphingolipid activator protein comprises a glycosylation site with conservative position (in N terminal sequence half part); but it is necessary (referring to Qi.X. and Grabowski that glycosylation is not its activity; G.A. (1998) Biochemistry37,11544-11554; Vaccaro, A.M., Ciaffoni, F., Tatti, M., Salvioli, R, Barca, A., Tognozzi, D. and Scerch, C. (1995) J.Biol.Chem.270,30576-30580).In addition, sphingolipid activator protein A has the second glycosylation site in C end half part.

When in solution, all sphingolipid activator proteins and sphingolipid activator protein sample albumen and domain comprise " sphingolipid activator protein folds (saposin fold) ".This is folding is the many α helical bundle motif that is characterised in that 3 conservative disulfide structures and several Amphiphilic peptides.Although there is this total sphingolipid activator protein foldable structure in solution, sphingolipid activator protein and sphingolipid activator protein sample protein have biological function in different bodies aspect strengthening specificity hydrolytic enzyme degraded lysosome sphingolipid (SL) and glycosyl sphingolipid (GSL).Due to this type of effect, sphingolipid activator protein in the control of lysosome sphingolipid and glycosyl sphingolipid metabolism, ply in the centre position (referring to Kishimoto, Y., Kiraiwa, M., and O'Brien, J.S. (1992) J.Lipid.Res.33,1255-1267; Fujibayashi, S., Kao, F.T., Hones, C, Morse, H., Law, M. and Wenger, D.A. (1985) Am.J.Hum.Genet.37,741-748; O'Brien, J.S., Kretz, K.A., Dewji, N., Wenger, D.A., Esch, F. and Fluharty, A.L. (1988) Science241,1098-1101).

The architectural feature of this type of sphingolipid activator protein is extremely important for different activate mechanisms.Because all these albuminoids have high sequence similarity, but lipid film is had to different mechanism of action, the specific biological function that therefore can infer sphingolipid activator protein and sphingolipid activator protein sample albumen is the interactional result of difference with biomembrane environment.In vitro; sphingolipid activator protein A strengthens acid beta-glucosidase activity in μ M concentration; but Saposin C defect causes glucosylceramide to be stored up with " Gaucher disease sample " phenotype (referring to Schnable; D.; Schroder; M. and Sandhoff, K. (1991) FEBS Lett.284,57-59; Rafi.M.A, deGala, G, Zhang, X.L. and Wenger, D.A. (1993) Somat.Cell Mol.Genet.19,1-7).The activation of sphingolipid activator protein B is by solubilising glycosyl sphingolipid substrate and be and be handed to lysosomal enzyme (referring to Furst, W. and Sandhoff, K., (1992) Biochim.Biophys.Acta1126,1-16) occurs.

Saposin C by inducible enzyme conformation change under acid pH promote acid beta-glucosidase activity (referring to Berent, S.L. and Radin, N.S. (1981) Biochim.Biophys.Acta664,572-582; Greenberg, P., Merrill, A.H., Liotta, D.C. and Grabowski, G A. (1990) Biochim.Biophys.Acta1039,12-20; Qi.X. and Grabowski, GA. (1998) Biochemistry37,11544-11554).This interaction of Saposin C and enzyme occurs on electronegative phospholipid surface.External and in vitro sphingolipid activator protein A and D bring into play respectively the degraded that strengthens galactosyl ceramide and ceramide/sphingomyelins (sphingomyelin) function (referring to Harzer, K., Paton; B.C., Christomanou, H.; Chatelut; M., Levade, T.; Hiraiwa; M. and O'Brien, J S. (1997) FEBS Lett.417,270-274; Klien, A., Henseler, M., Klein, C, Suzuki, K., Harzer, K. and Sandhoff, K. (1994) Biochem.Biophys.Res.Commun, 200,1440-1448).The patient who lacks independent sphingolipid activator protein B and C show respectively the metachromatic leukodystrophy of variant form (metachromatic leukodystrophy) and Gaucher disease (referring to Wenger, D.A, DeGala, G, Williams, C, Taylor, H.A., Stevenson, R.E., Pruitt, J.R., Miller, J., Garen, P.D. and Balentine, J.D. (1989) Am.J.Med.Genet.33,255-265) (referring to Christomanou, H., Aignesberger, A. and Linke, R.P. (1986) Biol.Chem.Hoppe-Seyler367,879-890).

The invention still further relates to and use the liposome that comprises Saposin C developer to be used to the method that comprises blood brain barrier through cell membrane.Non-invasive imaging technology can be used for monitoring distribution and the effect of liposome delivery system, thereby promotes to use the gene therapy of liposome or the assessment of therapeutic treatment and clinical practice.Developer can be used magnetic resonance, fluorescence or CT/PET as detection means.Yet successfully using the major obstacle of developer monitoring liposome delivery is the easiness detecting, availability and the easiness of sending and the efficiency of correlation technique.

About using liposome delivery developer, lipophilic molecules is normally suitable, although the present invention is not limited to, uses this quasi-molecule.Do not wish bound by theory, the lipophilic dyes that comprises lipotropy part can embed liposome membrane or rebuild in the lipid core of liposome structure.The example of this type of dyestuff known in the art is indole cyanines (indocarbocyanine) dyestuff DiI.DiI is the conventional fluorescence carbonyl cyanine dye for labelling lipid film.Other similar dyestuffs are as Honig, M.G. wait people, DiI and DiO:versatile fluorescent dyes for neuronal labeling and pathway tracing.Trends Neurosci.12:333-335,340-331, the DiA or the DiaO that describe in 1989.Can be used for other lipophilic dyes of the present invention and comprise PKH2, NeuroVue Green, PKH26, NeuroVue Red and NeuroVue Maroon, as by Fritzsch, Deng people Diffusion and Imaging proerties of Three New Lipophilic Tracers, NeuroVue Maroon, NeuroVue and Neurovue Green and their use for Double and Triple Labeling of Neuronal Profile, described in manuscript.Any dyestuff in this type of dyestuff can be individually or in combination for the present invention described herein.

Also for this area and to be suitable for of the present invention be the developer with two or more imaging character.This type of reagent makes researcher or clinician to detect the developer of using with multiple formation method.The example of this type of reagent is as by Li, and H., waits people, MR and Fluorescent Imaging of Low-Density Lipoprotein Receptors, Acad Radiol.2004; The so-called PTIR dyestuff that 11:1251-1259 (being incorporated to by reference herein) describes.This type of dyestuff comprises permission by fluorogen and Gd (III) part of nuclear magnetic resonance (MRI) or confocal fluorescent microscopic examination.Lipotropy side chain promotes dyestuff to the embedding in Lipid monolayer.Therefore, this type of dyestuff is suitable for for example liposome delivery system described herein of liposome delivery system.

Proton MR imaging provides following advantage: Noninvasive, tomography (tomographic) and high-resolution.In recent years, nuclear magnetic resonance (MRI) has appeared for the powerful tool in clinical setting, because its accurate volume drawing (volume rendering) that be Noninvasive and that produce experimenter.Conventionally referring to, U.S. Patent No. 6,962,686, Kayyem, waits people, and title is Cell-specific gene delivery vehicles.This type of favourable aspect makes MRI at medical imaging with as become the technology of selection for the imaging tool of biological experiment.Different from the optical microscope imaging technique of use based on dyestuff or fluorescein stain, MRI does not produce poisonous photobleaching by-product.In addition, different from light microscopy, MRI is not subject to approximately only having the light scattering of cell or the restriction of other optical aberrations in the surface of 100 microns.Described above those of reagent example with MRI character can be used for the present invention.

Therefore, exist promoting medicine or developer to pass or comprise by biomembrane the remarkable needs of the nontoxic reagent of sending or transporting of blood brain barrier.The present invention meets these needs.

Summary of the invention

The present invention relates to that for example medicine or therapeutic agent delivery are through biomembranous compositions and method by reagent, wherein said pack is contained in immobilized artificial membrane or embeds wherein, and promotes to send by synexin.More specifically, the present invention relates to for Contrast agent through the film of skin and mucosa or blood brain barrier and/or in the film of skin and mucosa or the transportation of blood brain barrier and the compositions of sending and method, wherein said pack is contained in liposome, and the Saposin C of use and liposome association promotes to send.

As described in this article, the present invention includes and for delivering drugs reagent, by biomembrane, comprise compositions and the method for blood brain barrier and cell membrane, wherein said method comprises uses compositions to film, and described compositions pharmaceutically comprises anionic phospholipid (having or do not have neutral phospholipid) in acceptable carrier, is included in safety and the pharmaceutical agent of effective dose and fusogenic protein or the polypeptide that is derived from Prosaposin in phospholipid.

In one embodiment, anionic phospholipid film is vesicle.In another embodiment, vesicle is liposome.Liposome is a kind of nano container of form, and for example nano-particle or liposome, be generally used for encapsulated drug.Also can use cationic phospholipid, as long as the total electrical charge of the liposome of gained is born.

In another embodiment of the invention, the pH of protein-lipid composition is acid.In another embodiment of the invention, the pH of compositions is approximately between 6.8 to 2.In another embodiment of the invention, the pH of compositions is approximately between 5.5 to 2.In another embodiment, pH approximately 5.5 to approximately between 3.5.

In another embodiment, with dried forms for example powder protein and lipid composition are provided.In another embodiment, use the dried forms of acid treatment protein and lipid composition.In one embodiment, described acid is acidic buffer or organic acid.In another embodiment, to be enough to the level of protonated at least a portion protein, add acid, wherein compositions has approximately 5.5 to approximately 2 pH.In another embodiment, to be enough to the level of protonated protein substantially, add acid, wherein compositions has approximately 5.5 to approximately 2 pH.

In other embodiments, then the pH that is enough to the acid-treated protein of protonated at least a portion protein and the dry powder of lipid composition has been used in neutralization substantially.In one embodiment, use in neutral pH buffer and pH.In one embodiment, with being enough to control in the big or small neutral pH buffer of liposome of gained and pH.In another embodiment, with being enough to control in the neutral pH buffer of size (so that the liposome of the average diameter with approximately 200 nanometers to be provided) of liposome of gained and pH.In another embodiment, liposome has the average diameter of 50 to 350 nanometers.In another embodiment, liposome has the average diameter of 150 to 250 nanometers.In another embodiment, in compositions, add buffer to provide approximately 5 to approximately 14, preferably approximately 7 to 14, more preferably from about 7 to approximately 12,7 to approximately 10 and more preferably from about 8 to approximately 10 whole compositions pH more preferably from about.

In one embodiment of the invention, use short chain lipid.Conventionally, the concentration of fusogenic protein or polypeptide is to be enough to pharmaceutical agent send into film and/or send the amount through film.In another embodiment, in external film the concentration of phospholipid calculate in molar ratio excessive with respect at least 5 times of the concentration of fusogenic protein or polypeptide.In another embodiment, in external film the concentration of phospholipid calculate in molar ratio excessive with respect at least 10 times of the concentration of fusogenic protein or polypeptide.In another embodiment, in external film the concentration of phospholipid calculate in molar ratio excessive with respect at least 15 times of the concentration of fusogenic protein or polypeptide.In one embodiment, in external film the concentration of phospholipid calculate in molar ratio excessive with respect at least 20 times of the concentration of fusogenic protein or polypeptide.In another embodiment, in external film the concentration of phospholipid calculate in molar ratio excessive with respect to approximately 10 times to approximately 50 times of the concentration of fusogenic protein or polypeptide or approximately 20 to approximately 30 times excessive.

In one embodiment, in body or in cell membrane system the concentration of phospholipid calculate in molar ratio excessive with respect at least 1 times of the concentration of short fusogenic peptide.In one embodiment, in body or in cell membrane system the concentration of phospholipid calculate in molar ratio excessive with respect at least 2 times of the concentration of short fusogenic peptide.In another embodiment, in body or in cell membrane system the concentration of phospholipid calculate in molar ratio excessive with respect at least 3 times of the concentration of short fusogenic peptide.In another embodiment, in body or in cell membrane system the concentration of phospholipid calculate in molar ratio excessive with respect to approximately 1 to approximately 10 times of the concentration of short fusogenic peptide or approximately 3 to 7 times excessive.

Do not wish bound by theoryly by any way, it is believed that synexin and immobilized artificial membrane associate by static and hydrophobicity and hydrophobic interaction, and lipid composition total electrical charge is for negative.

According to the present invention, by the biomembrane of targeting, included but not limited to the film (dermal membrane) of skin, film, blood brain barrier and the cell membrane of mucosa.

Other protein, polypeptide analog or polypeptide that preferred synexin comprises Saposin C and is derived from arbitrary Saposin C (SEQ.ID.NO.1 to 13), with and composition thereof.

In one embodiment, synexin comprises the sequence that is selected from Saposin C (SEQ.ID.NO.1 and SEQ.ID.NO.2) at least 8,10,12,14,16,18,20,22,24 or more continuous amino acid.In one embodiment, the peptide that synexin comprises following formula:

h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys-Glu-h-h-Asp-h-h-Asp-Lys-h-Cys-u-Lys-h-h

Wherein h=hydrophobic amino acid, comprises Val, Leu, Ile, Met, Pro, Phe and Ala; And wherein the uncharged polar amino acid of u=, comprises Thr, Ser, Tyr, Gly, Gln and Asn, and its mixture.

In another embodiment, synexin comprises one or more protein that are selected from other protein, polypeptide analog or the polypeptide that are derived from arbitrary Saposin C (SEQ.ID.NO.1 or SEQ.ID.NO.2), can use the polypeptide of following formula:

h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys-Glu-h-h-Asp-h-h-Asp-Lys-h-Cys-u-Lys-h-h，

Wherein h=hydrophobic amino acid, comprises Val, Leu, Ile, Met, Pro, Phe and Ala; And wherein the uncharged polar amino acid of u=, comprises Thr, Ser, Tyr, Gly, Gln and Asn, and its mixture.

In one embodiment, synexin comprises following sequence at least 8,10,12,14,16,18,20,22,24,30 or more continuous amino acid: Ser Asp Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro.At least 8,10,12,14,16,18,20,22,24,30 or more continuous amino acid that synexin comprises following sequence: Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro.

In another embodiment, synexin comprises sequence: Ser Asp Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro.In another embodiment, synexin comprises sequence: Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro.In another embodiment, synexin is 22 aggressiveness, comprises sequence: CEFLVKEVTKLIDNNKTEKEIL.

In another embodiment, synexin comprises Saposin C.In another embodiment, synexin comprise according to known method (specifically referring to, J.M.Shaw, op.cit; The people such as Basu, Proc.Natl.Acad.Sci.USA73,3178-3182 (1976); J.Steinbrechert, Biol.Chem.262,3703 (1987)) modify the Saposin C of the oxidation, acetylation (for example, formylated and acetylation), acetoacetylate or lactosylation (lactosylated) form that produce.

In one embodiment, phospholipid nano-capsule bubble is the anionic liposome that comprises pharmaceutical agent.In another embodiment, liposome is made by any lipid mixture that comprises anion long-chain lipid.In another embodiment, liposome is made by the mixture that comprises anion long-chain lipid, neutral long-chain lipid and neutral short chain lipid, and wherein the total electrical charge of the liposome of gained is born.

During the lipid of the nano-capsule bubble using, find that numerous lipids is suitable for building them in selecting for the preparation of the present invention.The useful especially those skilled in the art of being are known is suitable for any material or its combination prepared by liposome.The lipid using can be the lipid in natural, synthetic or semi-synthetic source.

In another embodiment, nano-capsule bubble is made by one or more biocompatibility lipids.In another embodiment, biocompatibility lipid is selected from fatty acid, lysolipin (lysolipid), phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, sphingolipid, glycolipid (glycolipid), glycolipid (glucolipid), sulfatide (sulfatides), glycosyl sphingolipid, phosphatidic acid, Palmic acid, stearic acid, arachidonic acid, oleic acid, the lipid with polymer, the lipid with sulfonation monosaccharide, the lipid with sulfonation disaccharide, the lipid with sulfonation oligosaccharide, the lipid with sulfonation polysaccharide, cholesterol, tocopherol, the lipid with the fatty acid of ether connection, the lipid with the fatty acid of ester connection, the lipid of polymerization, diacetyl phosphate ester, the two hexadecane esters of phosphoric acid, stearmide, cuorin, having length is the phospholipid of the fatty acid of 6 to 8 carbon, the synthetic phospholipid with asymmetric acyl chain, ceramide, nonionic lipid, sterin aliphatic acid ester (sterol aliphatic acid esters), the sterol ester of saccharic acid, the ester of saccharic acid, the ester of sugar alcohol, the ester of sugar, the ester of aliphatic acid, saponin, GLYCERYL DILAURATE, trilaurin, glycerol-1,3-dipalmitate, glycerol, glyceride, length is the alcohol of 10 to 30 carbon, 6-(5-cholestene-3 beta-yl oxygen)-l-sulfo--β-D-galactopyranoside, digalactosyl diglyceride, 6-(5-cholestene-3 beta-yl oxygen) hexyl-6-amino-6-deoxidation-l-sulfo--β-D-galactopyranoside, 6-(5-cholestene-3 beta-yl oxygen) hexyl-6-amino-6-deoxidation-l-sulfo--α-D-mannopyranose glycosides, 12-(((7'-diethylamino coumarin-3-yl) carbonyl) methylamino)-octadecanoid acid, N-[12-(((7'-diethylamino coumarin-3-yl) carbonyl) methylamino) octadecanoyl] the amino Palmic acid of-2-, cholesteryl (4'-trimethyl-ammonium) butyrate, N-succinyl DOPE, DAG, l, 2-bis-palmityls-sn-3-succinyl glycerol, l, 3-bis-palmityls-2-succinyl glycerol, 1-cetyl-2-palmityl phosphoglycerol ethanolamine, palmityl homocysteine, cation lipid, N-[l-(2,3-dioleoyl oxygen) propyl group]-N, N, N-trimethyl ammonium chloride, 1,2-dioleoyl oxygen-3-(trimethyl ammonium) propane, l, 2-dioleoyl oxygen-3-(4'-trimethyl-ammonium) bytyry-sn-glycerol, lysophosphatide, lysophosphatidic acid (lysobisphosphatidic acid, LBPA), half-lysophosphatidic acid (semi-LBPA), cuorin, the lipid with cationic polymer, alkyl phosphonates, hypophosphorous acid Arrcostab (alkyl phosphinate) and alkyl phosphite (alkyl phosphite).

In one embodiment, phosphatidylcholine is selected from DOPC, dimyristoyl phosphatidyl choline, two pentadecanoyl phosphatidylcholine, DLPC, dipalmitoyl phosphatidyl choline and distearoyl phosphatidylcholine; Wherein PHOSPHATIDYL ETHANOLAMINE is selected from DPPE and DOPE; Wherein sphingolipid is sphingomyelins; Wherein glycolipid is selected from ganglioside GMl and Ganglioside GM2; Wherein, in carrying the lipid of polymer, described polymer is selected from Polyethylene Glycol, chitin, hyaluronic acid and polyvinylpyrrolidone; Wherein sterin aliphatic acid ester is selected from cholesterol sulfate, cholesterol butyric ester, cholesterol isobutyrate, cholesterol cetylate, cholesterol ester stearic acid, lanosterol acetas, ergosterol cetylate and plant sterol n-butyric acie ester; Wherein the sterol ester of saccharic acid is selected from cholesterol glucosiduronate, lanosterol glucosiduronate, 7-DHC glucosiduronate, ergosterol glucosiduronate, cholesterol gluconate, lanosterol gluconate and ergosterol gluconate; Wherein the ester of saccharic acid and the ester of sugar alcohol are selected from lauroyl glucosiduronate, stearoyl glucosiduronate, myristoyl glucosiduronate, lauroyl gluconate, myristoyl gluconate and stearoyl gluconate; Wherein the ester of sugar and the ester of aliphatic acid are selected from Surfhope SE Cosme C 1216, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid, accharic acid and polyuronide; Wherein saponin is selected from Sarsasapogenin (sarsasapogenin), smilagenin (smilagenin), hederagenin (hederagenin), oleanolic acid (oleanolic acid) and .DELTA.20:22-3.beta.,12.beta.,14,21-tetrahydroxynorcholenic acid lactone (digitoxigenin); Wherein glyceride is selected from tripalmitin, distearin, glyceryl tristearate, two myristin, myristin; Wherein alcohol is that length is the alcohol of 10 to 30 carbon and is selected from n-decanol, lauryl alcohol, myristyl alcohol, spermol and n-octadecanol; Wherein, in carrying the lipid of cationic polymer, cationic polymer is selected from poly-D-lysine and poly arginine.

In another embodiment, lipid is selected from dipalmitoyl phosphatidyl choline, DPPE and DPPA.

In another embodiment, the pharmaceutical preparation comprising in liposome comprises biomolecule and/or organic molecule.This technology can be used for beauty treatment and medical application, wherein object be by activating agent send through film for example the film of skin, the film of mucosa, blood brain barrier or cell membrane.

The invention still further relates to by by macromole for example the transportation of gene, protein and other biological molecule or organic molecule through blood brain barrier, treat the method for disease, wherein said method comprises uses compositions, and described compositions comprises anionic liposome (having or do not have short chain lipid), is included in macromole therapeutic agent and the Saposin C of the safe and effective amount in liposome.

In other embodiments, the invention describes compositions, described compositions comprises with the short nexus of sphingolipid activator protein or liposome mixes or small nucleic acids molecule compound or that be conjugated to it for example short interfering nucleic acid (siNA), short interfering rna (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA) or short hairpin RNA (shRNA).

The invention still further relates to the method that can treat following disease: neuroblastoma, brain inflammation (cerebral inflammation), metachromatic leukodystrophy (MLD), Niemann-Pick, apoplexy, parkinson disease, Alzheimer, demyelination obstacle (demyelination disorder), retina neural sick (retinal neuropathy), Huntington Chorea, A.L.S., multiple sclerosis, neuro-AIDS, the brain cancer, brain or spinal cord injuries receptor, infantile autism, lysosomal storage disease, fragile X mental retardation, hereditary ataxia and blind, wherein said method comprises the step that produces liposome delivery system, in described liposome delivery system, liposome comprises acid long-chain lipid (adding or do not add neutral long-chain lipid and neutral short chain lipid) and Saposin C, Prosaposin and other protein that are derived from Saposin C or Prosaposin, polypeptide analog or polypeptide.Liposome can comprise therapeutic agent for example antiinflammatory, anti-apoptotic agent and neuroprotective drug or enzyme, protein or corresponding gene, the DNA that is accredited as the gene lacking in this type of disease or RNA sequence.

In one embodiment, provide compositions and method, it comprises and short polynucleotide or its precursor that merges anionic phospholipid film or liposome, the fusogenic protein that is derived from Prosaposin or polypeptide and pharmaceutically acceptable carrier combinations.

In one embodiment, provide and comprised and short anionic phospholipid film or the short interfering nucleic acid (siNA) of liposome, the fusogenic protein that is derived from Prosaposin or polypeptide and pharmaceutically acceptable carrier combinations or compositions and the method for its precursor of merging.In new compositions of the present invention, siNA can be merged to anionic phospholipid film or liposome (having the fusogenic protein or the polypeptide that are derived from Prosaposin) combination or compound or be conjugated to it with short, to form the compositions of sending in the cell that strengthens siNA.

The present invention also comprises compositions and the method that is used for the treatment of Gaucher disease, wherein said method comprises uses compositions, described compositions comprises the anionic liposome being all included in pharmaceutically acceptable carrier, acid β-glucosyl enzym and the Saposin C that is included in the safe and effective amount in liposome, wherein the pH of compositions be approximately 7,6.8,6.5,6,5.9,5.8,5.7,5.6,5.5,5.4,5.3,5.2,5.1,5.0 or lower and Saposin C by the surface association of static and hydrophobic interaction and liposome.Conventionally, the relative concentration of liposome is excessive in approximately 1 to 10 times of the concentration of Saposin C.In one embodiment, the pH of compositions is lower than approximately 6.8.In another embodiment, the pH of compositions is lower than approximately 6.0.In another embodiment, the pH of compositions is lower than approximately 5.5.In another embodiment, the pH of compositions is lower than approximately 5.0.

The invention still further relates to the compositions and the method that are used for the treatment of Peyer patches (Peyer's patches), mesenteric lymph node, bronchial lymph nodes, wherein said method comprises using of compositions, lipid, DNA or proteantigen, Saposin C, the Prosaposin that described compositions comprises anion long-chain lipid, long-chain neutral lipid and/or neutral short chain lipid, safe and effective amount and be derived from Saposin C or other protein, polypeptide analog or the polypeptide of Prosaposin.

The invention still further relates to compositions and method to tissue and cell imaging, wherein said compositions comprises liposome and the detectable developer that contains Saposin C, and described detectable developer is selected from the detectable reagent of MRI, fluorometric reagent, the detectable reagent of CT/PET, the reagent with a plurality of detection character or its combination.Reagent can be embedded to lipid film or be encapsulated in liposome.In another embodiment of the invention, Saposin C liposome complex can mix 1,2 or 3 kind of different reagent with different imaging character, so as can be by multiple different detection method the single administration for Saposin C liposome.

Other auxiliary reagents comprise fluorogen (such as fluorescein, dansyl, quantum dot etc.) and IR dyes (infrared dye) or metal, and it can be used for optics or photoimaging (for example, confocal microscopy and fluorescence imaging).

In another embodiment, the nanometer that compositions also comprises radionuclide, chelating agen, biotin, fluorogen, antibody, horseradish peroxidase, alkali phosphatase, nano-particle, quantum dot, anticarcinogen is dripped (nanodroplet), anticarcinogen or chemotherapeutant, liposome medicament, cytokine or is connected to its micromolecule toxin.

In another embodiment, the nanometer that visualization portion is selected from radionuclide, biotin, fluorogen, antibody, horseradish peroxidase, alkali phosphatase, nano-particle, quantum dot, detectable anticarcinogen is dripped, liposome medicament and cytokine.

Those skilled in the art are familiar with radionuclide, chelating agen and chelating agen-joint conjugate to be connected to compositions and the method for part of the present invention.Particularly, radionuclide, chelating agen and chelating agen-joint conjugate to the connection of part of the present invention can be used the difunctional linking group (isodigeranyl function linking group conventionally) being for example obtained commercially to realize easily, described difunctional linking group can be connected to the functional group in the non-interference position being present on compound, then can further be connected to that for example the nanometer of radionuclide, chemotherapeutant, anticarcinogen, nano-particle, quantum dot, anticarcinogen is dripped or micromolecule toxin.In this way, compound of the present invention can be used for suitable reagent to be transported to target site to profit, conventionally tumor or have the organ or tissue of cancerous cell.In another embodiment, by by biotinylated part and Succ-PEG-DSPE Qdot605 (Quantum Dot Corp.; Hayward, Calif) together incubation prepare part Qdot complex.

In one embodiment of the invention, the liposome that comprises traceable developer can be used for for example neuroblastoma of target tumor, thereby allows to measure tumor size, growth, position or transfer.

Above-mentioned general introduction of the present invention is not intended to describe each embodiment of the present invention or each realization.By reference to following detailed description and claim and accompanying drawing, favourable aspect of the present invention and achievement and more fully understanding will become obviously and easy to understand.

In whole description, unless otherwise noted, otherwise all temperature to be degree Celsius to provide, and all percentage ratio is percentage by weight.Whole publication mentioned in this article, for the compositions describing and openly can be combined with current invention disclosed and the object of method (described compositions and method are described in publication), is integrated with herein by reference.The publication of discussing is herein only that these background technologies should not be construed as to admit formerly to invent and disclosed the present invention in order to be disclosed in the background technology before applying date of the application.

Summary of drawings

In claim, defined the present invention can be better understood by reference to following accompanying drawing.Accompanying drawing needn't, by drawing in proportion, clearly illustrate principle of the present invention but focus on.

Fig. 1: the Clip-on model of the fusion of Saposin C induction: the Saposin C of liposome combination is clamped (clip) each other by hydrophobic interaction, and induce liposome to merge.

Fig. 2: the association of Saposin C and liposome vesicle: the folding conformational change of sphingolipid activator protein of finding in the Saposin C that liposome is combined.The film topology of Saposin C interacts and shows, the amphipathic helix on amino and c-terminus is embedded in lipid bilayer and the zone line of Saposin C is exposed to water.The zone line of Saposin C is exposed to water.

Fig. 3: the schematic diagram of the functional organization of the neural axon growth of Saposin C (neuritogenic), the activation of acidic beta glucosidase and lipid binding character.Except indicating the corner of prediction and the frame of disulfide bond, figure does not mean that and represents known physical arrangement.Residue 22 to 32 is extremely important for neurotrophic effect.Across the region of residue 42 to 61, for the acid β-glucosyl enzym activation effect of Saposin C, be vital, the existence of whole 3 disulfide bond is also very important for this function.In addition, higher structure is that to have the complete activity of Saposin C necessary.Lipid/lipid film interaction zone is positioned at NH ₂-and COOH-end regions.

Fig. 4: by Ca ²⁺(a) or Saposin C (b) in the size variation of BPS (cephalin acyl serine) liposomees of pH4.7 or 7.4 inductions.Medium auto-correlation function (Fair autocorrelation function), dust=0.0%, lubber line error <1%, room temperature.

Fig. 5 .NBD-DOPS and Saposin C are to the transhipment of the cerebellum of mouse brain.By tail intravenous administration NBD-DOPS-Saposin C proteoliposome (A, C, D) and the PBS (B, E, F) of FBV/N adult mice.At the latter 48 hours freezing little brain sections of preparation of injection.Use microscope (Zeiss Axioskop, 100X) to manifest the NBD green fluorescence of the DOPS for detection of (A) and (B).At Laser Scanning Confocal Microscope (LSM510, Zeiss) under, be used in and detect the NBD green fluorescence (C and E) of the Saposin C (D and F) in Purkinje cell (Purkinje cell) and anti-His antibody (rhodamine two resisting of puting together, red fluorescence) imaging.Bar rod (Bar): 20 μ m (C-F).Term: p=Purkinje cell; G=granular cell.

The fluorescence spectrum of (20 μ the M)/SapC-DOPS proteoliposome of the PTIR-316 in the PTIR-271 in Fig. 6 .PBS (20uM)/Saposin C-DOPS proteoliposome (Fig. 6 A) and PBS (Fig. 6 B).

Fig. 7. the picked-up in the sphingolipid activator protein-C-DOPS the pure man neuroblast oncocyte (CHLA-20) that comprises PTIR-271 and PTIR-316.Fig. 7 A shows the picked-up of PTIR-271 when by SapC-DOPS liposome delivery.Fig. 7 B shows the picked-up of PTIR-316 when by SapC-DOPS liposome delivery.Contrast liposome (using the SapC-DOPS liposome-treated without PTIR-271 or PTIR-316) is shown in Fig. 7 C and 7D.Red image represents the picked-up of dyestuff.Use Zeiss Axiovert-ApoTome microscope (63X and 40X oil mirror) to carry out visual: λ _eX/ λ _eM; Beam splitter (Beam splitter): 660; The B/W phase contrast of morphocytology (phase contrast).Axiovision software is for imaging.

Fig. 8. use Sap-C-DOPS liposome that GFP22siRNA is delivered in EGFP4T1 cell.GFP22siRNA is the double-stranded RNA that specificity suppresses 22 nucleotide of Green Fluorescent Protein Gene Expression.(people such as NJ Caplen, PNAS, 2001,98:9742-9747).Incubative time is 72 hours.20x, the exposure of 800 milliseconds; Photoshop: input level 27,1.19,164; Output level 255.Size 3X2.29 inch.The liposome that Fig. 8 A and 8C representative contain GFP22siRNA; The negative control of the non-reticent siRNA (the double-stranded RNA fragment by 22 nucleotide forms) that does not affect GFP expression is wherein used in Fig. 8 B and 8D representative.All RNA is purchased from QIAGEN.

Fig. 9 .GFP22siRNA is to the microphotograph of sending in neuroblastoma (CHLA-20) cancerous cell, and it shows (a) rhodamine-GFP22siRNA; (b) phase contrast and (c) (merged) photo of merging.

In the following description of exemplary, with reference to accompanying drawing, described accompanying drawing forms the application's a part, and wherein illustrates by way of example that can put into practice various embodiments of the present invention shows.Should be understood that and can use other embodiments, and can carry out 26S Proteasome Structure and Function change and not deviate from scope of the present invention.

Detailed Description Of The Invention

Before describing this compositions and method, the present invention be should understand and specific method, device and preparation are not limited to, because these, certainly, can change.Also should be understood that term used herein is just in order to describe particular, rather than be intended to limit scope of the present invention, scope of the present invention is only subject to the restriction of claims.

Must be pointed out, as herein with claims in use, unless context point out clearly, otherwise singulative " ", " a kind of " and " this ", " this " comprise plural indication thing.Unless otherwise defined, otherwise all technology used herein and scientific terminology have with the present invention under the meaning of the same meaning conventionally understood of those of skill in the art.Although can be used for practice or test the present invention to those any method, device and materials similar or that be equal to described herein, describe now method for optimizing, device and material.

Definition

Term administering " and " using " typically refer to patient used to biocompatible materials, comprise, for example, lipid and/or vesicle and abluent (flush agent).Therefore, " using " and " using " refers to, for example, to intravascular injection lipid and/or vesicle and/or abluent.Term administering " and " using " also can refer to send lipid and/or vesicle and/or abluent to object region.

As used herein, term " aminoacid " or " aminoacid sequence " refer to the fragment of oligopeptide, peptide, polypeptide or protein sequence or any these sequences, and refer to naturally occurring or synthetic molecule.When " aminoacid sequence " is used in reference to the aminoacid sequence of naturally occurring protein molecule in this article, " aminoacid sequence " do not represent aminoacid sequence to be defined in the complete natural aminoacid sequence relevant to described protein molecule with similar term.

Term " amphipathic lipids " means to have hydrophilic " head " group and hydrophobicity " afterbody " group and there is the molecule of the ability that forms film.

As used herein, term " anionic phospholipid film " and " anionic liposome " refer to immobilized artificial membrane or the liposome that comprises lipid components and have overall negative charge under physiological pH.

" anionic phospholipid " means to have the phospholipid of negative charge, comprises phosphate ester, sulfuric ester and the lipid based on glycerol.

" bioactivator " refers to such material, and described material can be used in nature the application for diagnosis or treatment, for example, for being used for diagnosing the illness in patient's existence or non-existent method and/or being used for treating the method for patient's disease.As used herein, " bioactivator " also refer to can be in vitro and/or body in the material of performance biological effect.Bioactivator can be the positively charged or electronegative of neutrality.The example of suitable bioactivator comprises diagnostic agent, pharmaceutical preparation, medicine, synthetic organic molecule, protein, peptide, vitamin, steroid and hereditary material, comprises nucleoside, nucleotide and polynucleotide.

Term " be included in ... in (interior) " refer to that pharmaceutical agent is encapsulated in immobilized artificial membrane, so as protection pharmaceutical agent avoid external environment.This term can with " encapsulation " Alternate.

" disappearance ", as this term is used in this article, refers to and causes the non-existent aminoacid of one or more amino acid residues or nucleotide or the variation of nucleotide sequence.

Term " derivant ", as used herein, refers to the chemical modification of peptide sequence or polynucleotide sequence.The chemical modification of polynucleotide sequence can comprise, for example, with alkyl, acyl group or amino, replaces hydrogen.Derivative polynucleotide encoding keeps the polypeptide of at least one biological function of natural molecule.Derivative polypeptide is the polypeptide of for example modifying by glycosylation or any other method, and described derivative polypeptide retains at least one biological function of its polypeptide being derived from.

Term " fusogenic protein or polypeptide ", as used herein, refers to and when being added to two bi-layer membranes that separate, can make them be fused into protein or the peptide of single film.Fusogenic protein forces cell or replica close contact and their is merged.

As used herein, term " insertion " or " interpolation " refer to and in aminoacid or nucleotide sequence, cause respectively one or more amino acid residues or nucleotide to the variation of the interpolation of the sequence of finding in naturally occurring molecule.

Term " lipid " and " phospholipid " are used interchangeably and refer to the structure that comprises lipid, phospholipid or derivatives thereof, comprise the multiple different structure arrangement that known lipid adopts in waterborne suspension.This class formation includes but not limited to lipid bilayer vesicle, micelle, liposome, emulsion, vesicle, lipid band (lipid ribbon) or lipid sheet (lipid sheet).In preferred embodiments, lipid is anionic liposome.Lipid can be used alone or with those skilled in the art for any being used in combination of the desired feature of application-specific is provided.In addition the technical elements that, lipid builds and liposome forms in this area, be know and this area in any method of commonly using can be used for the present invention.

" lipid composition " refers to the compositions that conventionally comprises lipid compounds in aqueous medium.Exemplary lipid composition comprises suspension, emulsion and vesicle." lipid formulations " refers to the lipid composition that also comprises bioactivator.

" liposome " refers to common spherical cluster (cluster) or the aggregation of amphiphilic compound (comprising lipid compounds), its conventionally with one or more layers concentric layer for example the form of bilayer exist.They also can be described as lipid vesicle (lipid vesicle) in this article.

Term " long-chain lipid " refers to that carbon chain lengths is about lipid of 13,14,15,16,17,18,19,20,21,22,23 or 24.In one embodiment, chain length is selected from 18,19 or 20 chain length.The example that can be used for lipid of the present invention can obtain on network address www.avantilipids.com.The representative example that can be used for long-chain lipid of the present invention includes but not limited to following lipid:

14:0PS1,2-bis-myristoyls-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt) (DMPS); 16:0PS1,2-bis-palmityls-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt) (DPPS); 17:0PS1, the two heptadecanoyl-sn-glycerol-3-[phosphoric acid-Serines of 2-] (sodium salt); 18:0PS1,2-distearyl-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt) (DSPS); 18:1PS1,2-bis-oleoyls-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt) (DOPS); 18:2PS l, 2-bis-sub-oleoyl-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt); 20:4PS1,2-bis-arachidonic acyl-sn-glycerol-3-[phosphoric acid-Serines] (sodium salt); 22:6PS1, the two two dodecahexaene acyl-sn-glycerol-3-[phosphoric acid-Serines of 2-] (sodium salt); 16:0-18:1PS1-palmityl-2-oleoyl-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt) (POPS); Sub-oleoyl-sn-glycerol-the 3-[of 16:0-18:2PS1-palmityl-2-phosphoric acid-Serine] (sodium salt); 16:0-22:6PS1-palmityl-2-bis-dodecahexaene acyl-sn-glycerol-3-[phosphoric acid-Serines] (sodium salt); 18:0-18:1PS1-stearoyl-2-oleoyl-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt); Sub-oleoyl-sn-glycerol-the 3-[of 18:0-18:2PS1-stearoyl-2-phosphoric acid-Serine] (sodium salt); 18:0-20:4PS1-stearoyl-2-arachidonic acyl-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt); 18:0-22:6PS1-stearoyl-2-bis-dodecahexaene acyl-sn-glycerol-3-[phosphoric acid-Serines] (sodium salt); 16:0PC1,2-bis-palmityls-sn-glycerol-3-phosphocholine (DPPC); 17:0PC1, the two heptadecanoyl-sn-glycerol-3-phosphocholines of 2-; 18:0PC1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC); 16:1PC (suitable) 1,2-bis-palmitoleoyls-sn-glycerol-3-phosphocholine; The anti-PC1 of 16:1,2-Dipalmitelaidoyl-sn-glycerol-3-phosphocholine; 18:1PC Δ 6 (suitable) 1,2-Dipetroselinoyl-sn-glycerol-3-phosphocholine; 18:2PC (suitable) 1, the sub-oleoyl-sn-of 2-bis-glycerol-3-phosphocholine; 18:3PC (suitable) 1,2-bis-Caulis et Folium Lini acyl-sn-glycerol-3-phosphocholines; 20:1PC (suitable) 1,2-Dieicosenoyl-sn-glycerol-3-phosphocholine; 22:1PC (suitable) 1,2-Dierucoyl-sn-glycerol-3-phosphocholine; 22:0PC1,2-bis-mountain Yu acyl-sn-glycerol-3-phosphocholines; 24:1PC (suitable) l, the neural acyl-sn-of 2-bis-glycerol-3-phosphocholine; 16:0-18:0PC1-palmityl-2-stearoyl-sn-glycerol-3-phosphocholine; 16:0-18:1PC1-palmityl-2-oleoyl-sn-glycerol-3-phosphocholine; Sub-oleoyl-sn-the glycerol-3-phosphocholine of 16:0-18:2PC1-palmityl-2-; 18:0-18:1PC1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine; Sub-oleoyl-sn-the glycerol-3-phosphocholine of 18:0-18:2PC1-stearoyl-2-; 18:1-18:0PC1-oleoyl-2-stearoyl-sn-glycerol-3-phosphocholine; 18:1-16:0PC1-oleoyl-2-palmityl-sn-glycerol-3-phosphocholine; 18:0-20:4PC1-stearoyl-2-arachidonic acyl-sn-glycerol-3-phosphocholine; 16:0-18:1PG1-palmityl-2-oleoyl-sn-glycerol-3-[phosphoric acid-rac-(1-glycerol)] (sodium salt) (POPG); 18:1PG1,2-bis-oleoyls-sn-glycerol-3-[phosphoric acid-rac-(1-glycerol)] (sodium salt) (DOPG); 18:1PA1,2-bis-oleoyls-sn-glycerol-3-phosphate salt (sodium salt) is (DOPA); 18:1PI1,2-bis-oleoyls-sn-glycerol-3-phosphate inositol (ammonium salt); 16:0 (D31)-18:1PI1-palmityl (D31)-2-oleoyl-sn-glycerol-3-phosphate inositol (ammonium salt); 18:1PE l, 2-bis-oleoyls-sn-glycerol-3-phosphate ethanolamine (DOPE); 18:2PE1, the sub-oleoyl-sn-of 2-bis-glycerol-3-phosphate ethanolamine.

As used herein, term " nucleic acid " or " nucleotide sequence " refer to nucleotide, oligonucleotide, polynucleotide or its any fragment." nucleic acid " refers to the string of at least two base-sugar-phosphoric acid combination.(polynucleotide and oligonucleotide difference are that it comprises 120 above monomer unit).Nucleotide is the monomer unit of nucleic acid polymers.This term comprises DNA (deoxyribonucleic acid) (DNA) and the part of the ribonucleic acid (RNA) that exists with oligonucleotide messenger RNA form, antisensenucleic acids, plasmid DNA, plasmid DNA or derive from viral hereditary material.Antisensenucleic acids is the polynucleotide that disturb DNA and/or RNA function.Term nucleic acid refers to the string of at least two base-sugar-phosphoric acid combination.Natural acid has phosphate backbone, and artificial nucleic acid can comprise the main chain of other types, but comprises identical base.Nucleotide is the monomer unit of nucleic acid polymers.This term comprises DNA (deoxyribonucleic acid) (DNA) and ribonucleic acid (RNA).RNA can exist with the form of tRNA (transfer RNA), siRNA (short interfering ribonucleic acid), snRNA (small nuclear RNA, snRNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA and ribozyme.DNA can exist with the form of the derivant of plasmid DNA, viral DNA, linear DNA or chromosomal DNA or these kinds.In addition, the DNA of this type of form and RNA can be strand, double-stranded, three chains or four chains.This term also comprises other variants of the phosphate ester main chain of PNA (peptide nucleic acid(PNA)), siNA (short interfering nucleic acid), thiophosphate and natural acid.

As used herein, term " pharmaceutical agent based on nucleotide " or " medicine based on nucleotide " refer to pharmaceutical agent or the medicine that comprises nucleotide, oligonucleotide or nucleic acid.

" patient " or " experimenter " refers to and comprises mammal by animal, preferred people.

As used herein, " pharmaceutical agent or medicine " refers to any chemistry or biologic material, compound or the compositions of the therapeutic effect that can induce expectation when patient is suitably used.Some medicines are sold with inactive form, and it changes into the metabolite with pharmaceutically active in vivo.For the purposes of the present invention, term " pharmaceutical agent " and " medicine " comprise medicine and the active metabolite of non-activity.

Term " pharmacy or treatment effective dose or amount " refers to the dosage level of the biological results that is enough to induction expectation.This result can be the alleviating or the change of any other expectation of biosystem of sign, symptom or the cause of disease of the sending of pharmaceutical agent, disease, and the accurate measuring of activating agent is certainly in the progress of patient's health, the disease for the treatment of etc.

As used herein, term " sphingolipid activator protein " refers to the family of the proteins and peptides that Prosaposin is derivative, includes but not limited to naturally occurring sphingolipid activator protein A, B, C and D and derivative protein and peptide and the peptide analogues of synthetic sphingolipid activator protein that shows short fusion activity.Saposin C and can be used for one embodiment of the invention from its derivative polypeptide.

Term " short chain lipid " refers to that carbon chain lengths is 4,5,6,7,8,9,10,11 or 12 lipid.In one embodiment, carbon chain lengths is 6,7,8,9 or 10.In one embodiment, carbon chain lengths is 6,7 or 8.The example of electronegative short chain lipid can obtain on the www.avantilipids.com of website.The example that can be used for short chain lipid of the present invention includes but not limited to following: 06:0PS (DHPS) 1,2-bis-hexanoyls-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt); 08:0PS1,2-bis-decoyls-sn-glycerol-3-[phosphoric acid-Serine] (sodium salt); 03:0PC1,2-bis-propionyl-sn-glycerol-3-phosphocholine; 04:0PC1,2-bis-butyryl-sn-glycerol-3-phosphocholine; 05:0PC1,2-bis-valeryls-sn-glycerol-3-phosphocholine; 06:0PC (DHPC) 1, DHPC; 07:0PC1,2-bis-oenanthyl-sn-glycerol-3-phosphocholine; 08:0PC1,2-bis-decoyls-sn-glycerol-3-phosphocholine; 09:0PC1,2-bis-nonanoyls-sn-glycerol-3-phosphocholine; 06:0PG1,2-bis-hexanoyls-sn-glycerol-3-[phosphoric acid-rac-(1-glycerol)] (sodium salt); 08:0PG1,2-bis-decoyls-sn-glycerol-3-[phosphoric acid-rac-(1-glycerol)] (sodium salt); 06:0PA1,2-bis-hexanoyls-sn-glycerol-3-phosphate salt (sodium salt); 08:0PA1,2-bis-decoyls-sn-glycerol-3-phosphate salt (sodium salt); 06:0PE1,2-bis-hexanoyls-sn-glycerol-3-phosphate ethanolamine; 08:0PE1,2-bis-decoyls-sn-glycerol-3-phosphate ethanolamine.

As used herein, term " short interfering nucleic acid ", " siNA ", " short interfering rna ", " siRNA ", " short interfering nucleic acid molecule ", " short interference oligonucleotide molecules " or " the short interfering nucleic acid molecule of chemical modification " refer to can be for example by disturbing " RNAi " with sequence-specific mode mediate rna or gene silencing suppresses or any nucleic acid molecules of down-regulation of gene expression or virus replication.In exemplary, siNA is the double-stranded polynucleotide molecule that comprises the complementary sense and antisense region of oneself, wherein antisense district inclusion with to lower nucleotide sequence in the target nucleic acid molecule of expression or the nucleotide sequence of its part complementation, there is adopted district inclusion corresponding to the nucleotide sequence of (that is, being same as substantially) described target nucleic acid sequence or its part in sequence." siNA " refers to that it is short length double-strandednucleic acid (or optionally its longer precursor) and the small RNA that there is no unacceptable toxicity in target cell, for example siRNA.The length of the interior useful siNA of the present invention is optimum about 21 to the 23bp long length that turn in certain embodiments.Yet useful siNA comprises that the length of siRNA does not exist specific restriction.For example, can siNA is and be handed to cell with precursor forms at first, described precursor forms be significantly different from when being delivered to target cell or has afterwards and bring into play the final or form processing of the siNA of active for gene silencing.The precursor forms of siNA can for example comprise precursor sequence element, and described precursor sequence element is processed when sending or after sending, degraded, change or cutting, thereby is created in the active siNA in cell with mediated gene silencing.Therefore, in certain embodiments, the useful siNA in the present invention for example will have approximately 100-200 base pair, 50-100 base pair or be less than the forebody length of about 50 base pairs, and it will produce the activated siNA through processing of tool in target cell.In other embodiments, useful siNA or siNA forebody length are about 10 to 49bp, 15 to 35bp or about 21 to 30bp.

" vesicle " refers to spherical entity, and its feature is to exist one or more layers wall or the film that forms one or more interior spaces conventionally.Can be for example from lipid (comprising various lipid described herein), proteinaceous material, polymeric material (comprising natural, synthetic and semisynthetic polymer) or surfactant preparation vesicle.Preferred vesicle is to comprise from the wall of lipid preparation or the vesicle of film.In these preferred vesicles, lipid can exist with the form of monolayer or bilayer, and monolayer or bilayer lipid can be used for forming one or more layers monolayer or bilayer.In the situation that surpassing one deck monolayer or bilayer, monolayer or bilayer can be concentric.Lipid can be used for forming unilamellar vesicle (comprising one deck monolayer or bilayer), few layer vesicle (comprise about 2 or about 3 layers of monolayer or bilayer) or multilamellar vesicle (comprise and surpass about 3 layers of monolayer or bilayer).Similarly, the vesicle of preparing from protein or polymer can comprise one or more layers concentric walls or film.The wall of the vesicle of preparing from protein or polymer or (uniformly) that film can be homogenizing substantially or they can be (semi-porous) porous or half porous.Vesicle described herein comprises and being commonly referred to such as liposome, micelle, vesicle (bubble), microvesicle capsule, microsphere, by this type of entity of vesicle of the coated vesicle of lipid, polymer, albumen and/or surfactant, microvesicle capsule and/or microsphere, microsphere, aeroge (aerogel), clathrate (clathrate) combination etc.The interior space of vesicle can be filled with liquid (comprise, for example, waterborne liquid), gas, gaseous precursors and/or solid or solute material, comprises, for example, targeting part and/or bioactivator (as expected).

Fusogenic protein or polypeptide

In one embodiment, the invention provides the immobilized artificial membrane that comprises one or more lysosome fusogenic proteins or polypeptide.In another embodiment, one or more lysosome fusogenic proteins or polypeptide are included in anionic liposome.In another embodiment, anionic liposome also comprises pharmaceutical agent.

Be used for the albumen that suitable lysosome fusogenic protein of the present invention and polypeptide include but not limited to sphingolipid activator protein family, for example Saposin C.What also comprise is the congener of Saposin C, and wherein said congener is because of coding Saposin C and have the polypeptide of the biologic activity similar to Saposin C and the degeneracy of the genetic code of peptide analogues has at least 80% sequence homology.

The example of peptide or peptide analogues comprises:

Ser-Asp-Val-Tyr-Cys-Glu-Val-Cys-Glu-Phe-Leu-Val-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu-Asp-Ala-Phe-Asp-Lys-Met-Cys-Ser-Lys-Leu-Pro(SEQ.ID.No.1)；

Val-Tyr-Cys-Glu-Val-Cys-Glu-Phe-Leu-Val-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu-Asp-Ala-Phe-Asp-Lys-Met-Cys-Ser-Lys-Leu-Pro(SEQ.ID.No.2)，

With its derivant, analog, congener, fragment and mixture.

What also comprise is the polypeptide of following formula:

h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys-Glu-h-h-Asp-h-h-Asp-Lys-h-Cys-u-Lys-h-h，

Wherein h=hydrophobic amino acid, comprises Val, Leu, Ile, Met, Pro, Phe and Ala; And the uncharged polar amino acid of u=, comprise Thr, Ser, Tyr, Gly, Gln and Asn.

Be used for the protein that suitable lysosome fusogenic protein of the present invention and polypeptide include but not limited to sphingolipid activator protein family, preferably Saposin C.What also comprise is the congener of Saposin C, and wherein said congener is because of coding Saposin C and have the polypeptide of the biologic activity similar to Saposin C and the degeneracy of the genetic code of peptide analogues has at least 80% sequence homology.

As used herein, term " peptide analogues " refers to such peptide, its on aminoacid sequence with the natural different conservative amino acid replacement that is only of peptide, such as Leu to the displacement of Val or Arg to displacement of Lys etc., or be to be positioned at locational one or more non-conservative amino acid replacements, disappearance or the insertion of the biologic activity (the short Fusogenic properties of peptide in this case) of not destroying peptide.As used herein, peptide analogues also can comprise one or more amino acid analogues (simulating the molecule of amino acid whose structure) and/or the natural amino acid of finding in the molecule except peptide or peptide analogues, as its sequence partly or entirely.

" analog " means displacement or the change in the aminoacid sequence of peptide of the present invention, described displacement or change the short Fusogenic properties that can not adversely affect peptide.Therefore, analog can comprise such peptide, described peptide have to be provided as herein SEQ ID NO:1 and 2 peptide substantially same aminoacid sequence and wherein one or more amino acid residues used chemically similar conservative aminoacid substitutions.The example of conservative substitution comprise nonpolar (hydrophobicity) residue for example isoleucine, valine, leucine or methionine to another amino acid whose displacement.Similarly, the present invention relates to polarity (hydrophilic) residue for example between arginine and lysine, between glutamine and agedoite and the displacement between glycine and serine.In addition, also relate to alkaline residue for example lysine, arginine or histidine to another amino acid whose displacement or acidic residues for example aspartic acid or glutamic acid to another amino acid whose displacement.

The formation of immobilized artificial membrane and liposome

The film that the present invention utilizes anionic phospholipid film to realize the mediation of sphingolipid activator protein merges, thereby specific pharmaceutical preparation or developer are sent through film skin or mucosa or through blood brain barrier or other cell membrane.This type of anionic phospholipid film is generally used for preparing liposome.The small vesicle that liposome is comprised of concentric lipid bilayer and, as used herein, refer to the vesicles being formed by the amphipathic lipids being arranged in spherical bilayer.In structure, the size and shape of liposome can change at long tube to the scope of spheroid, and size changes to the scope of millimeter at hundreds of dusts.Regardless of overall shape, bilayer is generally organized as airtight concentric thin layer, and aqueous layer is adjacent layer separately by each thin layer.Vesicle size conventionally at diameter about 20 to about 30, in the scope of 000nm.

Liquid film between thin layer is generally approximately 3 to 10nm.Several different methods for the preparation of different liposome form has been described in periodical and patent documentation.The special summary and the information that about liposome, form, can referring to the summary of being write by Pagano and Weinstein (referring to Ann.Rev.Biophysic.Bioeng., 7,435-68 (1978) and Ann.Rev.Biophysic.Bioeng., 9,467-508 (1980)).

In one embodiment, anionic phospholipid film is vesicle.In another embodiment, vesicle is liposome.Liposome is for example form of nano-particle or liposome of nano container (nanocontainer), is generally used for the encapsulation of medicine.Liposome preferably has the average diameter of approximately 200 nanometers.In another embodiment, liposome has the average diameter of 50 to 350 nanometers.In another embodiment, liposome has the average diameter of 150 to 250 nanometers.

Liposome to target tissue for example the specific delivery of proliferative cell group, tumor tissue, inflammatory tissue, Inflamed tissue and infected tissue can be suitable for therapeutic agent delivery to the liposome size of described target tissue to realize by selection.For example, the liposome that has an average diameter of 180nm may not accumulate in solid tumor; There is the liposome of average diameter of 140nm in the periphery accumulation of identical solid tumor, and the liposome of average diameter with 110nm is in periphery and the core accumulation of this solid tumor.

About relating to the embodiment of vesicle, can with regard to the final use of concrete expectation, adjust (comprising for example diagnosis and/or therapeutic use) size of vesicle.The size of vesicle can be preferably approximately 30 nanometers (nm) to approximately 300 microns (μ m) on diameter, and all combinations within the scope of this and sub-combination (subcombination).More preferably, vesicle has about 100nm to the diameter of approximately 10 μ m, and about 200nm to the average diameter of approximately 7 μ m be preferred.About concrete purposes intravascular way for example, comprise the nuclear magnetic resonance of vascular system, vesicle preferably diameter is not more than approximately 30 μ m, and less vesicle is preferred, and for example, diameter is not more than the vesicle of approximately 12 μ m.In some preferred embodiment, the diameter of vesicle can be approximately 7 μ m or less, and the vesicle with approximately 5 μ m or less average diameter is preferred, and the vesicle with approximately 3 μ m or less average diameter is preferred.

If desired, can by several different methods (for example comprise shake, microemulsified, vortex, extrude, filtration, supersound process, homogenize (homogenization), multigelation circulation, under pressure by determining extruding and similar method of big or small hole) adjust the size of liposome.

Except above-mentioned lipid, protein properties and/or poly-compounds or replace, compositions described herein can comprise one or more stabilizing materials (stabilizing material).The example of this type of stabilizing material is biological example compatible polymer.Stabilizing material can be used for helping the formation of vesicle and/or guarantees gas or the abundant encapsulation of gaseous precursors.Even for relatively do not allow, non-dispersive gas for example perfluoropropane or sulfur hexafluoride, when by one or more stabilizing materials when being filled with the formation of vesicle of gas and gaseous precursors, can obtain the vesicle of improvement.This compounds can help improve stability and the integrity of vesicle aspect size, shape and/or other attributes of vesicle.

Term " stable " or " through stable ", as used herein, expression vesicle can be resisted degraded substantially, for example comprises that the gas of imitated vesicle structure or encapsulation or the forfeiture of gaseous precursors continue useful a period of time.Conventionally, for vesicle of the present invention, there is the shelf life of expectation, conventionally under home condition, keep its prototype structure volume at least about 90% time of at least about 2 to 3 weeks.In a preferred form, be at least about 1 month the stabilization time of the expectation of vesicle, more preferably at least about 2 months, and more preferably at least about 6 months, more preferably about 18 months and more preferably reach about 3 years.Vesicle described herein, comprises the vesicle that is filled with gas and gaseous precursors, in disadvantageous condition, can be even also stable under for example higher or lower than the temperature and pressure experiencing under home condition.

The stability of vesicle described herein can be at least in part owing to the material of manufacturing vesicle, comprise for example above-mentioned lipid, polymer and/or protein, and conventionally needn't use other stabilizing material, although optionally can do like this and can preferably do like this.This type of other stabilizing material and its feature are more at large described hereinafter.

The material of structure vesicle is biocompatibility lipid, protein or polymeric material preferably, and in this type of material, biocompatibility lipid is preferred.In addition, due to the easiness (comprise, can prepare vesicle before being about to use) of preparation, easily this type of vesicle of in situ preparation.

It as the biocompatible polymer of preparing the stabilizing material of the vesicle that is filled with gas and gaseous precursors, can be the polymer in natural, semi-synthetic (modified is natural) or synthetic source.As used herein, term polymer refers to and comprises two or more repeated monomer units, preferably 10 or the compound of more repeated monomer units.As used herein, the semisynthetic polymer of phrase (or modified natural polymer) refers to, with the natural polymer of some mode chemical modification.Be suitable for exemplary natural polymer of the present invention and comprise naturally occurring polysaccharide.This type of polysaccharide comprises for example arabinan, levan, fucosan, galactan, polygalacturonic acid, glucosan, mannan, xylan (for example, for example inulin), Gum levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectin, comprises amylose, pulullan polysaccharide, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, dextrosan, pustulan, chitin, agarose, Keratin, chrondroitin (chondroitan), dermatan, hyaluronic acid, alginic acid, xanthan gum, starch and various other natural homopolymer or heteropolymers, for example, comprise following aldose, ketose, the homopolymer of one or more of acid or amine or heteropolymer: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, lucose, mannose, gulose, idose, galactose, talose, Erythrulose (erytirulose), ribulose, xylulose, psicose, fructose, sorbose, Tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, agedoite, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucosaccharic acid, galacturonic acid, mannuronic acid, glucamine, galactosamine and neuraminic acid, and its naturally occurring derivant.Therefore, suitable polymer comprises for example protein, for example albumin.Exemplary semi synthetic polymer comprises carboxymethyl cellulose, hydroxy methocel, hydroxypropyl emthylcellulose, methylcellulose and methoxyl group cellulose.Be suitable for exemplary synthetic polymer of the present invention and comprise that polyethylene (for example, Polyethylene Glycol, polyoxyethylene and polyethylene terephthalate (polyethylene terephthlate)), polypropylene (for example, polypropylene glycol), polyurethane (for example, polyvinyl alcohol (PVA), polrvinyl chloride and polyvinylpyrrolidone), polyamide (for example comprises nylon, polystyrene, polylactic acid, fluorinated hydrocarbon, perfluorocarbon, politef), and polymethyl methacrylate, and its derivant.When present disclosure and information known in the art United States Patent (USP) 5 for example, 205, in 290 (its disclosure is integrated with in full herein by reference with it) information that describe and that mention in conjunction with time, once use present disclosure, for the preparation of vesicle, to use polymer will be obviously as the method for stable compound to those skilled in the art.

Conventionally, size of population that can be based on them and the character of laminate structure will be divided into 3 classes (referring to the new york academic science meeting of in December, 1977, " Liposomes and Their Use in Biology and Medicine ") for liposome of the present invention.4 classification comprise multilamellar vesicle (MLV), small-sized unilamellar vesicle (SUV), large-scale unilamellar vesicle (LUV) and huge unilamellar vesicle (GUV).SUV and LUV, by definition, only have a bilayer, and MLV comprises many concentric bilayers.The spherical unilamellar vesicle (ULV) with low polydispersity can spontaneous formation in charged mixture of phospholipids.The formation of this type of low polydispersity ULV needs the process that sharply raises or dilute suddenly of temperature conventionally.In some cases, spontaneous low polydispersity ULV is high stability on inspection within a period of time and when dilution, and this shows that it has the great potential becoming for the package carrier of drug delivery or gene therapy.Referring to Nieh, wait people Low-Polydispersity Phospholipid Unilamellar Ellipsoidal Vesicles and Their Interaction with Helical Domains of Saposin C, manuscript.

The size, composition and the electric charge that depend on them, liposome is shown extensively various feature.For example, the liposome with the unsaturated lipids of little percentage ratio tends to slightly more can see through, yet that the liposome that mixes cholesterol or other sterin tends to is harder and compared with impermeable.Depend on hydrophilic radical, liposome can be positive, negative or neutral on electric charge.For example, the lipid based on choline is given the electric charge of overall neutral, lipid based on phosphoric acid and sulphuric acid contribution negative charge, and the lipid based on glycerol is conventionally electronegative, and sterin is normally neutral but have charged group in solution.For lipid of the present invention, be anion and neutral lipid.

The many methods relevant to the preparation of liposome composition are obtainable.Therefore, any that can use many conventional liposome technologies of preparing prepared liposome, and described technology it will be apparent to those skilled in the art that.This type of technology comprises such as solvent dialysis, not crusher (French press), extrusion molding (utilizing or do not utilize freeze thawing), reverse phase evaporation, simple freeze-thaw method, supersound process, chelating dialysis (chelate dialysis), homogenize, solvent infusion, microemulsified, spontaneous formation, solvent evaporation, solvent dialysis, French press (French pressure cell) technology, controlled detergent dialysis (controlled detergent dialysis) etc., relates to separately and prepare in a different manner vesicle.Referring to for example, the people such as Madden, Chemistryand Physics of Lipids, 199053,37-46, its disclosure is complete to be by reference incorporated to herein.Suitable freeze-thaw technology is described in the international application series No.PCT/US89/05040 for example submitting on November 8th, 1989, and its disclosure is complete to be by reference incorporated to herein.About the preparation of liposome, the method that relates to freeze-thaw technology is preferred.Can for example carry out the preparation of liposome in saline solution, phosphate-buffered aqueous solution or sterilized water at solution.Also can shake or the distinct methods of vortex is prepared liposome by relating to.This can be for example by using for example Wig-L-Bug (Crescent Dental of mechanical shaking device, Lyons, I11.), by Degussa AG, Frankfurt, the Mixomat that Germany sells, by Espe Fabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, the Capmix that Oberay Germany sells, by Vivadent, the Silamat Plus that Lechtenstein sells or by Quayle Dental, Sussex, the Vibros that England sells realizes.Also can use for example Microfluidizer (Microfluidics, Woburn, Mass.) of conventional microemulsified equipment.

Also can use spray drying method to prepare vesicle.By utilizing the method, can be in aqueous environments premixing lipid, then sprayed dry to produce the vesicle that is filled with gas.Can under the headroom of the gas of expecting, be stored in vesicle.

The many liposome technologies of preparing that are applicable to prepare vesicle through transformation are discussed in for example U.S. Patent No. 4,728,578; UK Patent Application GB2193095A; United States Patent (USP) 4,728,575; U.S. Patent No. 4,737,323; International application series No.PCT/US85/01161; The people such as Mayer, Biochimica et Biophysica Acta, the 858th volume, pp.161-168 (1986); The people such as Hope, Biochimica et Biophysica Acta, the 812nd volume, pp.55-65 (1985); U.S. Patent No. 4,533,254; The people such as Mayhew, Methods in Enzymology, the 149th volume, pp.64-77 (1987); The people such as Mayhew, Biochimica et Biophysica Acta, the 755th volume, pp.169-74 (1984); The people such as Cheng, Investigative Radiology, the 22nd volume, pp.47-55 (1987); International application series No.PCT/US89/05040; U.S. Patent No. 4,162,282; U.S. Patent No. 4,310,505; U.S. Patent No. 4,921,706; With Liposome Technology, Gregoriadis, G, ed., I volume, pp.29-31,51-67 and 79-108 (CRC Press Inc., Boca Raton, Fla.1984), its disclosure is separately complete to be by reference incorporated to herein.

Alternatively, can in the preparation of compositions, comprise one or more antibacterial agents and/or antiseptic, for example sodium benzoate, quaternary ammonium salt, Sodium Azide, methyl parahydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid, ascorbyl palmitate, Butylated hydroxyanisole (butylated hydroxyanisole), butylated hydroxytoluene, methaform, dehydroacetic acid, ethylenediamine, thioglycerol, Potassium Benzoate, potassium metabisulfite, potassium sorbate, sodium sulfite, sulfur dioxide and organic mercury salt.When stable vesicle is for for example invading under environment in blood vessel or during intraperitoneal imaging, such sterilizing (it also can for example complete by radiation by other conventional methods) will be essential.Based on present disclosure, the suitable method of sterilizing will be obvious for technical staff.

The same with the preparation of lipid and/or vesicle, can obtain numerous technology and prepare lipid formulations.For example, can prepare lipid and/or vesicle formation from the mixture of lipid compounds, protein and bioactivator.In this case, prepare as mentioned above lipid composition, wherein compositions also comprises bioactivator.Therefore, for example, can in the situation that existing, prepare bioactivator micelle.

As recognized by those skilled in the art, can be by any lipid and/or vesicle and/or lipid and/or vesicle formation lyophilizing to store, and for example utilize aqueous medium (for example sterilized water, phosphate buffered solution or saline solution) to rebuild by vigorous stirring.For the lipid that prevents from producing due to lyophilizing and/or coagulation or the fusion of vesicle, can usefully comprise the additive that prevents that this type of fusion or coagulation from occurring.Operable additive comprises for example PEG400 of sorbitol, mannitol, sodium chloride, glucose, trehalose, polyvinylpyrrolidone and PEG (PEG).This type of and other additives are described in document, American Pharmacopeia for example, USP XXII, NF XVII, The United States Pharmacopeia, The National Formulary, United States Pharmacopeial Convention Inc., 12601Twinbrook Parkway, Rockville, in Md.20852, its disclosure is complete to be by reference incorporated to herein.The preparation of lyophilizing has advantages of the longer shelf life conventionally.

Conventionally, lipid mixture of the present invention comprises anion long-chain lipid.In one embodiment, the lipid mixture for the synthesis of the liposome that comprises Saposin C comprises: 1) anion long-chain lipid, 2) neutral long-chain lipid and 3) short chain lipid.Short chain lipid can be neutral or anion.In another embodiment, lipid mixture only comprises anion long-chain lipid and neutrality or anion short chain lipid.Following table for example understands and can be used for the synthetic liposome that comprises Saposin C to realize the example of combination of the phospholipid of object of the present invention.Can use method described herein to the polypeptide that adds Saposin C or Saposin C in the combination of following lipid.Lower list 1 illustrates the combination that can be used for putting into practice phospholipid of the present invention.Example is not exhaustive, and is just intended to illustrate possible embodiment of the present invention.

Table 1

Table 1. can be used to form the combination according to the long-chain of the liposome that comprises fusogenic protein of the present invention and short-chain phospholipid with the polypeptides in combination of Saposin C or Saposin C.

Observe, in liposome complex, the existence of Saposin C albumen makes liposome membrane stabilization removal and by membrane reconstitution, thereby causes utilizing the limited shelf life of the liposome delivery system of this protein.Referring to the people such as Mu-Ping Nieh, Low-Polydispersity Phospholipid Unilamellar Ellipsoidal Vesicles and Their Interaction with Helical Domains of Saposin C; 2005.The invention solves this problem.One embodiment of the invention comprise the use of the short chain lipid of at least one type.The interpolation of short chain lipid causes stabilisation and the increase of liposome shelf life of film, thereby has increased practicality and the availability of the therapeutic agent based on liposome.

An example for the synthesis of the lipid mixture of Saposin C liposome is, the mixture that comprises electronegative lipid DOPS (DOPS), wherein adds a small amount of neutral long-chain lipid dipalmitoyl phosphatidyl choline (DPPC) and neutral short chain lipid DHPC (DHPC).Referring to for example, the people such as Nieh, Low-Polydispersity Phospholipid Unilamellar Ellipsoidal Vesicles and Their Interaction with Helical Domains of Saposin C, manuscript.Can use known in the art in electric charge and length corresponding lipid arbitrarily.The sample that contains this lipid composition that is mixed with a small amount of Saposin C can stabilization removal, but for the system with the Saposin C of higher concentration, large aggregation can be from the solution precipitation of system, and this shows the stabilization removal of film.DOPS/DPPC/DHPC sample is stable within the period of 24 months, and this shows that the interpolation of neutral long-chain lipid and short chain lipid strengthens the stability of aggregation.Yet, can use according to invention described herein the combination in any of long-chain and short chain lipid.

Electronegative long-chain lipid of the present invention can be any long-chain phospholipid, and it has length is approximately 14 to approximately 24 carbon, or length is the carbochain of approximately 18 to approximately 20 carbon.Can on www.avantilipids.com, obtain the limit catalogue of lipid.Which kind of lipid those skilled in the art know can be used for the present invention.Although can use the combination in any of long-chain and short chain lipid, the liposome that some combination results are more stable.For example, although be not intended to limit the present invention, following aspect can instruct the selection of the compositions that forms liposome: when using the long-chain that length is approximately 20 to approximately 24 carbon, the short chain lipid with the length of approximately 6 to approximately 8 carbon can be used to improve liposome stability.When using the long-chain length of approximately 14 to approximately 18 carbon, the short chain lipid with the length of approximately 6 to approximately 7 carbon can be used for improving liposome stability.Although the more stable liposome of this type of combination results of lipid, also can successfully be used other combinations, and be not intended to abandon other combinations.Table 2 for example understands the example of the phospholipid combination that can be used for the more stable liposome of generation.Yet this type of example is not intended to imply that other combinations of phospholipid can not be for the present invention.

Table 2

Table 2., based on phospholipid chain length, can be used for the example of the combination of long-chain of the present invention and short-chain phospholipid.The invention is not restricted to following combination.

In addition the existence of saturated hydrocarbons or do not have the stability that affects liposome on lipid chain.For example, use and to have approximately 18 or the lipid of the chain length of more carbon, phospholipid can be saturated or unsaturated, preferably undersaturated.The long-chain lipid for example for shorter long-chain lipid with approximately 14 to approximately 16 carbon, lipid can be undersaturated, but the use of saturated lipid produces the performance of the present invention improving.

The example of suitable drugrlipid ratio is as follows.The neutral phospholipid of selecting in compositions is about 1:10 (approximately 10% neutral phosphor lipid) or about 1:5 (approximately 20% neutral phospholipid) or 1:1 (50% neutral phospholipid) to the mol ratio of the electronegative phospholipid of selecting.The long-chain phospholipid of selecting in compositions is about 4:1 (approximately 20% short chain) to the mol ratio of the short chain lipid of selecting, and can be for about 10:1 (approximately 10% short chain) is to about 3:1 (approximately 33% short chain).Long-chain is as follows to the ratio of short chain example in one embodiment: [neutral long-chain lipid]+[acid long-chain lipid])/[neutral short chain lipid] is approximately 4.As another example, in one embodiment, in mixture DOPS to the mol ratio of DPPC at about 10-8:1, or in the scope of about 7-6:1 or about 5-3:1 or about 1-2:1, and ([DPPC]+[DOPS])/DHPC=approximately 4.For suitable lipid of the present invention, can be selected from any lipid providing on known in the art or www.avantilipids.com.

Table 3

Table 3. from the hydrodynamic radius (Hydrodynamic radii) of the DLS data of DOPS/DPPC/DHPC aggregation in solution (nm), wherein [DOPS]+[DPPC])/DHPC=4.Only have the sample of DOPS/DPPC=10 just to show bimodal distribution.

In order to make many medicines there is treatment potential, they must be delivered to position suitable in health, and medicine must have the ability that reaches required tissue.Liposome can form the basis that continues drug release and be delivered to the specific part of particular cell types or health.The therapeutic use of liposome also comprises that send is conventionally poisonous medicine when free form.In liposome form, drug toxicity is closed, thereby can avoid the region to the tissue of this medicaments insensitive and targeting selection.Also can by liposome therapeutic for discharge medicine within the period extending, thereby reduce frequency of administration.In addition, liposome also can be provided for forming the method for the aqueous dispersion be conventionally not suitable for hydrophobicity that intravenous sends or amphipathic medicine.

Liposome of the present invention can comprise one or more and be trapped in pharmaceutical agent and/or the developer between aqueous interior or bilayer, or in bilayer, comprises described reagent by hydrophobic molecule is captured in.Several technology can be used for using liposome by the host tissue of the drug targeting selection of encapsulation and avoid sensitive organization.This type of technology comprises handles the size of liposome, their clean surface charge and their route of administration.

Also can send liposome of the present invention by passive route of delivery.Liposome passive sent and comprised and use various route of administration, for example intravenous, subcutaneous, intramuscular and local approach.Each approach produces difference on the location of liposome.

Liposome of the present invention is also desirable for delivering therapeutic agents or developer through blood brain barrier.The present invention relates to can be by the liposome containing therapeutic agent for being delivered to this type of reagent the method for CNS, and wherein said pack is contained in the liposome of the variant that contains above-mentioned lipid and Saposin C, Prosaposin or sphingolipid activator protein.Can by intravenous injection, intramuscular injection, nasal delivery there or arbitrarily other intravascular delivery method (method of using this area conventionally to accept) use the liposome that comprises therapeutic agent.

Be not wishing to be bound by theory, a possible mechanism that how to mediate film fusion about sphingolipid activator protein is to change by protein conformation.In the middle of the derivative protein of Prosaposin, sphingolipid activator protein A and Saposin C show the aminoacid homogeneity/similarity of top.By calculating, predict that two kinds of protein are all folded into amphipathic helix Shu Jixu.Usually, sphingolipid activator protein is folding is that 5 amphipathic alpha-helixs are folded to the common supersecondary structure of single globular domain and are common for two kinds of protein.In one embodiment, the folding spiral along being positioned at center on aminoterminal carries out, and for its spiral 2 and 3, from a side, piles up and spiral 4 and 5 is piled up from opposite side.This folding interface that can be provided for membrane interaction.

Sphingolipid activator protein mediation, it is believed that it is two step process with the mechanism of the fusion of anionic phospholipid film.In first step, the positively charged aminoacid (alkaline form) of sphingolipid activator protein, lysine (Lys) and arginine (Arg), and the electrostatic interaction between electronegative immobilized artificial membrane causes the association (referring to Fig. 1) between these two kinds.In second step, in the molecule between the spiral of the sphingolipid activator protein of two vicinities, hydrophobic interaction makes two films enough close, thereby makes the fusion of film that (referring to Fig. 2) occur.

Therefore, according to the present invention, sphingolipid activator protein, particularly the association of Saposin C and lipid conventionally need to approximately 5.5 or lower pH scope, because the initial association of Saposin C and film produces by the positively charged alkaline amino acid residue of Saposin C and the electrostatic interaction of anionic membrane.Therefore, high expectations basic amino acid exists with its protonated form, to obtain a large amount of electrostatic interactions.

Alternatively, the related fusion of the sphingolipid activator protein family of egg-derived white matter and peptide can not have this lower pH scope restriction, thereby the pH scope of other synexins and peptide can be in physiological pH (approximately 7 pH) to lower pH.

Bioactivator

According to the present invention, by bioactivator for example pharmaceutical agent be included in anionic phospholipid film or liposome, to carry out the lower or through the transportation of blood brain barrier or other cell membrane to film skin or mucosa and/or to it of sphingolipid activator protein mediation.Activating agent can be large biomolecule, includes but not limited to lipid, particularly ceramide, steroid, fatty acid, triacylglycerol, gene and protein, DNA, RNA or siRNA.Activating agent also can be comprised of little organic molecule.As used herein, " pharmaceutical agent " means to provide beauty treatment or the treatment any materials of benefit or the mixture of material when by Saposin C liposome delivery.

Exemplary bioactivator or the medicine that can send by system of the present invention can include but not limited to analgesic, anesthetics, antifungal, antibiotic, antiinflammatory, vermifuge (anthelmintics), antidote, Bendectin, antihistaminic, antihypertensive, antimalarial, antimicrobial drug (antimicrobials), psychosis, antipyretic, antimicrobial drug (antiseptics), anti-arthritic, antitubercular agent, cough medicine, antiviral agents, cardioactive medicine (cardioactive drug), cathartic, chemotherapeutant, corticosteroids (steroid class), antidepressant, tranquilizer, diagnostic aid (diagnostic aid), diuretic, enzyme, expectorant, hormone, sleeping pill, mineral, supplementary (nutritional supplements), parasympathomimetic agent, potassium supplement, tranquilizer, sulfa drugs, analeptic, sympathomimetic (sympathomimetics), sedative, urinary system anti-infective (urinary antiinfective), vasoconstrictor, vasodilation, vitamin, xanthine derivative etc.

Folium Digitalis Purpureae medicine

The preferred embodiment of pharmaceutical agent comprises Folium Digitalis Purpureae medicine for example digoxin, Digitoxin, digoxigenin and cerberigenin.This type of medicine is all mainly as cardiac drug.

Steroidal compounds (Steroidal Compounds)

Steroidal compounds forms the pharmaceutical agent of another kind of preferred kind.The example of steroid drugs reagent is testosterone (17 beta-hydroxy androstane-4-en-3-ketone), main male steroid.Its primary treatment purposes is to treat the defective endocrine function of testis.Estradiol (female steroid-l, 3,5 (10)-triolefin-3,17-isoallopregnane-3β) is also preferred steroid drugs reagent.Estradiol and its ester derivant are specified for treating menopause and cause the symptom of other situations that endogenous estrogen production lacks.Progesterone is also preferred steroid drugs reagent.Progesterone is mainly used in suppressing rutting period or the rutting period of making is synchronous and control habitual abortion and diagnosis and treatment menoxenia.Preferred steroid drugs reagent in addition comprises 3-hydroxyl-5 alpha-pregnane-20-ketone, 3-beta-hydroxy-pregnant steroid-5-alkene-20-ketone and related compound.

Nonsteroidal and-inflammatory drug (NSAID)

The example of NSAID comprises piroxicam (4-hydroxy-2-methyl-N-2-pyridine radicals-2H-l, 2-benzothiazine-3-carboxylic acid amides 1, 1-dioxide), diclofenac, ibuprofen, ketoprofen, Pethidine, dextropropoxyphene, nalbuphine, pentazocine, buprenorphine, aspirin, indomethacin, diflunisal, acetaminophen, naproxen, fenoprofen, piroxicam, sulindac, tolmetin, meclofenamic acid, zomepirac, penicillamine, Phenylbutazone, oxyphenbutazone, chloroquine, oxychloroquine, azathiaprine, cyclophosphamide, levamisole, prednisone, prednisolone, betamethasone, triamcinolone and methylprednisolone and indomethacin (l-(4-chlorobenzene formacyl)-5-methoxyl group-2-methyl-lH-indole-3-acetic acid).

Based on amino acid whose medicine

Medicine based on protein and peptide and other also can be used as according to pharmaceutical agent of the present invention based on amino acid whose medicine.The problem relevant to conventional delivery strategies for protein and peptide medicine is known.This type of medicine Orally administered conventionally because of the degraded in gastrointestinal tract with do not absorb impracticable.Therefore, parenteral approach remains main route of delivery.

Based on amino acid whose medicine cephalosporin for example, conventionally have and be less than approximately 5000, be preferably less than approximately 2500, be more preferably less than approximately 1000 molecular weight.Protein and peptide medicine have conventionally at least about 100 daltonian molecular weight, more typically approximately 200 to 40,000 daltonian molecular weight.The example of the peptides and proteins in this magnitude range includes but not limited to, luteinizing hormone releasing hormone, somatostatin, Kallidin I, goserelin, pituitary growth hormone, buserelin, platelet derived growth factor, triptorelin, gonadorelin, asparaginase, nafarelin, Bleomycin Sulphate, leuprorelin, chymopapain, somatotropin releasing factor, parathyroid hormone (PTH), cholecystokinin, chorionic-gonadotropin hormone, insulin, thyroliberin (ACTH), calcitonin erythropoietin, glucagon, hyaluronidase, interferon is interferon-alpha for example, interleukin is IL-1 for example, throtropin releasing hormone, menotropin, pituitary hormone (Urofollitropin (folliculus hGH for example, hMG, hCG, FSH etc.), melanotropin, gonadotropin-releasing hormone, oxytocin, vassopressin, streptokinase, tissue plasminogen activator, angiotensin-ii antagonist, Kallidin I intensifier B, bradykinin antagonist, Kallidin I intensifier C, enkephalin, insulin like growth factor, prostaglandin antagonist, tumor necrosis factor, epidermal growth factor (egf), granulose (amylin), lipotropin and thyrotropin.

The example of preferred peptide pharmaceutical agent be parathyroid hormone (PTH) (referring to people such as Harper, Eds., Review of Physiological Chemistry, the 16th edition, Lange Medical Publications, Los Altos, Calif. (1977) is p.468).Similarly, also separated approximately 34 fragments that amino acid residue forms of having been held by N and complete biologic activity of finding its displaying PTH are (referring to people such as Potts, in Parathyroid Hormone and Thyrocalcitonin (Calcitonin), R.V.Talmage, Deng people, Eds.Excerpta Medica, in New York (1968)).The sequence of polypeptide slightly changes between mammalian species.According to the present invention, PTH is intended to comprise human parathyroid hormone and other variants and 34 amino acid whose fragments.PTH in the Self homeostasis adjustment (homeostatic control) of calcium and phosphate metabolism, be used as regulatory factor (referring to, for example, Parsons, Deng people " Physiology and Chemistry of Parathyroid Hormone " in Clinics in Endocrinology and Metabolism, I.MacIntyre, Ed.Saunders, Philadelphia (1972) pp.33-78).The primary treatment purposes of PTH is to treat osteoporosis.PTH has also been used as blood calcium regulator.

In one embodiment, calcitonin is also preferred peptide pharmaceutical agent.Calcitonin be the polypeptide that contains 32 amino acid residues (referring to people such as Harper, Eds., Review of Physiological Chemistry, the 16th edition, Lange Medical Publications, Los Altos, Calif. (1977), p.469).According to the present invention, calcitonin is intended to comprise all calcitonins, comprises calcitonin and other variants of people, mammal and fish.Calcitonin is regulate the hormone of calcium and be used to treat osteoporosis, hypercalcemia and Paget (Paget's disease).

Other preferred protein medicine is cytokine IL-10.IL-10 is by the auxiliary subgroup of TH2, and the mononuclear cell of B cell subgroup and LPs-activation produces.IL-10 suppresses to reply relevant some immunologic functions to skin immunization, thus the inhibition stimulation relevant to the transdermal delivery of medicine sometimes and the generation of inflammation.More specifically, the release of the IFN-α of the cascade of the cell activation of the initial immunne response that causes skin is suppressed by IL-10.IL-10 also suppresses the synthetic numerous proinflammatory cytokines of macrophage, and breeds by lowering II class MHC expression inhibiting T cells with antigenic specificity.

Medicine based on nucleic acid

Conventionally, part is because of the stability to them and send relevant problem, and the medicine based on nucleic acid is only obtained limited success as therapeutic agent.Pharmaceutical agent based on nucleotide comprises the phosphodiester bond of the degraded sensitivity that nuclease is caused conventionally.This type of degraded is the major obstacles as the pharmaceutical agent of the sequence-dependent integrity of its identification specificity by oligonucleotide or nucleic acid.Therefore, naturally occurring oligonucleotide and nucleic acid conventionally must carry out chemical modification so that their nuclease-resistants, if described nuclease degradation in vivo they or select absent-mindedly suitable condition to degrade even in vitro them.Yet, the drug delivery system of the application of the invention, this is by optional.

Medicine based on nucleotide of the present invention comprises fit (aptamer), antisense compounds and triple helical medicine.Medicine based on nucleotide conventionally has and is greater than approximately 350 molecular weight and can reaches approximately 100 bases.The example of the medicine based on nucleotide comprises two and trinucleotide, GS375 for example, and it is the similar thing of dinucleotide (Gilead Sciences, Inc., Foster City, Calif.) with the potential therapeutic activity of resisiting influenza virus.

In one embodiment, the pharmaceutical pack based on nucleotide is containing one or more therapeutic genes.The therapeutic gene being encapsulated in liposome can be any common treatment gene for expression treatment agent and diagnostic agent.Exemplary treatment gene comprises the Brain Derived Neurotrophic Factor (BDNF) that is used for the treatment of neurodegenerative diseases, apoplexy or brain trauma; Be used for the treatment of Parkinsonian tyrosine hydroxylase and/or aromatic amino acid decarboxylase; β-glucuronidase; Hexosaminidase A; Be used for the treatment of the herpes simplex virus thymidine kinase of the cerebral tumor or coding for the gene of the antisense RNA of EGF-R ELISA; The lysosomal storage disease that is used for the treatment of Tay-Sachs and other lysosomal storage diseases substitutes enzyme (replacement enzyme); Coding is used for the treatment of the gene of antisense RNA of the brain composition of acquired immune deficiency syndrome (AIDS) (AIDS).Except therapeutic gene, plasmid DNA also can comprise DNA sequence before or after therapeutic sequence, and this type of extra section of plasmid can promote the tissue-specific transcription in the specific cells of plasmid in brain, can promote translation and/or the stabilisation of enhancing of the mRNA of therapeutic gene, and can make transgenic can be in brain cell episomal replication.Conventionally, therapeutic gene will comprise at least 100 nucleotide or have higher than 30,000 daltonian molecular weight.Preferably therapeutic gene is included in plasmid or in other suitable carriers, and is encapsulated in the inside compartment of liposome or nano container.

Can therapeutic gene be encapsulated in liposome according to the drug pack method of any known.For example by supersound process, freezing/to melt, evaporation and extruding via membrane filter.

The number that is encapsulated in the therapeutic gene in liposome can be 1 to many, and this depends on disease to be treated.Limiting factor can be the diameter that is encapsulated in the therapeutic gene in liposome.By using polycation protein for example histone, protamine or poly-D-lysine, the size of the plasmid DNA that comprises thousands of nucleotide can be compressed to the structure of the diameter with 10 to 30nm.The volume of the liposome of 100nm diameter is respectively 1000 times and 35 times of volume of the fine and close spheroid of DNA of 10nm and 30nm.Therefore, the several genes of the homologous genes of many copies or a plurality of copies can be encapsulated in liposome.

Bioactivator comprises oligomer for example (1) antisense compounds and (2) other biological active oligomeric thing.The two strands of the oligonucleotide that as used herein, term " antisense compounds " especially comprises single stranded antisense oligonucleotides (DNA, DNA sample, RNA, RNA sample) or comprises antisense orientation or construct, antisense PNA, ribozyme and the EGS (describing hereinafter) of self hybridization.Antisense compounds can be brought into play its effect by several different methods.Such method is endogenous nucleic acid enzyme such as the RNA enzyme H in eukaryote or the RNA enzyme P in prokaryote or the dsRNA enzyme in RNAi approach of antisense mediation to the guiding of target nucleic acid (people such as Chiang, J.Biol.Chem., 1991,266,18162; The people such as Forster, Science, 1990,249,783).The sequence of recruiting RNA enzyme P is called as external guide sequence (External Guide Sequences), is abbreviated as " EGS " people such as (, Proc.Natl.Acad.Sci.USA, 1997,94,8468) Guerrier-Takada.

The biological activity oligomer of another kind of type is the RNA-RNA crossbred molecule of scalable gene expression.Double-stranded RNA can be described to siRNA in some cases.In order to describe embodiment of the present invention, siRNA is the combination of antisense strand and sense strand, and each chain has is enough to show for example length-specific of stability and target-specific of the character of expectation, for example approximately 8 to 30, approximately 12 to 27, approximately 17 to 25 or approximately 19 to 23 nucleotide are long.The complementary pair of such oligonucleotide can be flush end or can comprise extra nucleotide in appointing on one or both ends of its 5' or 3' end.In addition, they can comprise other molecules or molecular structure on its 3' or 5' end, for example the phosphate group on 5' end.The preferred group of compound of the present invention is included in the phosphate group on the 5' end of antisense strand compound.Other preferred compounds are also included in the phosphate group on the 5' end of sense strand compound.Preferred compound can comprise for example overhanging of two bases on 3' end of extra nucleotide.

Term " other biological active oligomeric thing " especially comprises fit and molecule bait.As used herein, this term means any oligonucleotide (comprising RNA or PNA), described oligonucleotide (1) is for having this animal needing that preventive effect is provided, appeasing effect or therapeutical effect and (2) by non-antisense mechanism, by except with nucleic acid hybridization certain methods work.

In one embodiment, bioactivator is fit or molecule bait.Fit is by the single stranded oligonucleotide of the machine-processed binding specificity part except Watson-Crick base pairing.Fit common targeting is such as protein and be not designed for bind nucleic acid (people such as Ellington, Nature, 1990,346,818).

Molecule bait be on simulation nucleic acid with the factor short double-strandednucleic acid in the site of protein bound (comprising through himself single-chain nucleic acid of design " folding back ") for example.This type of bait expection competitive inhibition factor; That is because factor molecule is bonded to excessive bait, so the concentration being bonded to corresponding to the factor in the cell site of bait reduce, thereby produce therapeutical effect, appease effect or preventive effect.The method of identifying and build bait molecule is described in such as belonging in the people's such as Edwards U.S. Patent No. 5,716,780.

The biological activity oligomer of another kind of type is the RNA-DNA crossbred molecule that can instruct the gene conversion of endogenous nucleic acid people such as (, Science, 1996,273,1386) Cole-Strauss.Can any aforementioned biological activity oligomer be formulated in liposome of the present invention and for preventative or therapeutic object.

Embodiments more of the present invention, select to have antisense part as the first area of oligonucleotide and have justice part as the single stranded oligonucleotide of the second area of oligonucleotide.First and second region is by nucleotide joint (strings of the one or more nucleotide together that are linked in sequence) or by non-nucleotide joint area or by together with being connected of nucleotide and non-nucleotide structure.In each structure of this class formation, oligonucleotide, when folding back himself, at least complementary between first area (antisense part) and second area (having adopted part).Therefore oligonucleotide can have palindrome in its structure, wherein first area (the antisense part of 5' to 3' direction) and second area (3' to 5' direction have adopted part) complementation.

In other embodiments, the present invention includes oligonucleotide/protein compositions.Said composition has oligonucleotide composition and protein component.Oligonucleotide composition comprises at least one oligonucleotide (antisense or have MODN, but preferably antisense oligonucleotide (being the oligonucleotide of antisense for target nucleic acid)).It is the protein of a part for RISC complex that the protein component of compositions comprises at least one formation RNA induction silencing complex.Oligonucleotide composition also can comprise antisense strand and sense strand oligonucleotide.

RISC is ribonucleoprotein complex, the protein that it comprises oligonucleotide composition and Argonaute family.Although we are not wishing to be bound by theory, Argonaute albumen is class protein, has shown that some albumen wherein comprise the process being associated with post-transcriptional silencing before PAZ and Piwi domain and participation.The Argonaute family of protein includes, but are not limited to elF2Cl and elF2C2 (depending on species).ElF2C2 is also referred to as people GERp95.Yet we are not wishing to be bound by theory, at least antisense oligonucleotide chain combination to protein component to form RISC complex.In addition, complex can also comprise sense strand oligonucleotide.

Oligomeric compounds of the present invention can be used and can comprise structural detail for example inside or end protrusion or ring with the form of strand, two strands, ring-type or hair clip oligomeric compounds.Once the system of being introduced into, oligomeric compounds of the present invention can cause the effect of one or more enzymes or protein, realizes the modification to target nucleic acid.

A non-limiting example of such protein is RISC complex.RISC complex is used for realizing the cutting of RNA target, thereby has greatly strengthened the efficiency of the inhibition of oligonucleotide mediated gene expression.Inferred other ribonuclease for example the enzyme in RNAMeiIIIHe ribonuclease l family there is similar effect.

In another embodiment, oligomeric compounds of the present invention is included in the single stranded antisense oligonucleotides of combination in RISC complex, the double-stranded antisense of oligonucleotide/have justice to or comprise antisense part and have the single stranded oligonucleotide of justice part.Each of this compounds or compositions is for inducing the adjusting of effective and specific gene function.By introduce duplex structure for example double-stranded RNA (dsRNA) molecule in many species, shown that this type of specificity of gene function regulates, and shown that this specificity regulates the reduction of effective and special antisense mediation of the function of induced gene or its related gene product.This phenomenon has generation and it is believed that having evolution with virus defense and transposon silencing contacts in plant and animal.

Fit (or nucleic acid antibody) is in conjunction with the strand of specific molecular target or double-stranded DNA or single stranded RNA molecule.Conventionally, fit by for example effect of protein of Inhibitory molecules target, by being combined in the storehouse of the target circulating in blood, work.Fit example comprises Gilead antithrombase inhibitor GS522 and its derivant (Gilead Science, Foster City, Calif; Also referring to the people such as Macaya (1993) Proc.Natl.Acad.Sci.USA90:3745-9; The people such as Bock (1992) Nature (London) 355:564-566; With people (1993) Biochem.32:1899-904 such as Wang).Similarly, as known in the art, siRNA (siRNA molecule) can be used for the present invention.Referring to Fig. 8 and 9.

For the disease causing because of unsuitable gene expression, the specificity of the expression of this genoid stops or minimizing has represented desirable therapy.By and large, can by with mRNA in come-at-able sequence or transcript pre-mRNA process the generation that the single chain deoxynucleotide of the sequence complementation in necessary sequence or gene itself or the hybridization of ribose Deoxydization nucleotide suppress, reduce or close specific gene product.This example of Gene Handling is commonly referred to antisense or anti-gene inhibition (antigene inhibition).

Antisense compounds is the oligonucleotide that produces the mRNA of specified protein and make to produce this mRNA or stop this mRNA to produce in conjunction with being responsible for through being designed for.Antisense compounds can be by suppressing in body to cause or the formation of one or more protein of involved in diseases provides treatment function.Report, the diffusion of the disease relevant with the retroviral infection factor with virus to the antisense compounds inhibition of some gene messenger RNA or virus sequence complementation (referring to, for example, the people such as Matsukura (1987) Proc.Natl.Acad.Sci.USA84:7706, and the reference material of quoting herein).Other research worker are reported, and the formation that oligonucleotide can pass through triple helical is in conjunction with duplex DNA, thereby inhibition is transcribed and/or DNA is synthetic.

Antisense compounds comprises antisense RNA or DNA, strand or double chain oligonucleotide or its analog; it can and stop transcribing and/or the translation of the polypeptide of RNA processing and/or coding of this mRNA kind with independent mRNA kind specific hybrid, thereby the minimizing of amount of polypeptide that realizes each own coding is (referring to people Proc.Natl.Acad.Sci.U.S.A.86:10006-10010 (1989) such as Ching; The people Ann.Int.Med.113:604-618 (1990) such as Broder; The people FEBS Letters274:53-56 (1990) such as Loreau).

Triple helical compound (also referred to as three chain medicines) is the oligonucleotide in conjunction with double chain DNA sequence, and be intended to selectivity and suppress for example transcribing of viral gene (for example gene of HIV and herpes simplex virus) and oncogene of Disease-causing gene, that is, they stop the generation of protein in nucleus.This type of medicine is directly combined with the double-stranded DNA in the genome of cell, forms triple helical, thus stop cell generation target protein (referring to, for example, U.S. Patent No. 5,176,996, the people such as Hogan, on January 5th, 1993).

The chemical modification of the modification of phosphodiester bond or oligonucleotide end can appreciable impact oligonucleotide (for example, antisense compounds and triple helical medicine) locus specificity.Therefore, can this class oligonucleotide of chemical modification; Thereby strengthen overall combination stability, increase the stability for chemical degradation, increase oligonucleotide is transported to the speed in cell and gives molecular chemical reaction.Structure can be used for the conventional method of the various oligonucleotide of antisense therapy and by people (1988) Cancer Res.48:2659-2668 such as the people such as vander Krol (1988) Biotechniques6:958-976 and Stein, is summarized.

Therefore, fit, antisense compounds and triple helical medicine also can comprise nucleotide subsitution, interpolation, disappearance or swivel base (transposition), as long as be maintained with the specific hybrid of relevant target sequence or the association functional character as oligonucleotide.For example, some embodiments by application than the similar thing of thiophosphate of more nuclease-resistant degraded of their naturally occurring di-phosphate ester homologue (thereby expection has the interior persistency of higher body and larger potential) (referring to, the people such as Campbell (1990) J.Biochem.Biophys.Methods20:259-267).Also the phosphoramidic acid ester derivant of known oligonucleotide is in conjunction with complementary polynucleotide and have the additional capabilities of holding covalently bound ligand species, and can be used for method of the present invention (referring to the people such as Froehler (1988) Nucleic Acids Res.16 (11): 4831).

In addition, for example wherein sugar or base be can be used for the present invention by the nucleotide analog of chemical modification to nucleotide analog.The analog form of purine and pyrimidine is common known form in this area, and many forms are wherein as chemotherapeutant.

The end modified process useful that the absolute cellular uptake speed of change cell type specificity, pharmacokinetics, nucleus permeability (nuclear permeability) and oligonucleotide drug reagent is also provided.For example; displacement on 5' and 3' end comprises that reactive group (its allow pharmaceutical agent and other kind covalent cross-linkings) based on nucleotide and large group (bulky group) (it has improved the picked-up of cell) are (referring to Oligodeoxynucleotides:Antisense Inhibitors of Gene Expression; (1989) Cohen; Ed., CRC Press; Prospects for Antisense Nucleic Acid Therapeutics for Cancer and AIDS, (1991), Wickstrom, Ed., Wiley-Liss; Gene Regulation:Biology of Antisense RNA and DNA, (1992) Erickson and Izant, Eds., Raven Press; With Antisense RNA and DNA, (1992), Murray, Ed., Wiley-Liss. about the conventional method relevant to antisense compounds, referring to Antisense RNA and DNA, (1988), D.A.Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Can be by delivery of polynucleotides to cell to express extraneous nucleotide sequence, to suppress, eliminate, to increase or to change the expression of endogenous nucleotide sequence, or with impact not to the natural relevant particular physiological characteristic of cell.Polynucleotide can be that its existence or expression in cell changes cytogene or the expression of RNA or the sequence of function.The polynucleotide that are delivered can depart from endogenous substance of heredity and stay in Cytoplasm or nucleus.Alternatively, DNA can recombinate with endogenous substance of heredity (part that becomes endogenous substance of heredity).Restructuring can cause that DNA passes through homology or chromosomal DNA is inserted in non-homogeneous restructuring.

Gene expression inhibitor based on polynucleotide comprises any polynucleotide that contain sequence, and existence or the expression of described sequence in cell causes the degraded of gene or the function of suppressor gene, transcribes or translate in sequence-specific mode.Expression inhibitor based on polynucleotide can be selected from: the DNA expression cassette of siRNA, microRNA, RNA interfering or RNAi, dsRNA, ribozyme, antisense polynucleotides and coding siRNA, microRNA, dsRNA, ribozyme or antisensenucleic acids.SiRNA comprises duplex structure, and described duplex structure contains 15 to 50 base pairs conventionally, preferably 19 to 25 base pairs and have with the target gene of intracellular expression or RNA is same or same nucleotide sequence almost.SiRNA can comprise the polynucleotide of two annealing or form the single polynucleotide of hairpin structure.MicroRNA (miRNA) is the long little non-coding polynucleotide of approximately 22 nucleotide, and it directly destroys or translate the mRNA target that suppresses them.Antisense polynucleotides comprises the sequence with gene or mRNA complementation.Antisense polynucleotides includes but not limited to: morpholine, 2'-O-methyl polynucleotide, DNA, RNA etc.The polymerization in vitro of expression inhibitor based on polynucleotide, restructuring, comprises chimeric sequences or their derivant.Expression inhibitor based on polynucleotide can comprise ribonucleotide, deoxyribonucleotide, synthetic nucleotide or suitable combination arbitrarily, to suppress target RNA and/or gene.

Polynucleotide can comprise encoded for expressing expression cassette complete or partially protein or RNA.Expression cassette refers to polynucleotide natural or that restructuring produces, and it can expressed sequence.The coding region that expression cassette comprises genes of interest and any other sequences that affect the expression of aim sequence.The sequence that expression cassette generally includes promoter (permission transcription initiation) and transcribes.Optionally, expression cassette can include but not limited to, transcriptional enhancer, non-coding sequence, splicing signal, transcription stop signals and polyadenylation signal.Rna expression box generally includes the sequence of translation initiation codon (permission translation initiation) and the one or more protein of coding.Optionally, expression cassette can include but not limited to, translation termination signal, poly adenosine sequence, internal ribosome entry site (IRES) and non-coding sequence.Polynucleotide can comprise does not bring into play specific function but for the sequence of the generation of these polynucleotide in target cell.This type of sequence includes but not limited to that polynucleotide copy or select necessary sequence in host organisms.

In certain embodiments of the invention, as above pointed, the short nexus of sphingolipid activator protein or liposome are used for promoting sending of the nucleic acid molecules larger than conventional siNA (the large Nucleic acid precurser that comprises siNA).For example, method and composition herein can be used for strengthening the sending of larger nucleic acid of " precursor " of the siNA of representative expectation, wherein precusor amino acids can be before being delivered to target cell, in delivery process or after sending cut or otherwise processed, thereby be formed on the active siNA that in target cell, regulator gene is expressed.For example, siNA precursor polynucleotide can be chosen as to cyclic single strand polynucleotide, the stem in the sense and antisense district that it has two or more ring structures and comprises self complementation, wherein antisense district comprise with target nucleic acid molecule in nucleotide sequence or the nucleotide sequence of its part complementation, and You Yi district has the nucleotide sequence corresponding to target nucleic acid sequence or its part, and in wherein can body or external processing ring-type polynucleotide with produce can mediate rna i active siNA molecule.

In mammalian cell, the dsRNA that is longer than 30 base pairs can activate dsRNA dependant kinase PKR and 2'-5'-oligoadenylate synthetase (conventionally by interferon-induced).The PKR activating is by phosphorylation translation factor eukaryotic initiation factors 2. α. and (eIF2. α .) suppresses general translation, and 2'-5' oligoadenylate synthetase causes that by activator RNA enzyme L non-specific mRNA degrades.Size less (referring to especially non-precursor forms) due to siNA of the present invention, is less than 30 base pairs conventionally, approximately 17 to 19,19 to 21 or 21 to 23 base pairs the most commonly, so they have avoided activation interferon response.

Contrary with the nonspecific effect of long dsRNA, siRNA can mediate selected gene silence in mammlian system.The hairpin RNA with becate and 19 to 27 base pairs (in stem) is the expression of the gene of the sequence homology in selective silence and double-stranded stem also.It is reticent with mediation selected gene that mammalian cell can convert short hairpin RNA to siRNA.

RISC mediation has the cutting with the single stranded RNA of the sequence of the antisense strand complementation of siRNA duplex.The cutting of target RNA occurs in the centre with the region of the antisense strand complementation of siRNA duplex.Study and show, when the 3' that comprises two nucleotide overhangs, the siRNA duplex activity of 21 nucleotide is the highest.In addition, with 2'-deoxidation (2'-H) or 2'-O-methyl nucleotide, replacing 1 or 2 siRNA chains completely, to have eliminated RNAi active, yet report, and with Deoxydization nucleotide (2'-H), replacing the 3'-end siRNA nucleotide of overhanging can tolerate.

Research shows, and with deoxyribonucleotide, replacing the section of overhanging of the 3' with the 21 aggressiveness siRNA duplexs that the 3' of 2 nucleotide overhangs does not have adverse effect to RNAi activity.Report, with deoxyribonucleotide, replacing on each end of siRNA at the most 4 nucleotide can be by well tolerable, yet utilizes the displacement completely that deoxyribonucleotide carries out to cause without RNAi active.

Alternatively, can be using siNA as being sent by the single or multiple transcription products of encoding single or multiple siNA and instruct its polynucleotide carrier of expressing to express in target cell.In this type of embodiment, the double-stranded part for the treatment of the whole transcription product of the siRNA that expresses in target cell can be for example 15 to 49bp, and 15 to 35bp or approximately 21 to 30bp is long.In exemplary, the nucleotide section that wherein the double-stranded part of the siNA of two chain pairings is not limited to match completely, and can comprise the not mating section causing because of mispairing (corresponding nucleotide is not complementary), protrusion (lacking corresponding complementary nucleotide on a chain), overhang etc.Can comprise the degree that mating section does not disturb siNA to form to them.In more detailed embodiment, " protrusion " can comprise 1 to 2 unpaired nucleotide, and wherein the double-stranded region of the siNA of two chain pairings can comprise approximately 1 to 7, or approximately 1 to 5 protrusions.In addition " mispairing " part comprising in the double-stranded region of siNAs, can exist with approximately 1 to 7 or approximately 1 to 5 number.The most common ground in the situation that of mispairing, one of nucleotide is guanine, and another is uracil.This type of mispairing can be for example has the sudden change of C to T, G to A in the corresponding DNA of adopted RNA or its mixing owing to coding, but also can relate to other reasons.In addition, in the present invention, wherein the double-stranded region of the siNA of two chains pairing can be included in protrusion in the roughly number range of appointment and the part of mispairing.

The end structure of siNA of the present invention can be (overhanging) flush end or viscosity, as long as siNA keeps the activity of the expression of its reticent target gene.The 3' that viscosity (overhanging) end structure is not only limited to as reported by other researcheres overhangs.On the contrary, can comprise the 5' structure of overhanging, as long as it can be for example by the reticent effect of RNAi induced gene.In addition, the number of the nucleotide of overhanging is not limited to 2 or 3 nucleotide of report, and can be arbitrary number, as long as overhang, does not damage the active for gene silencing of siNA.For example, overhang and can comprise approximately 1 to 8 nucleotide, more commonly approximately 2 to 4 nucleotide.The total length with the siNA of sticky end structure be expressed as the length of double-stranded part of pairing and the right length that comprises the strand of overhanging at two ends and.For example, be that two ends all have under the exemplary cases of the 19bp double-stranded RNA of overhanging of 4 nucleotide, total length is expressed as 23bp.In addition, because the sequence of overhanging can have low specificity to target gene, so its needn't with target-gene sequence complementary (antisense) or same (having justice).In addition, as long as siNA can maintain its gene silencing effect to target gene, it for example comprises low-molecular-weight structure (for example for example tRNA, rRNA or viral RNA of natural RNA molecule, or artificial RNA molecule) in the part of overhanging on an end so.

In addition, the end structure of siNA can have stem-ring structure, wherein the end of a side of double-strandednucleic acid by joint nucleic acid for example joint RNA connect.The length of double-stranded region (stem-loop section) can be for example 15 to 49bp, and common 15 to 35bp, and more commonly approximately 21 to 30bp is long.Alternatively, as being for example approximately 15 to 49bp by the length of the double-stranded region of the whole transcription product of the siNA expressing in target cell, 15 to 35bp or approximately 21 to 30bp is long.When using joint section, the length of butt joint is not particularly limited, as long as it does not hinder the pairing of stem.For example, for the inhibition of the restructuring between the stable pairing of stem and the DNA of this part of encoding, blank area can have clover leaf tRNA structure.Even if joint has the length of the pairing that hinders stem, still can for example build the blank area that comprises intron, so that intron is cut to the course of processing of mature rna at precursor RNA, thus the pairing of permission stem.The in the situation that of stem ring siRNA, arbitrary end (head or tail) without the RNA of ring structure can have low molecular weight RNA.As mentioned above, this type of low molecular weight RNA can comprise natural RNA molecule for example tRNA, rRNA or viral RNA or artificial RNA molecule.

SiNA also can comprise have with target nucleic acid molecule in nucleotide sequence or the strand polynucleotide of the nucleotide sequence of its part complementation (for example, when this kind of siNA molecule need to be at siNA molecular memory during at the nucleotide sequence corresponding to target nucleic acid sequence or its part), wherein strand polynucleotide also can comprise terminal phosphate group, such as 5'-phosphoric acid (referring to such as people such as Martinez, Cell, the people such as 110:563-574 (2002) and Schwarz, Molecular Cell, 10:537-568 (2002)) or 5', 3'-diphosphonic acid.

As used herein, term siNA molecule is not limited to only comprise the molecule of naturally occurring RNA or DNA, but also comprises nucleotide and the non-nucleotide of chemical modification.In certain embodiments, short interfering nucleic acid molecule of the present invention lacks the nucleotide containing 2'-hydroxyl (2'-OH).In certain embodiments, the existence that short interfering nucleic acid does not need to have the nucleotide of 2'-hydroxyl carrys out mediate rna i and therefore, short interfering nucleic acid molecule of the present invention does not optionally comprise any ribonucleotide (nucleotide for example, with 2'-OH).Yet, need at ribonucleotide, not support this type of siNA molecule of RNAi can there is joint or group, part or the chain other connections or that associate of the connection of the nucleotide that comprises the one or more 2'-OH of having groups at siNA molecular memory.Optionally, siNA molecule can comprise ribonucleotide on approximately 5,10,20,30,40 or 50% nucleotide position.

As used herein, term siNA is intended to be equal to for describing other terms of the nucleic acid molecules that can mediate sequence-specific RNA i, the oligonucleotide of modifying such as short interfering rna (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), short hairpin RNA (shRNA), short interference oligonucleotide, short interfering nucleic acid, short interference, siRNA, the PTGS RNA (ptgsRNA) etc. of chemical modification.

In other embodiments, for siNA molecule of the present invention, can comprise sense and antisense sequence or region separately, wherein said sense and antisense region is covalently connected by nucleotide or non-nucleotide linkers, or by ionic interaction, hydrogen bond, Van der Waals interaction, hydrophobic interaction and/or accumulative facies mutual effect, noncovalently connects alternatively.

" antisense RNA " is the RNA chain having with the sequence of target gene MRNA complementation, and it it is believed that by conjunction with target gene MRNA induction RNAi." have adopted RNA " and have the sequence with antisense RNA complementation, and antisense RNA complementary with it annealing forms siRNA.Used RNA synthesizer synthesize routinely this type of antisense and have adopted RNA.As used herein, term " RNAi construct " is the general terms using in whole description, and it comprises siRNA (siRNA), hairpin RNA and can be cut in vivo other RNA kinds that form siRNA.RNAi construct herein also can comprise expression vector (also referred to as RNAi expression vector), and described expression vector can be created in the transcript that forms dsRNA or hairpin RNA in cell and/or the transcript that can produce in vivo siRNA.Optionally, siRNA comprises strand or double-stranded siRNA.

SiHybrid molecule is the double-strandednucleic acid with the function similar to siRNA.Be different from double stranded rna molecule, siHybrid comprises RNA chain and DNA chain.Preferably, RNA chain is antisense strand, because it is the chain in conjunction with said target mrna.The siHybrid producing by the hybridization of DNA and RNA chain has the complementary portion of hybridization and preferred at least one 3' end of overhanging.

The siNA that can use from two oligonucleotide assembling the present invention that separate, wherein a chain is that sense strand and another chain are antisense strands, wherein antisense and sense strand be self complementation (that is, each chain comprise with another chain in the nucleotide sequence of nucleotide sequence complementation; For example wherein antisense strand and sense strand form duplex or duplex structure, and for example wherein double-stranded region is approximately 19 base pairs).Antisense strand can comprise with target nucleic acid molecule in nucleotide sequence or the nucleotide sequence of its part complementation, and sense strand can comprise the nucleotide sequence corresponding to target nucleic acid sequence or its part.Alternatively, can be from single oligonucleotide assembling siNA, wherein the sense and antisense region of self complementation of siNA is by connecting based on nucleic acid or the joint based on non-nucleic acid.

In other embodiments, for can be the hair clip in sense and antisense region or the polynucleotide of asymmetric hair clip secondary structure that there is duplex, asymmetrical duplex, there is self complementation according to the siNA sending in method and composition cell of the present invention, the wherein nucleotide sequence in antisense district inclusion and the target nucleic acid molecule separating or the nucleotide sequence of its part complementation, and have adopted district inclusion corresponding to the nucleotide sequence of target nucleic acid sequence or its part.

The non-limiting example of the chemical modification that can produce in siNA includes but not limited to key between thiophosphate nucleotide, 2'-deoxyribonucleotide, 2'-O-methyl ribonucleotides, 2'-deoxidation-2'-fluorine ribonucleotide, " universal base " nucleotide, " non-annularity " nucleotide, 5-C-methyl nucleotide and end glyceryl and/or reversing deoxidation mixing without base residue.This type of chemical modification when for different siNA construct, keeps RNAi active in being presented at cell, and increases significantly the serum stability of this compounds simultaneously.

In non-limiting example, the nucleotide of chemical modification to being introduced in nucleic acid molecules overcomes in the potential restriction of the intrinsic body internal stability of the natural RNA molecule sent in outer seedbed and bioavailability provides powerful instrument.For example, the use of the nucleic acid molecules of chemical modification can make it possible to produce given therapeutic effect with the specific nucleic acid molecule of low dosage more, because the nucleic acid molecules of chemical modification tends to have longer serum half-life.In addition, some chemical modification can improve by targeting specific cells or tissue and/or the cellular uptake that improves nucleic acid molecules the bioavailability of nucleic acid molecules.Therefore, for example, even if the activity of the nucleic acid molecules of chemical modification (is compared reduction with natural acid molecule, when comparing with complete-RNA nucleic acid molecules), the gross activity of the nucleic acid molecules of modification is because of the stability of molecule that improves and/or to send the activity of comparable natural molecule still higher.Different from the siNA of natural unmodified, the siNA of chemical modification also can make the probability that activates interferon activity in people be decreased to minimum.

SiNA molecule described herein, the antisense district of siNA molecule of the present invention can comprise key between thiophosphate nucleotide on the 3' end in described antisense district.Any embodiment Zhong， antisense district of the siNA molecule of describing in this article can comprise key between approximately 1 to approximately 5 thiophosphate nucleotide on the 5' end in described antisense district.In any embodiment of the siNA molecule of describing in this article, the 3' terminal nucleotide of siNA molecule of the present invention is overhang and can be included in ribonucleotide or the deoxyribonucleotide of chemical modification on sugar, base or the main chain of nucleic acid.In any embodiment of the siNA molecule of describing in this article, 3' terminal nucleotide is overhang and can be comprised one or more universal base ribonucleotides.In any embodiment of the siNA molecule of describing in this article, 3' terminal nucleotide is overhang and can be comprised one or more non-annularity nucleotide.

For example, in non-limiting example, the invention describes in a siNA chain, have approximately 1,2,3,4,5,6,7,8 or more thiophosphate nucleotide between the short interfering nucleic acid (siNA) of chemical modification of key.In another embodiment, the invention describes in two siNA chains, all have approximately 1,2,3,4,5,6,7,8 or more thiophosphate nucleotide between the short interfering nucleic acid (siNA) of chemical modification of key.Between thiophosphate nucleotide, key can be present in one of siNA duplex or two oligonucleotide chains, for example, be present in sense strand, antisense strand or two chains.SiNA molecule of the present invention can comprise key between one or more thiophosphate nucleotide on 3' end, 5' end or the 3' of sense strand, antisense strand or two chains and 5' end.For example, exemplary siNA molecule of the present invention can comprise approximately 1 to approximately 5 or more (for example, approximately 1,2,3,4,5 or more) key between thiophosphate nucleotide continuously on the 5' end of sense strand, antisense strand or two chains.In another non-limiting example, exemplary siNA molecule of the present invention can comprise for example, between one or more (, approximately 1,2,3,4,5,6,7,8,9,10 or more) pyrimidine phosphorothioate phosphate ester nucleotide key in sense strand, antisense strand or two chains.In another non-limiting example, exemplary siNA molecule of the present invention can comprise for example, between one or more (, approximately 1,2,3,4,5,6,7,8,9,10 or more) purine thiophosphate nucleotide key in sense strand, antisense strand or two chains.

SiNA molecule can comprise circular nucleic acid molecule, wherein said siNA is approximately 38 to approximately 70 (for example in length, approximately 38,40,45,50,55,60,65 or 70) individual nucleotide, (for example have approximately 18 to approximately 23, approximately 18,19,20,21,22 or 23) individual base pair, wherein ring-type oligonucleotide forms the dumbbell-shaped structure with approximately 19 base pairs and 2 rings.

Ring-type siNA molecule comprises two cyclic group orders, and wherein one or two loop section of siNA molecule is biodegradable.For example, design like this ring-type siNA molecule of the present invention so that the vivo degradation of the loop section of siNA molecule can produce and have 3' end and overhang and for example comprise the double-stranded siNA molecule that the 3' terminal nucleotide of approximately 2 nucleotide is overhang.

Be present in siNA molecule, preferably be present in the antisense strand of siNA molecule, but the nucleotide that is also optionally present in the modification in both of sense strand and/or antisense strand and sense strand comprises the nucleotide of the modification with the character similar to naturally occurring ribonucleotide or feature.For example, the invention describes to comprise and (for example there is Northern conformation, the false rotating ring (Northern pseudorotation cycle) of Northern, referring to for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984) the siNA molecule of the nucleotide of modification.Similarly, be present in siNA molecule of the present invention, preferably be present in the antisense strand of siNA molecule of the present invention, but be also optionally present in the nucleotide nuclease-resistant degraded of sense strand and/or antisense strand and the sense strand chemical modification in both, keep the ability of mediate rna i simultaneously.The non-limiting example with the nucleotide of northern configuration comprises lock nucleic acid (LNA) nucleotide (for example, 2'-O, 4'-C-methylene-(D-RIBOSE base) nucleotide); 2'-methoxy ethoxy (MOE) nucleotide; 2'-methyl-sulfo--ethyl, 2'-deoxidation-2'-fluorine nucleotide, 2'-deoxidation-2'-chlorine nucleotide, 2'-azido nucleotide and 2'-O-methyl nucleotide.

The sense strand of double-stranded siNA molecule can have for example deoxidation base portion (inverted deoxybasic moiety) of reversing of end cap part on the 3' of sense strand end, 5' end or 3' and 5' end.

SiNA also can further comprise the nucleotide/non-nucleotide joint of nucleotide, non-nucleotide or mixing that siNA You Yi district is connected to the antisense district of siNA.In one embodiment, nucleotide joint can be in length, to be greater than 2 nucleotide, for example, in length, be the joint of approximately 3,4,5,6,7,8,9 or 10 nucleotide.In another embodiment, nucleotide joint can be aptamer.As used herein, " fit " or " aptamer " means the nucleic acid molecules of specific binding target molecule, and wherein said nucleic acid molecules has the sequence that comprises the sequence of being identified by target molecule in its natural surroundings.Alternatively, fit can be the nucleic acid molecules of binding target molecule, the not natural bind nucleic acid of wherein said target molecule.Target molecule can be any molecules of interest.For example, the fit ligand binding domains that can be used for conjugated protein, thus stop the interaction of naturally occurring part and described protein.This is non-limiting example and those skilled in the art will recognize that, can use common known technology in this area easily to produce other embodiments.[referring to, for example, the people such as Gold, Annu.Rev.Biochem., 64:763 (1995); Brody and Gold, J.Biotechnol., 74:5 (2000); Sun, Curr.Opin.Mol.Ther., 2:100 (2000); Kusser, J.Biotechnol., 74:27 (2000); Hermann and Patel, Science287:820 (2000); And Jayasena, Clinical Chemistry, 45:1628. (1999).

Non-nucleotide joint for example can comprise, without nucleotide base, polyethers, polyamine, polyamide, peptide, carbohydrate, lipid, poly-hydrocarbon (polyhydrocarbon) or other poly-compounds (Polyethylene Glycol for example has the Polyethylene Glycol of 2 Zhi100Ge ethylene glycol units).Concrete example comprises by Seela and Kaiser, Nucleic Acids Res., 18:6353 (1990) and Nucleic Acids Res., 15:3113 (1987); Cload and Schepartz, J.Am.Chem.Soc, 113:6324 (1991); Richardson and Schepartz, J.Am.Chem.Soc, 113:5109 (1991); The people such as Ma, Nucleic Acids Res., 21:2585 (1993) and Biochemistry32:1751 (1993); The people such as Durand, Nucleic Acids Res., 18:6353 (1990); The people such as McCurdy, Nucleosides & Nucleotides, 10:287 (1991); The people such as Jschke, Tetrahedron Lett., 34:301 (1993); The people such as Ono, Biochemistry, 30:9914 (1991); The people such as Arnold, International Publication case No.WO89/02439; The people such as Usman, International Publication case No.WO95/06731; The people such as Dudycz, International Publication case No.WO95/11910 and Ferentz and Verdine, J.Am.Chem.Soc, the example that 113:4000 (1991) describes.Any group or compound that " non-nucleotide " also represents to mix at one or more nucleotide units place nucleic acid chains (comprising sugar and/or phosphoric acid displacement) and allow their enzymatic activity of base displaying of reservation.Described group or compound can be without bases, and it does not comprise generally acknowledged nucleotide base, for example adenosine, guanine, cytosine, uracil or thymidine conventionally on the C1 position of for example sugar.

Can be comprised by the synthetic of siNA molecule of the present invention of chemical modification: (a) two of siNA molecule complementary strands is synthetic; (b) make two complementary strands annealing together being suitable for obtaining under the condition of double-stranded siNA molecule.In another embodiment, utilize solid phase oligonucleotide to synthesize two complementary strands of siNA molecule.In another embodiment, utilize solid phase series connection oligonucleotide to synthesize two complementary strands of siNA molecule.

(for example use scheme synthetic oligonucleotide known in the art, the part of the oligonucleotide of the oligonucleotide of some modification or shortage ribonucleotide), such as people such as Caruthers, 1992, Methods in Enzymology211, 3-19, the people such as Thompson, the open case No.WO99/54459 of International PCT, the people such as Wincott, 1995, Nucleic Acids Res.23, 2677-2684, the people such as Wincott, 1997, Methods Mol.Bio., 74, 59, the people such as Brennan, 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Patent No. 6, 001, in 311, describe.RNA comprises that the synthetic of some siNA molecule of the present invention defer to such as people such as Usman, 1987, J.Am.Chem.Soc, 109,7845; The people such as Scaringe, 1990, Nucleic Acids Res., 18,5433; With the people such as Wincott, 1995, Nucleic Acids Res.23,2677-2684, the people such as Wincott, 1997, Methods Mol.Bio., the conventional method of describing in 74,59.

Heterocyclic drug

Heterocyclic drug, the heterocyclic drug that particularly comprises at least one azacyclo-can be used as pharmaceutical agent in method described herein.For example, Yohimbine (yohimbine) is the indole alkaloid of blocking-up α-2-adrenoreceptor.Its secondary role is at it, to reduce element on kidney can in activity, increase cholinergic activity.This combination has caused Yohimbine in the treatment of the male erectile dysfunction of some type and the purposes in diagnostic classification.

Other examples of heterocyclic drug include but not limited to that morphine, methotrexate (were called methotrexate in the past; N-[4-[[(2; 4-diaminourea-6-pteridyl)-methyl] methylamino] benzoyl]-Pidolidone), lorazepam (the chloro-5-of 7-(the chloro-phenyl of o-)-l; 3-dihydro-3-hydroxy-2H-l; 4-benzodiazepine-2-ketone), Ismipur, (1,7-dihydro-6H-purine-6-thioketone monohydrate), 5-fluorouracil, nicotine, nicotinic acid and nicotinic acid (niacin).

The preparation of pharmaceutical agent and sending

Compositions of the present invention comprises short albumen or the polypeptide that merges sphingolipid activator protein conventionally, described albumen or polypeptide and anionic liposome associate, described liposome comprises at least one anion long-chain lipid, there is or do not have at least one neutral long-chain lipid, with at least one neutrality or anion short chain lipid, the pharmaceutical agent of the safe and effective amount that comprises the effect for expecting or developer (being all included in the pharmaceutically acceptable carrier with suitable pH).The safe and effective amount of activating agent is defined as and can in patient, causes the beauty treatment of expectation or the amount of therapeutic effect.In the present invention, skilled experienced doctor has the knowledge about suitable administration ratio (dosing ratio).

At any suitable dosage of using given in the situation that, certainly depend on known factor, for example the pharmacodynamic properties of certain drug reagent and its mode of administration and approach; Receptor's age, general health, metabolism, body weight and impact other factors of replying to compound; The effect of the type of the treatment of simultaneously carrying out, the frequency for the treatment of and expectation.

In one embodiment, the present invention includes safe and effective amount approximately 5.5 or the aqueous buffer solution of lower pH in the pharmaceutical agent of expectation, described pharmaceutical agent is included in anionic liposome.Preferred fusogenic protein or polypeptide are extremely about 100nM (nanomole) of the about 20nM of concentration, and preferably approximately 40 Saposin Cs to about 50nM, are then introduced into liposome-pharmaceutical agent mixture.The relative concentration of liposome is in the concentration excess of fusogenic protein or polypeptide, and with respect to the concentration of Saposin C calculate in molar ratio approximately 1 to 10 times excessive, or, approximately 3 to 7 times excessively (that is calculated at least Saposin C of 1:10: liposome) in molar ratio.In this embodiment, can in liposome composition, add at least one to there is the developer of at least one imaging character.Alternatively, in this embodiment, pharmaceutical agent can substitute with developer.

In one embodiment, the electronegative long-chain lipid that liposome comprises at least one type is DOPS (DOPS) for example.Liposome can be made by any lipid mixture of the anion long-chain lipid that comprises appropriate amount.In a specific embodiment, liposome for example, for example, for example, is made by comprising anion long-chain lipid (DOPS or GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG)), neutral long-chain lipid (dipalmitoyl phosphatidyl choline (DPPC) or dimyristoyl phosphatidyl choline (DMPC)) and the mixture of neutral short chain lipid (DHPC).The overall charge of liposome that is derived from the gained of lipid mixture is born.Short-chain phospholipid can be also electronegative.

Then can to topical application or by method described herein, use such compositions to its hetero-organization or brain and CNS.In U.S. Patent No. 6,099,857, Gross, on August 8th, 2000 and U.S. Patent No. 5,766,626, Gross, has provided other examples of preparing this lipoid plastid-fusion rotein complex (wherein activating agent is included in liposome) on June 16th, 1998 (it is incorporated to herein by reference).

Transdermal delivery

Can be by pharmaceutical agent-chemical modifier complex described herein through dermal administration.Through dermal administration, generally include delivering drugs reagent so that its percutaneous enters patient's body circulation.Skin site comprises for the region of anatomy through skin drug administration, comprises forearm, abdominal part, breast, the back of the body, buttocks, mastoid region etc.

Transdermal delivery was realized by a period of time that the source of complex is exposed to patient's skin and extends.Transdermal patch has provides the extra favourable aspect of the controlled delivery of pharmaceutical agent to health (referring to Transdermal Drug Delivery:Developmental Issues and Research Initiatives; Hadgraft and Guy (eds.); Marcel Dekker; Inc., (1989); Controlled Drug Delivery:Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); With Transdermal Delivery of Drugs, 1-3 volume, Kydonieus and Berner (eds.), CRC Press, (1987)).This type of dosage form can be by dissolving pharmaceutical agent, Saposin C and anionic liposome, disperse or otherwise being incorporated in suitable medium and for example producing in elastomeric matrices material (elastomeric matrix material).Also can utilize absorption enhancer to increase compound flowing through skin.This mobile speed can be by providing rate controlling membranes or compound being dispersed in polymeric matrix or gel and being controlled.

Passive through skin drug delivery

Being permitted eurypalynous transdermal patch can be used in method described herein.For example, can prepare simple paster (adhesive patch) from base material and acrylate adhesive.Pharmaceutical agent-chemical modifier complex and any reinforcing agent (enhancer) can be formulated in viscosity Casting solution (adhesive casting solution) and it is fully mixed.Solution direct pouring is evaporated on base material and by casting solvent in baking box, thereby leave adhesive film.Can stick release liner (release liner) with completion system.

Alternatively, can be by polyurethane substrates patch for delivering drugs reagent-chemical modifier complex.The layer of this patch comprises backing (backing), polyurethane medicine/reinforcing agent substrate, film, adhesive and release liner.By using curing polyurethane at room temperature prepolymer, preparation polyurethane substrates.Can the firm elastomeric formation of the viscosity on base material by direct pouring to adding water, alcohol and complex to cause in prepolymer.

Other embodiments of the present invention can be utilized hydrogel matrix patch.Conventionally, hydrogel matrix can comprise alcohol, water, medicine and several hydrophilic polymer.This hydrogel matrix can be mixed between the backing and tack coat (adhesive layer) of transdermal patch.

For passive delivery system; conventionally by being placed in the film between reservoir (reservoir) and skin; by spreading from monolithic devices (monolithic device) or controlling rate of release (referring to United States Patent(USP) Nos. 4 by the rate controlled barrier that skin itself is used as delivery system; 816,258; 4,927,408; 4,904,475; 4,588,580,4,788,062).The speed of drug delivery will depend in part on the character of film.For example, conventionally high than the speed through skin barrier through the speed of the drug delivery of the film in health.Complex is delivered to from device the speed limit film (rate-limiting membranes) that the speed of film is the most advantageously placed between reservoir and skin by use and controls.Suppose that skin is fully permeable (that is, being greater than the speed by film via the absorption of skin) for complex, film is by for controlling the medicine-feeding rate (dosage rate) of patient experience so.

Can permeability degree, character and the machinery relevant to construction device of complex based on expectation consider to select suitable permeable membrane material.Exemplary permeable membrane material comprises for example polydimethylsiloxane (silicone rubber), ethylene vinyl acetate copolymer (EVA), polyurethane, polyurethane-polyether copolymer, polyethylene, polyamide, polrvinyl chloride (PVC), polypropylene, Merlon, politef (PTFE), cellulosic material Triafol T and nitric acid/cellulose acetate and hydrogel HEMA (HEMA) for example for example of numerous natural and synthetic polymer.

The device characteristic that depends on expectation, can be included in other in device, for example, treat other conventional ingredients of product.For example, according to compositions of the present invention, also for example can comprise one or more antiseptic or antibacterial, methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride etc.This type of pharmaceutical composition also can comprise other active component for example antimicrobial, particularly antibiotic, anesthetics, analgesic and pruritus.

Topical therapeutic

Another aspect of the present invention provides the local delivery of pharmaceutical composition.This therapeutic scheme is suitable for the general of pharmaceutical agent to be used or topical therapeutic,, is applied directly to pathological tissue or illing tissue that is.

Conventionally; topical formulations can comprise for pharmaceutical agent-chemical modifier complex being directly delivered to the preparation of affected skin; it comprises described complex (conventionally with approximately 0.001% to 10%; preferred approximately 0.01 to approximately 10%; 0.1 to approximately 5% and most preferably from about 1 to approximately 5% concentration more preferably from about) and avirulent pharmaceutically acceptable topical carrier (referring to Dermatological Formulations:Percutaneous Absorption; Barry (ed.); Marcel Dekker Inc., (1983); About the standard dose of conventional medicine reagent, referring to, for example, Physicians Desk Reference (1992 editions); With American Medical Association (1992) Drug Evaluations Subscriptions).

Can by by pharmaceutical agent-chemical modifier complex with in local desiccation preparation, liquid preparation, cream preparation and aerosol preparations normally used conventional medicine diluent and the incompatible topical formulations of preparing of vehicle group.Ointment and ointment be use or oleaginous base for example, by adding suitable thickening agent and/or gellant, prepares.This type of substrate can comprise for example for example Oleum Arachidis hypogaeae semen or Oleum Ricini of liquid paraffin or vegetable oil of water and/or oil.The thickening agent that can use according to the character of substrate comprises soft paraffin, aluminium stearate, cetostearyl alcohol (cetostearyl alcohol), propylene glycol, Polyethylene Glycol, lanoline, hydrogenated lanolin, Cera Flava etc.Available aqueous or oleaginous base preparation lotion, it also comprises one or more following materials conventionally: stabilizing agent, emulsifying agent, dispersant, suspending agent, thickening agent, coloring agent, spice etc.Can form powder by means of any suitable powder substrate (powder base) such as Talcum, lactose, starch etc.Available aqueous matrix or the non-aqueous substrate preparation drop that also comprises one or more dispersants, suspending agent, solubilizing agent etc.

Dosage form for local application complex of the present invention comprises powder, spray, ointment, paste, ointment, lotion, gel, solution, patch and inhalant.Can under aseptic condition, reactive compound be mixed with pharmaceutically acceptable carrier and with any antiseptic, buffer agent or the propellant that may need.

Ointment, paste, ointment and gel also can comprise excipient for example animal and plant fat, oil, wax, paraffin, starch, Tragacanth, cellulose derivative, Polyethylene Glycol, silicone, bentonite, Talcum and zinc oxide or its mixture.Powder and spray also can comprise for example mixture of lactose, Talcum, aluminium hydroxide, calcium silicates and polyamide powder or this type of material of excipient.Spray can comprise for example for example butane and propane of fluorochlorohydrocarbon and the unsubstituted hydro carbons of volatility of conventional propellant extraly.

Transmucosal delivery (Transmucosal Delivery)

Although many discussions herein concentrate on technical for transdermal delivery, but the also transportation of the enhancing of the film by mucosa (such as the film of gastrointestinal tract, Sublingual, cheek, nose, lung, vagina, cornea and eye) and send (referring to the people such as Mackay (1991) Adv.Drug Del.Rev, 7:313-338) applicable to pharmaceutical agent of method of the present invention.Especially, between skin and the film of mucosa, there are many similaritys.For example, the film right and wrong in oral cavity are cornified.Yet the film in oral cavity is similar to skin, because both layerings, and the former is comprised of epilamellar polygon cell (form and be positioned at surperficial squamous cells).

Through mucous membrane (that is, Sublingual, cheek and vagina) drug delivery provides the direct metabolism that effectively enter and reduce liver and intestinal wall flora (flora) of active substance to body circulation.Transmucosal drug dosage form (for example, tablet, suppository, ointment, gel, pessulum (pessary), membrane (membrane) and powder) conventionally keep contact with the film of mucosa and fast disintegrate and/or dissolving to allow general absorption immediately.

Through cheek, use (buccal administration)

In order to be delivered to the film in cheek or Sublingual, conventionally will use oral formulations for example lozenge, tablet or capsule.The method of manufacturing this type of preparation is known in this area, includes but not limited to, pharmaceutical agent-chemical modifier complex is added into prefabricated tablet; Cold pressing inert filler, binding agent and pharmaceutical agent-chemical modifier complex or containing the material of described complex (as U.S. Patent No. 4,806, described in 356, being incorporated to by reference herein) and encapsulation.

Another kind of oral formulations is for example, to be applied to oral mucosal preparation together with adhesive agent (cellulose derivative hydroxypropyl cellulose), for example, as U.S. Patent No. 4,940, described in 587 (being incorporated to by reference this paper).This cheek adhesion preparation, when being applied to the mucosa of cheek, allows pharmaceutical agent-chemical modifier complex to the controlled release of normal sensation in the mouth through buccal mucosa.

Per nasal/use through lung

In order to be delivered to the film of nose and/or lung, conventionally can use aerosol preparations.Term " aerosol " comprises the suspended phase of any gas load of pharmaceutical agent-chemical modifier complex, and it can be inhaled into bronchioles or nasal meatus.Especially, aerosol comprises the float of microdroplet of the compounds of this invention of gas load, and this can produce in metered dose inhaler or nebulizer or in aerosol apparatus (mist sprayer).Aerosol also comprises the dry powder composite of the pharmaceutical agent-chemical modifier complex being suspended in air or other vector gas, and described dry powder composite can be sent from inhaler device by sucking.

Through sending of blood brain barrier

The present invention also can be used for transporting pharmaceutical preparation or developer passes blood brain barrier.Can use in this area the method for describing, intramuscular, intravenous, ophthalmic or nasal administration comprise for example liposome of DOPS of Saposin C (or its variant or peptide) and electronegative long-chain lipid, to be delivered to CNS, and brain particularly.Via using of nasal cavity, for example cause entering olfactory sensation CSF, then enter PBF, similar to Intraventricular infusion (ICV).As above at large described, it will be understood by those skilled in the art that, can in the compositions of above-mentioned liposome, comprise other neutral long-chain lipids and/or short chain lipid (neutrality or electronegative), to improve stability or the effectiveness of whole compositions.

As the example of successful transportation to CNS, inventor proves, by means of the complex that utilizes DOPS liposome, Saposin C can be transported to cultured mouse cortical neurons and the hippocampal neuron into cultivation.Sphingolipid activator protein-C liposome that use contains long-chain anionic phospholipid, can be transported to Saposin C in endosome and lysosome compartment.The method can be used for treating sacred disease, for example comprises, wherein the pathology of disease and the sacred disease of progress are facilitated in the accumulation of MVB.For example, in PSAP-/-mice (wherein finding the formation of MVB in neuron and cerebral tissue), by afterbody, inject and use DOPS-Saposin C liposome and cause the accumulation of this class formation to reduce.Referring to Fig. 5 and 7.

In another embodiment, the reagent of many targeting blood-brains is conjugated to the surface of liposome.Suitable targeting agent comprises insulin, transferrins, insulin like growth factor or leptin, because this type of peptide all has the endogenous RMT system in BBB, it is upper that this system is also present in BCM, and this type of endogenous peptide can be used as " transportable peptide (transportable peptide) ".Alternatively, the surface of liposome can be conjugated with 2 kinds different " transportable peptides ", the endogenous BBB receptor of a kind of peptide targeting and the endogenous BCM peptide of another kind of peptide targeting.The latter can be specific to the specific cells in brain, for example neuron, glial cell, pericyte, smooth muscle cell or microglia.Targeting peptides can be the endogenous peptide part of receptor, the analog of endogenous ligands or in conjunction with the simulating peptide Mab of the same receptor of endogenous ligands.TfR (TfR) specificity simulating peptide monoclonal antibody is described in detail in United States Patent (USP) 5,154,924 as the purposes of BBB " transportable peptide "; 5,182,107; 5,527,527; 5,672,683; In 5,833,988 and 5,977,307.The purposes as BBB " transportable peptide " for the Mab of insulin human receptor (HIR) has been described.

For the reagent of targeting blood barrier (blood-barrier) being conjugated to the agent of puting together of surface of liposome, can be that agent for example sphingomyelins, Polyethylene Glycol (PEG) or other organic polymers are puted together in any known polymerization.In one embodiment, PEG puts together agent.In one embodiment, the molecular weight of puting together agent is between 1000 to 50,000DA.In one embodiment, puting together agent is difunctional 2000DA PEG, and it at one end comprises lipid and comprises maleimide base group at the other end.The lipid end of PEG is in conjunction with the surface of liposome, the carrier of maleimide base group bind receptor monoclonal antibody specific or other targeting blood brain barrier.In one embodiment, 5 to 1000 targeting vectors are conjugated to each liposome.The liposome of puting together 25 to 40 targeting vectors of having an appointment on it is provided in one embodiment.

Although used liposome to describe the present invention as preferred nano container, those skilled in the art will recognize that, can use other nano containers.For example; available nano-particle or any other molecule nano container with the diameter that is less than 200nm substitute liposome, and described nano-particle or nano container can encapsulated dnas and protected nucleic acid to avoid nuclease degradation when preparation is still in blood or the transhipment of the intracellular region chamber from blood to target cell.Similarly, PEG chain also available multiple other polymeric materials for example sphingomyelins substitute, described polymeric material is connected to surface and the performance dual purpose of liposome or nano container: for puting together of " transportable peptide " provides support, and the blood of retard formulation is removed and optimization blood plasma pharmacokinetics.In addition, the present invention relates to gene to thering is any cell mass of particular target receptor or sending of organ.

The treatment of the Gaucher disease that the short proteins and peptides that merges sphingolipid activator protein of utilization carries out

In addition, Saposin C is that in glucosylceramide body, to be hydrolyzed to ceramide necessary.The shortage of epidermis glucocerebrosidase causes the glucosylceramide of change extremely relevant to the skin barrier that is characterised in that Gaucher disease to the ratio of the ratio of ceramide and this change.It is believed that, Saposin C is for by maintaining in horny layer the physiological concentration of glucosylceramide and ceramide, to form epidermis permeability barrier be vital.According to this model, by it, the stabilization removal effect to film mediates in the effect of Saposin C in stimulating glucocerebrosidase.Therefore, in thering is the patient of epidermis ceramide glucoside azymia, the local application of Saposin C-liposome complex that wherein liposome comprises acidic beta glucosidase (mixture is included in pharmaceutically acceptable carrier) can be used for fused cell film, to promote that glucosylceramide is hydrolyzed to ceramide, thereby contribute to the adjusting of skin barrier formation and function.Such composition can for example be formulated as ointment, lotion, solution or gel.Carrier can comprise for example pharmaceutically acceptable lubricant, emulsifying agent, thickening agent, solvent, antiseptic, coloring agent and spice.

Saposin C lysosome is as for using the delivery system of developer

In another embodiment of the invention, the liposome that comprises Saposin C can be used for sending the developer that at least one has one or more different imaging character simultaneously.This type of reagent can be used nuclear magnetic resonance, fluorescence or CT/PET to detect character.One or more developers can be mixed simultaneously or are encapsulated in the liposome that comprises Saposin C, so as the liposome that comprises Saposin C of single-population can be used for by multiple developer (with or not together with pharmaceutical agent) be delivered to the tissue of expectation.

In other embodiments of the present invention, the contrast agent based on liposome of the present invention also can comprise for example conventional contrast agent of other contrast agent, and it can be used for increasing the effect of the contrast agent of MRI.Many these type of contrast agent are known to those skilled in the art and comprise paramagnetism and superparamagnetism contrast agent.

Be suitable for exemplary paramagnetic contrast medium of the present invention and (for example comprise stable free radical, stable NO free radical (nitroxides)) and the compound that comprises transition elements, lanthanide series and actinides, it can exist or it can covalently or non-covalently be connected to chelating agent (comprising its lipophilic derivatives) or proteinaceous macromole with salt form if desired.

Preferred transition elements, lanthanide series and actinides comprise Gd (III), Mn (II), Cu (II), Cr (III), Fe (II), Fe (III), Co (II), Er (II), Ni (II), Eu (III) and Dy (III).More preferably, described element comprises Gd (III), Mn (II), Cu (II), Fe (II), Fe (III), Eu (III) and Dy (III), particularly Mn (II) and Gd (III).

If desired, this dvielement can exist with salt form, manganese salt for example, for example for example managanese gluconate and hydroxyapatite manganese of the organic salt of manganese chloride, manganese carbonate, manganese acetate and manganese; And iron salt for example, for example sulfide of ferrum and ferric salt ferric chloride for example.

If desired, this dvielement can for example be bonded to chelating agent (comprising its lipophilic derivatives) or proteinaceous macromole covalently or non-covalently.Preferred chelating agent comprises for example diethylene-triamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1, 4, 7, 10-tetraazacyclododecanand-N, N', N', N' " tetraacethyl (DOTA), 1, 4, 7, 10-tetraazacyclododecanand-N, N', N " triacetic acid (DO3A), 3, 6, 9-tri-azepines-12-oxa--3, 6, 9-tricarboxylic methylene-10-carboxyl-13-phenyl-tridecanoic acid (B-19036), hydroxybenzyl EDDA (HBED), N, N'-two (pyrrole tremble base-5-phosphoric acid) ethylenediamine, N, N'-diacetin (DPDP), l, 4, 7-7-triazacyclononane-N, N', N " triacetic acid (NOTA), 1, 4, 8, 1l-tetraazacyclododecane tetradecane-N, N', N ", N' " tetraacethyl (TETA), kryptands (being macro ring complex) and deferoxamine.More preferably, chelating agent is EDTA, DTPA, DOTA, DO3A and kryptands, most preferably DTPA.Its preferred lipotropy complex comprises the alkyl derivative of chelating agent EDTA, DOTA etc., EDTA-DDP for example, that is, N, N'-is two-(carboxyl-decyl amino methyl-N-2,3-dihydroxypropyl)-ethylenediamine-N, N'-oxalic acid; EDTA-ODP, i.e. N, N'-is two-(carboxyl-octadecyl amino-methyl-N-2,3-dihydroxypropyl)-ethylenediamine-N, N'-oxalic acid; EDTA-LDPN, N'-pair-(carboxyl-lauryl amino methyl-N-2,3-dihydroxypropyl)-ethylenediamine-N, N'-oxalic acid etc.; The U.S. series case No.887 for example submitting on May 22nd, 1992, the complex of describing in 290 (its disclosure is complete to be by reference incorporated to herein).Preferred proteinaceous macromole comprises albumin, collagen protein, poly arginine, poly-D-lysine, poly histidine, gamma globulin and betaglobulin.More preferably, proteinaceous macromole comprises albumin, poly arginine, poly-D-lysine and poly histidine.

Therefore, suitable complex comprises Mn (II)-DTPA, Mn (II)-EDTA, Mn (II)-DOTA, Mn (II)-DO3A, Mn (II)-kryptands, Gd (III)-DTPA, Gd (III)-DOTA, Gd (III)-DO3A, Gd (III)-kryptands, Cr (III)-EDTA, Cu (II)-EDTA or ferrum-deferoxamine, particularly Mn (II)-DTPA or Gd (III)-DTPA.

NO free radical is paramagnetic contrast medium, and it increases Tl and T2 relaxation rate (relaxation rate) by a unpaired electronics in NO free radical molecule.Given compound is relevant to the number of not sharing electron in paramagnetism core or molecule at least in part as the paramagnetism efficiency of MRI contrast agent, especially square relevant to the number of sharing electron not.For example, gadolinium has 7 unpaired electronics, and NO free radical molecule only has 1 unpaired electronics; Therefore gadolinium is normally more than the stronger MRI contrast agent of NO free radical.Yet effectively correlation time (effective correlation time), (estimating another important parameter of the efficiency of contrast agent) gave the relaxation property (relaxivity) of the potential increase of NO free radical.When effective correlation time and proton Larmor frequency (Larmour frequency) extremely approach, relaxation rate can significantly increase.When for example when paramagnetic contrast medium being connected to large structure and slowing down tumble rate (tumbling rate), it will more slowly roll, thereby more effectively shift energy, accelerate the relaxation of water proton.Yet in gadolinium, the Study of Electron Spin Relaxation Time time is very fast and will limit the degree of the relaxivity of spin correlation time increase slowly.Yet for NO free radical, electron spin correlation time is more favourable and can obtain by the spin correlation time of this quasi-molecule that slows down the huge increase of relaxivity.The target of the improvement of the spin correlation time that liposome of the present invention slows down for realization and the relaxivity causing is desirable.Although do not wish to be subject to any specific theory of operation to fetter, think, because NO free radical can be designed to the periphery (for example, by producing its alkyl derivative) of coated liposome, correlation time that therefore can optimization gained.In addition, the contrast agent of the present invention of gained can be counted as magnetic spheres, and it is to make the maximized geometric configuration of relaxivity.

If desired, NO free radical can carry out alkylation or other are derivative, and for example NO free radical 2,2,5,5-tetramethyl-l-pyrrolidinyl oxygen, free radical and 2,2,6,6-tetramethyl-l-piperidines oxygen, free radical (TMPO).

Be suitable for exemplary superparamagnetism contrast agent of the present invention and comprise metal-oxide and sulfide, ferrimagnetism or ferromagnetic compound for example pure iron, magnetic iron oxide (for example Magnetitum), the γ-Fe of experience magnetic domain (magnetic domain) ₂o ₃, Mn ferrite (ferrite), Conjugate ferrite and Ni ferrite.

Contrast agent (contrast agent) for example above-mentioned paramagnetism and superparamagnetism contrast agent can be used as in microsphere or the microsphere that comprises contrast agent in composition.They can be captured in to the inner space of microsphere, as solution, use together with microsphere, or mix the stable compound that forms microsphere body wall.

For example, if desired, can send paramagnetism or superparamagnetism reagent to mix alkylation in stable compound (the particularly lipid wall of microsphere) or the form of other derivants.Especially, NO free radical 2,2,5,5-tetramethyl-1-pyrrolidinyl oxygen, free radical and 2,2,6,6-tetramethyl-l-piperidines oxygen, free radical can for example, form additive compound with long-chain fatty acid by many different connections (acetoxyl group) on the position of the ring not occupied by methyl.This type of additive compound is highly susceptible to mixing the stable compound of stable compound, particularly lipid in nature, and described stable compound forms the wall of microsphere of the present invention.

Can in contrast agent, use similarly the mixture of any or multiple paramagnetism reagent and/or superparamagnetism reagent.

If desired, also can use altogether dividually above-mentioned paramagnetism and superparamagnetism reagent.

The liposome using in the present invention not only can be used as for example effective carrier of iron oxides of superparamagnetism reagent, but also shows the effect of having amplified magnetic susceptibility contrast agent (susceptibility contrast agent).Superparamagnetism contrast agent comprises metal-oxide, and iron oxides (but comprising manganese oxide) particularly for example experiences the iron oxides that comprises not commensurability manganese, cobalt and nickel of magnetic domain.This type of reagent is nano-particle or micron particle and has high body susceptibility (bulk susceptibility) and transverse relaxation speed.Larger granule, for example the diameter of 100nm, has the R2 relaxivity more much higher than R1 relaxivity, but less granule, for example 10 to 15nm diameter, has lower a little R2 relaxivity, but have balance many R1 and R2 values.Minimum granule for example single-crystal iron oxide particle (diameter 3 is to 5nm) has lower R2 relaxivity, but may have best balanced R1 and R2 relaxation rate.Also can prepare the core that ferritin encapsulates the superparamagnetism ferrum with high relaxation rate.Find, for effect and the safety that can increase the MRI contrast agent based on iron oxides of this type of routine through stable liposome of the present invention.

It is favourable that developer is mixed to liposome is included in the picked-up of pharmaceutical agent wherein and sends for mensuration.In addition, this type of reagent also can allow the imaging of organizational structure, or the in the situation that of cancer, allows to measure degree or the tumor growth shifting.In one embodiment of the invention, the liposome that contains Saposin C can shift medicine and developer through biomembrane.In another embodiment, multiple developer can be mixed to liposome membrane, maybe can use have a plurality of imaging character developer (for example, above with embodiment in the PTIR reagent described).Either method allows clinician or researcher to utilize multiple detection method by the single administration of liposome composition.

Developer can be used nuclear magnetic resonance, fluorescence or PT/CAT device.Can put into practice nuclear magnetic resonance (MRI) contrast-enhancing agents or radiosiotope use in vivo by several different methods.For example, Li, waits people, United States Patent (USP) 6,569, and 451 (being incorporated to by reference herein) are instructed such method, by the method, can by the liposome particles of polymerization for delivery of contrast agents, for example, use the contrast agent of nuclear magnetic resonance.

In one embodiment of the invention, mr angiography agent (for example extra small superparamagnetism iron oxides (USPIO) nano-particle) can be encapsulated in to the aqueous interior of liposome.MRI scanning can be used the chelate of gadolinium or manganese.Yet the nonphagocytic labelling detecting for MR needs liposome encapsulation and sends the contrast agent of sufficient quantity.Tumour-specific liposome can be used for reagent to be delivered to tissue, thereby helps to use MRI to carry out earlier detection and better visual.Also can, by the double carrier as medicine and contrast agent by liposome of the present invention, use sending and absorbing of contrast Enhanced MR micro-imaging assessment targeted drug.For example, can, by COMBIDEX (Advanced Magnetics, MA, the size of 0nm) (the molecular imaging agent of using MRI to detect), be encapsulated in the liposome of being made by DOPS (DOPS).Then this lipoid plastid is sent effectively to the pure man neuroblastoma cell.This have been described in detail in embodiments of the invention 3.

If desired, two or more different ions of use capable of being combined.As those skilled in the art recognize that, when using present disclosure, can be by the various combination of fat-soluble compound and paramagnetic ion for changing the relaxation behavior of the contrast agent of gained.Find, paramagnetic ion and fat-soluble compound complex of the present invention are the extremely effectively contrast-enhancing agents (contrast enhancement agent) for nuclear magnetic resonance.

Fat-soluble compound of the present invention can be individually or in combination with each other, with one or more paramagnetic ions combinations, as the contrast agent of nuclear magnetic resonance.According to present disclosure, exemplary paramagnetic ion comprises transition elements, lanthanide series (rare earth element) and actinides ion, and this it will be apparent to those skilled in the art that.Preferred paramagnetic ion comprises the paramagnetic ion that is selected from Cr3, Co2, Mn2, Ni2, Fe3, Fe2, La3, Cu2, Gd3, Ce3, Tb3, Pr3, Dy3, Nd3, Ho3, Pm3, Er3, Sm3, Tm3, Eu3, Yb3 and Lu3.More preferably, paramagnetic ion is selected from Mn2, Fe3 and Gd3, most preferably Mn2.

Can obtain multiple contrast agent, for strengthening contrast in tissue in nuclear magnetic resonance.Some in the most frequently used contrast agent are chelate for example Gd-DTPA, Gd-DTPA-BMA and Gd-DOTA of gadolinium.The molecular size of most of current obtainable contrast media formulations is less.In one embodiment, contrast agent is selected from iodine, gadolinium and Magnetitum.

In addition, fluorescent imaging agent can be mixed in liposome of the present invention, thereby other detection method is provided.For example, can use NBD, rhodamine, above-mentioned PTIR labelling or other known fluorometric reagents.Any fluorescent labeling being obtained commercially or fluorescently-labeled dyestuff (lipophilic or containing lipotropy part) for example above-mentioned dyestuff can be used for the present invention.Hui, the people such as L. have described such method, can be by PTIR contrast agent for label L DL granules in described method, it is incorporated to herein by reference.Hui, the people such as L., MR and Fluroescent Imaging of Low Density Lipoproteing Receptors, Acad Radiol2004; 11:1251-1259.The total concentration of the fluorometric reagent in liposome composition is approximately 1% to approximately 5%, or approximately 2% to approximately 4%.In the middle of fluorometric reagent, the mark of launching long wavelength's (red fluorescence) is more background in PTIR271 and 316 bodies that produce still less for example.Blue and green wavelength has higher background signal.Inventor shows, and the PTIR271 that mixes the liposome that comprises sphingolipid activator protein-C has minimum background and the clear signal of surveying.Fig. 7 illustrates the picked-up of the DOPS liposome that contains PTIR271 and 316.

It is liquid containing iodine compound, suitably iodate or poly-iodophenyl (polyiodophenyl) derivant be as containing diodone.Suitable material comprises Iopromide, ioxitalamic acid salt (Ioxitalamate), ioxaglic acid salt, iopamidol, iohexol, Iotralon, metrizamide or Ultravist (Ultravist).Meanwhile, contrast agent can be used as the solvent of the mixture of lyophilized products (lyophilisate).Can be by the contrast agent that contains gadolinium or contain Magnetitum for magnetic resonance tomography (MRT).Suitably the granule of 30mg to 90mg lyophilizing is blended in the cytostatic drug of requirement, is then dissolved in the contrast agent of 3ml to 6ml.

New formulation and its use make it possible under the help without indirect method, use XRF method, directly describe sufficient thromboembolism, and the tumor imaging that makes to have blood vessel is rest image; Can use the radiography medicine that contains gadolinium or Magnetitum of measuring combined sequence with flow-coded, and can describe thromboembolism by means of magnetic resonance, body section radiography; The accessible concentration of cytostatic drug in tumor tissues (cytostatic drug) is compared with other administration forms greatly to be increased (reaching 20 times); And simplified application, increased again safety (having avoided antidromicity mistake perfusion (retrograde faulty perfusion)) simultaneously.

Finally, can use the developer for computerized tomography (computed tomography, CT scan) or positron emission tomography (positron emission tomography, PET).The radionuclide image agent of the most often using comprises radioiodine and indium.Utilize the imaging of CT scan can utilize for example iron chelate of heavy metal.In addition, positron emission tomography (PET) can be used the positron emitter of oxygen, nitrogen, ferrum, carbon or gallium.The example that is used for the radionuclide of formation method comprises: ⁴³k, ⁵²fe, ⁵⁷co, ⁶⁷cu, ⁶⁷ga, ⁶⁸ga, ⁷⁷br, ⁸¹rb, ⁸¹kr, ⁸⁷sr, ⁹⁹tc, ¹¹¹in, ¹¹³in, ¹²³i, ¹²⁵i, ¹²⁷cs, ¹²⁹cs, ¹³¹i, ¹³²i, ¹⁹⁷hg, ²⁰³pb and ²⁰⁶bi.The developer that can use method known to those skilled in the art that this type of can be detected by CT/PET mixes sphingolipid activator protein-C liposome.

Those skilled in the art can use known technology by sphingolipid activator protein-C conjugation of polypeptides to radionuclide.For example, Magerstadt, M. (1991) Antibody Conjugates And Malignant Disease, CRC Press, Boca Raton, Fla.; And Barchel, S.W. and Rhodes, B.H., (1983) Radioimaging and Radiotherapy, Elsevier, New York, N.Y. (it is incorporated to herein separately by reference) instructs various therapeutic and diagnostic radionuclide to the amino acid whose of antibody to put together.This type of reaction can be used for radionuclide to be conjugated to sphingolipid activator protein-C peptide or to be conjugated to sphingolipid activator protein-C peptide by suitable joint.

Tumor-homing (Tumor Homing) and imaging

As described in this article, the present invention also provides sensitive, quantitative and simple tumor-homing nano-capsule bubble, and it comprises phospholipid and Saposin C peptide.In one embodiment, when the developer providing herein is further provided, peptide of the present invention can be used for conventional imaging.In another embodiment, the nano-capsule bubble that comprises developer of the present invention can be used for the routine monitoring of growth of cancers.Go back to the nest nano-capsule bubble of the novel tumor that described technology can be used as innovation, for making human or animal's tumor imaging and measuring its size.

In one embodiment, nano-capsule bubble comprises Saposin C (fragment 80AA), DOPC (DOPS) and developer be fluorogen for example for SapC, little lysosomal protein (for example Fe granule, radiosiotope and quantum dot substitute the probe of its available other types for MRI, PET and SPECT detection).

Nano-capsule bubble is stable and manufactures relatively simple.Primary evidence shows, fluorescence nano-capsule bubble is effectively transported into tumor in vivo.Other evidence hint, nano-capsule bubble can be suitable for the far-ranging cancer of targeting (comprising metastatic carcinoma) and make described cancer imaging.For example, our fluorogen-nano-capsule bubble provides for observing and measure the formation method fast and accurately of variation of the tumor size of mice alive.Tissue of the present invention is gone back to the nest, imaging nano-capsule bubble makes researcher and doctor can measurement range position and the size of tumor (comprising hiding tumor, metastatic tumo(u)r) widely.

In another embodiment, the fragment that can go back to the nest of Saposin C is mixed in the nano-capsule bubble that comprises cancer target and tumor-killing function.In another embodiment, provide the preparation without the tumor-homing nano-capsule bubble of anti-tumor activity, said preparation is desirable for tumor monitoring and imaging.

The peptide of synthetic several Saposin Cs is to detect their activity in nano-capsule bubble.Result shows, (for example, DOPS) the nano-capsule bubble of the SapC peptide-phospholipid without tumor-killing effect is provided.According to tumor imaging experiment, the sequence in SapC (E24-K41) shows best tumor-homing function.Short sequence from 10 amino acid residues of F32 to K41 in this region also shows tumor-homing function.In addition, for example E6-P40, K41-G80 and S67-G80 show tumor-homing function to other SapC peptides.In some embodiments, the amphipathic character of SapC peptide is that its tumor-homing function is necessary.In one embodiment, described peptide comprises one of a plurality of sequences that are selected from MCS, LCS and VCS in peptide sequence.In certain embodiments, for example Lys or Cys are that tumor-homing effect is necessary to aminoacid.

The invention still further relates to for organizing compositions and the method with cell imaging, wherein said compositions comprises the liposome that contains Saposin C and is selected from the detectable developer of the detectable reagent of MRI, fluorometric reagent, the detectable reagent of CT/PET, the reagent with a plurality of detection character or its combination.In another embodiment of the invention, Saposin C liposome complex can mix 1,2 or 3 kind of different reagent with different imaging character, to can use multiple different detection method by the single administration of Saposin C liposome.

Other reagent comprise fluorogen (for example, fluorescein, dansyl, quantum dot etc.) and IR dyes or metal, and it can be used for optics or photoimaging (for example, confocal microscopy and fluorescence imaging).

In another embodiment, the nanometer that compositions also comprises radionuclide, chelating agen, biotin, fluorogen, antibody, horseradish peroxidase, alkali phosphatase, nano-particle, quantum dot, anticarcinogen is dripped, anticarcinogen or chemotherapeutant, liposome medicament, cytokine or be connected to the micromolecule toxin on it.

In another embodiment, the nanometer that imaging moiety is selected from radionuclide, biotin, fluorogen, antibody, horseradish peroxidase, alkali phosphatase, nano-particle, quantum dot, detectable anticarcinogen is dripped, liposome medicament and cytokine.

Those skilled in the art are familiar with for radionuclide, chelating agen and chelating agen-joint conjugate being connected to compositions and the method for part of the present invention.Especially, can use easily for example commercially available difunctional linking group (isodigeranyl function linking group conventionally) that radionuclide, chelating agen and chelating agen-joint conjugate are connected to part of the present invention, described difunctional linking group can be connected to the functional group in the non-interference position being present on compound, then it be further connected to for example the nanometer of radionuclide, chemotherapeutant, anticarcinogen, nano-particle, quantum dot, anticarcinogen is dripped or micromolecule toxin.Like this, can be by compound of the present invention for suitable reagent being transported to target site (be generally tumor or there is the organ or tissue of cancerous cell).In another embodiment, by by biotinylated part and Succ-PEG-DSPE-Qdot605 (Quantum Dot Corp.; Hayward, Calif) together incubation prepare part Qdot complex.

In one embodiment of the invention, can be by the liposome that comprises traceable developer for target tumor neuroblastoma for example, thus allow to measure tumor size, growth, position or transfer.

In another embodiment, the peptide that comprises the sequence of approximately 9 to approximately 20 continuous amino acids with following formula for preparing the peptide of tumor-homing nano-capsule bubble:

EKEILDAFDKMCSKLPK

EKEILDAFDKMCSKLP

FDKMCSKLPK

FDKMCSKLP

EVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLP

KSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSG

SPELVCSMLHLCSG

In another embodiment, the peptide that comprises the sequence with one or more following formulas for preparing the peptide of tumor-homing nano-capsule bubble:

EKEILDAFDKMCSKLPK

EKEILDAFDKMCSKLP

FDKMCSKLPK

FDKMCSKLP

EVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLP

KSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSG

SPELVCSMLHLCSG

In one embodiment, peptide is approximately 9 to approximately 20 aminoacid.In another embodiment, peptide is approximately 14 to approximately 20 aminoacid.In another embodiment, peptide is approximately 16 to approximately 20 aminoacid.In another embodiment, peptide is approximately 16 to approximately 19 aminoacid.

The optimization of tumor-homing peptide:

In one embodiment, for preparing the peptide of tumor-homing nano-capsule bubble, comprise the peptide with following formula:

XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaPheAspLysMetCysSerLysLeuProXaa

Wherein the Xaa on position 1 is Asn or does not exist; Wherein the Xaa on position 2 is Lys or does not exist; Wherein the Xaa on position 3 is Thr or does not exist; Wherein the Xaa on position 4 is Glu or does not exist; Wherein the Xaa on position 5 is Lys or does not exist; Wherein the Xaa on position 6 is Glu or does not exist; Wherein the Xaa on position 7 is Ile or does not exist; Wherein the Xaa on position 8 is Leu or does not exist; Wherein the Xaa on position 9 is Asp or does not exist; Wherein the Xaa on position 10 is Ala or does not exist; And wherein the Xaa on position 20 is Lys or does not exist.

In another embodiment, for preparing the peptide of tumor-homing nano-capsule bubble, comprise the peptide with following formula:

XaaXaaXaaXaaXaaXaaXaaPheAspLysMetCysSerLysLeuProXaa

Wherein the Xaa on position 1 is Glu or does not exist; Wherein the Xaa on position 2 is Lys or does not exist; Wherein the Xaa on position 3 is Glu or does not exist; Wherein the Xaa on position 4 is Ile or does not exist; Wherein the Xaa on position 5 is Leu or does not exist; Wherein the Xaa on position 6 is Asp or does not exist; Wherein the Xaa on position 7 is Ala or does not exist; And wherein the Xaa on position 17 is Lys or does not exist.

In another embodiment, for preparing the peptide of tumor-homing nano-capsule bubble, comprise the peptide with following formula:

XaaXaaXaaGluLysGluIleLeuAspAlaPheAspLysMetCysSerLysLeuProXaa

Wherein the Xaa on position 1 is Asn or does not exist; Wherein the Xaa on position 2 is Lys or does not exist; Wherein the Xaa on position 3 is Thr or does not exist; And wherein the Xaa on position 20 is Lys or does not exist.

In another embodiment, the peptide that comprises the sequence of approximately 9 to approximately 20 continuous amino acids with following formula for preparing the peptide of tumor-homing nano-capsule bubble:

AsnLysThrGluLysGluIleLeuAspAlaPheAspLysMetCysSerLysLeuProLys

In another embodiment, the peptide that comprises the sequence of approximately 14 to approximately 19 continuous amino acids with following formula for preparing the peptide of tumor-homing nano-capsule bubble:

AsnLysThrGluLysGluIleLeuAspAlaPheAspLysMetCysSerLysLeuProLys

In one embodiment, the peptide that comprises following formula for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity:

SDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVV

DTYGSSILSILLEEVSPELVCSMLHLCSG

EVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLP

EKEILDAFDKMCSKLPK

NKTEKEILDAFDKMCSKLPK

FDKMCSKLPK

KSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSG

SPELVCSMLHLCSG

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be selected from:

ser-asp-val-tyr-cys-glu-val-cys-glu-phe-leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys-ser-leu-ser-glu-glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly 80

glu-val-cys-glu-phe-leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 35

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be selected from:

val-cys-glu-phe-leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

cys-glu-phe-leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

glu-phe-leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

phe-leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro

ala-phe-asp-lys-met-cys-ser-lys-leu-pro

phe-asp-lys-met-cys-ser-lys-leu-pro

leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys

phe-asp-lys-met-cys-ser-lys-leu-pro-lys

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be selected from:

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 17

phe-asp-lys-met-cys-ser-lys-leu-pro-lys 10

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 16

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 15

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 14

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 13

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 12

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 11

ala-phe-asp-lys-met-cys-ser-lys-leu-pro 10

phe-asp-lys-met-cys-ser-lys-leu-pro 9

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 16

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 15

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 14

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 13

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 12

ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 11

phe-asp-lys-met-cys-ser-lys-leu-pro-lys 10

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be selected from:

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 17

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 17

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 16

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 15

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 14

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 13

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 12

ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 11

phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 10

Xaa-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 17

Xaa-Xaa-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 16

Xaa-Xaa-Xaa-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 15

Xaa-Xaa-Xaa-Xaa-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 14

Xaa-Xaa-Xaa-Xaa-Xaa-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 13

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 12

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 11

phe-asp-lys-met-cys-ser-lys-leu-pro-lys 10

Xaa-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 17

Xaa-Xaa-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 16

Xaa-Xaa-Xaa-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 15

Xaa-Xaa-Xaa-Xaa-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 14

Xaa-Xaa-Xaa-Xaa-Xaa-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 13

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 12

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 11

phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 10

In one embodiment, Xaa is hydrophobic amino acid, comprises val, leu, and ile, met, pro, phe and ala, or do not exist.

In another embodiment, the Xaa on slot # is uncharged polar amino acid, comprises thr, ser, and tyr, gly, gln and asn, or do not exist.

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be selected from:

ser-asp-val-tyr-cys-glu-val-cys-glu-phe-leu-val-lys-glu-val-thr-lys-leu-ile-asp-asn-asn-lys-thr-glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys-ser-leu-ser-glu-glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly 80

lys-ser-leu-ser-glu-glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly 40

ser-leu-ser-glu-glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

leu-ser-glu-glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

ser-glu-glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

glu-glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

glu-cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

cys-gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

gln-glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

glu-val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

val-val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

val-asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

asp-thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

thr-tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

tyr-gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

gly-ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

ser-ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

ser-ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

ile-leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

leu-ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

ser-ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

ile-leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be selected from:

leu-leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

leu-glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

glu-glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

glu-val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

val-ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be selected from:

ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly 14

pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

leu-val-cys-ser-met-leu-his-leu-cys-ser-gly

val-cys-ser-met-leu-his-leu-cys-ser-gly

cys-ser-met-leu-his-cys-ser-gly

In another embodiment, for preparing the peptide of the tumor-homing nano-capsule bubble without anti-tumor activity, be:

ser-pro-glu-leu-val-cys-ser-met-leu-his-leu-cys-ser-gly 14

Labelling

Compositions of the present invention optionally comprises one or more labellings; The detectable labelling of optics for example, for example fluorescence or luminescent marking, and/or non-optical detectable labelling, for example magnetic mark.Many fluorescent labelinies are known in the art, (for example include but not limited to quantum dot, hydrophobicity fluorogen, coumarin, rhodamine and fluorescein) and green fluorescent protein (GFP) and variant (for example, cyan fluorescent protein and yellow fluorescence protein) thereof.Referring to for example, Haughland (2002) Handbook of Fluorescent Probes and Research Products, the 9th edition or current online edition, both can be from Molecular Probes, and Inc. obtains.Similarly, multiple donor/acceptor and the combination of fluorogen/quencher of using for example cancellation based on FRET (fluorescence resonance energy transfer) (FRET), the cancellation based on non-FRET or wavelength to shift results molecule are known.Example combinations comprises cyan fluorescent protein and yellow fluorescence protein, terbium chelate and TRITC (four RITCs), lanthanide series (for example, europium or terbium) chelate and allophycocyanin (APC) or Cy5, europium cryptate and allophycocyanin, fluorescein and tetramethyl rhodamine, IAEDANS and fluorescein, EDANS and DABCYL, fluorescein and DABCYL, fluorescein and fluorescein, BODIPY FL and BODIPY FL and fluorescein and QSY7 dyestuff.Non-fluorescence acceptor for example DABCYL and QSY7 and QSY33 dyestuff has and eliminates the special favourable aspect that is excited the background fluorescence causing by direct (that is, nonsensitized) acceptor.About the description of FRET dyestuff, referring to for example, belong to the people's such as Mathies U.S. Patent No. 5,668,648,5,707,804,5,728,528,5,853,992 and 5,869,255.

Purposes about quantum dot as the labelling of biomolecule, referring to, for example, the people such as Dubertret (2002) Science298:1759; Nature Biotechnology (2003) 21:41-46; With Nature Biotechnology (2003) 21:47-51.In description of the present invention, this type of quantum dot can be used for any object nucleic acid of labelling, for example RNA interfering.

On other optics, detectable labelling also can be used for the present invention.For example, gold bead grain can be used as labelling and can with white light source, detect by resonant light scattering.Referring to, for example, http://www.geniconsciences.com.Suitable non-optical detectable labelling is also known in the art.For example, magnetic mark can be used for the present invention (the 3nm superparamagnetism CI oxide for example, serving as a mark and NMR detection; Referring to for example, Nature Biotechnology (2002) 20:816-820).

Can utilize the technology of having set up in this area, in building-up process or by reaction after synthetic, labelling is introduced to nucleic acid.For example, can for example select in advance or random nucleotide position in the enzymatic of nucleic acid or chemosynthesis process, fluorescently-labeled nucleotide is mixed to RNA or DNA.Alternatively, can be on the position of selecting at random or in advance by reaction after synthetic, fluorescent labeling (be for example added into RNA or DNA, can be with terminal amine or free sulfhydryl groups chemical synthetic oligonucleotide on the position of selecting in advance, and by fluorogen being coupled to oligonucleotide with reacting of amine or sulfydryl).Fluorescently-labeled reagent for nucleic acid is obtained commercially; For example, the many kinds of test kits for fluorescent labeling nucleic acid can be from Molecular Probes, Inc. (www.probes.com) obtains, test kit for random labelling double-stranded RNA can be from Ambion, Inc. (www.ambion.com, the Silencer.TM.siRNA labelling kit) obtains.Can introduce quencher by similar technology.

Described the building-up process neutralization of Liao automatization, by reaction after synthetic, labelling has been connected to peptide.

Labelling and/or quencher can be introduced to oligonucleotide, for example, for example, by using the controlled glass column of cell size to introduce for example quencher (, 4-dimethylaminoazobenzene-4'-sulfonyl part (DABSYL)).For example in ，Ke automatization building-up process, at the 3' of oligonucleotide end, add quencher; When the site connecting is primary amine group, can use the succinimide ester of 4-(4'-dimethylamino phenylazo) benzoic acid (DABCYL); And when the site connecting is sulfydryl, can use 4-dimethylamino phenylazo-phenyl-4'-maleimide (DABMI).Similarly, can use by use the fluorescein phosphoramidite of fluorescein substituted nucleosides, or the fluorescein dT phosphoramidite of introducing fluorescein part via introns (spacer) on thymidine ring by use is introduced oligonucleotide by fluorescein.For fluorescein is partly connected to terminal position, iodacetyl amido fluorescein can be coupled to sulfydryl.Can use 5'-tetrachloro-fluorescein phosphoramidite in automatization's building-up process, to introduce Tetrachlorofluorescein. (TET).Other reactive fluorogen derivants and its connection site separately comprise the succinimide ester that is coupled to amino 5-carboxyl rhodamine-6G (RHD); Be coupled to the iodoacetamide of the tetramethyl rhodamine of sulfydryl; Be coupled to the isothiocyanate of amino tetramethyl rhodamine; Or be coupled to the sulfonyl chloride of the texas Red of sulfydryl.If desired, can for example utilize the oligonucleotide of high pressure liquid chromatography or additive method purification labelling.

Similarly, can utilize any method known in the art substantially to detect the signal (absorption for example, being produced by fluorescent labeling and/or fluorescent emission) from labelling.For example, and the detection of polychrome detection, FRET (comprise, for example, time resolution or TR-FRET between lanthanide chelate donor and fluorescent dye acceptor; Referring to for example, Journal of Biomolecular Screening (2002) 7:3-10) etc., be known in the art.In brief, FRET (FRET (fluorescence resonance energy transfer)) is non-radiative energy transfer phenomena, wherein has two fluorogens of overlapping transmitting and excitation spectrum, when enough near time, the energy that the dipolar interaction of experience by resonance dipole induction produces shifts.This phenomenon is generally used for researching and analysing the combination of thing such as nucleic acid, protein etc.FRET is that Range-dependent excited state interacts, another fluorogen of the transmitting of one of them fluorogen and next-door neighbour (closely to the variation that is enough to occur observable transmitting) excite coupling.Some fluorogens that excite interact to form excimer (excimer), and it for example, for showing the excited state dimer (, having the phospholipid of pyrene sn-2 acyl chain like thing) of the emission spectra changing; Referring to, for example, Haughland (2003) Handbook of Fluorescent Probes and Research Products the 9th edition, can obtain from Molecular Probes.The discussion being easily understood of FRET is found in handbook and the reference material of quoting herein.

As another example, can use fluorescence polarization.In brief, this type of fluorescence in conjunction with the enforcement of measuring in, by common little, fluorescently-labeled molecule such as the part, antigen etc. with relatively fast spin correlation time for much bigger molecule such as receptor protein, antibody etc. in conjunction with thering is the spin correlation time much slow.The combination of the molecule of little labelling and larger molecule significantly increases the spin correlation time (having reduced the amount of rotation) of tagged kind (being the complex of labelling), and it surpasses the spin correlation time of the molecule of the unconjugated labelling dissociating.This has corresponding effect to detectable polarization level.Especially, the complex of labelling presents the fluorescence polarization more much higher than the molecule of unconjugated labelling.

As those skilled in the art recognize that, any lipid compounds and the preparation that comprises lipid compounds (comprising lipid and contrast media formulations) lyophilizing can be stored, and for example, in for example aqueous medium (sterilized water or phosphate buffered saline(PBS)), rebuild by means of vigorous stirring.For coagulation or the fusion of the lipid that prevents from being caused by lyophilization, can usefully in preparation, comprise additive in case here class merge or coagulation.Useful additive comprises sorbitol, mannitol, sodium chloride, glucose, trehalose, polyvinylpyrrolidone and Polyethylene Glycol (for example PEG400).This type of and other additives are described in document, for example be described in American Pharmacopeia, USP XXII, NF XVII, The United States Pharmacopeia, The National Formulary, United States Pharmacopeial Convention Inc., 12601Twinbrook Parkway, Rockville, in Md.20852, its disclosure is complete to be by reference incorporated to herein.The preparation of lyophilizing has the favourable aspect of longer shelf life conventionally.

If desired, contrast agent of the present invention also can comprise suspending agent.Preferred suspending agent comprises Polyethylene Glycol, lactose, mannitol, sorbitol, ethanol, glycerol, lecithin, Tween-81, sorbitan monooleate and albumin.As those skilled in the art recognize that, also can use different sugar and other polymer for example polyethylene, polyvinylpyrrolidone, propylene glycol and polyoxyethylene.The agent of paramagnetism acidylate mr angiography for example the amount of Mn-DDP-EDTA calculate by weight can be approximately 1% to 75% for preparing total composition of paramagnetism mr angiography agent emulsion.

The present invention can be used for conventionally to imaging patients, and/or especially for diagnosing the existence of patient's illing tissue.Formation method of the present invention can be undertaken by following: to patient, use contrast agent of the present invention, then use nuclear magnetic resonance art scan patients to obtain the visual image of any illing tissue in patient's interior zone and/or this region.Patient's region means whole patient, or patient's specific region or part.

Any dissimilar MR imaging apparatus can be used for putting into practice the present invention, the particular type of device or model are not vital for method of the present invention.The mr imaging technique using is conventional and is described in for example Kean, D.M. and M.A.Smith, in Magnetic Resonance Imaging:Principles and Applications (Williams and Wilkins, Baltimore1986) (its disclosure is complete to be by reference incorporated to herein).The mr imaging technique relating to includes but not limited to nuclear magnetic resonance, NMR (NMR), NMR spectral method and electron spin resonance (ESR).Preferred imaging pattern is NMR.

As recognized by the skilled person, can be by different way such as in blood vessel, per os, per rectum etc., use various dosage forms to patient's administration of contrast agents.Preferably, by intravascular method, use.Useful dosage to be administered and the concrete pattern of using will depend on age, body weight and concrete animal and its region to be scanned, and particular contrast agent of the present invention to be used.Conventionally, with lower horizontal initial dose, then increase dosage until reach the contrast enhancing of expectation.As general guide, common every kg of patient body weight is used about 0.1mg to fat-soluble compound of the present invention and approximately 1 to the approximately 50 micromolar paramagnetic ion of about 1g, although can use higher and lower amount.Similarly, as general guide, when by lipid or suspending agent during for preparation, it can use the amount of the whole preparation of calculating by weight approximately 0.5 to approximately 50% conventionally, although also can use higher and lower amount.

When carrying out method of the present invention, can be individually or with other diagnostic agents, therapeutic agent or other agent combination use contrast agent.These type of other reagent comprise excipient for example flavoring agent or coloured material.

In one embodiment, in suspection, to suffer from the people of propagation of cell mass be useful especially to described method.It also can be used for other imaging techniques and device, as described in this article.Imaging can be used similar compositions to start using in advance of medicine, to determine best liposome size or the bio distribution of following the tracks of afterwards the liposome that carries medicine in injection.Conventionally, compositions is injected into people's blood vessel.Imaging be included in injection described compositions after imaging at least 10 hours or firm once injection after imaging.Can use compositions with the device that is selected from the injection of intravenous injection device, conduit, intravenous drip and the injection of peritoneal injection device.Can use known method to set up lipid dosage range, described scope can comprise the dosage of 0.10 to 0.50 mM of lipid of every kg body weight.

The cell mass that liposome specific delivery to target tissue is for example bred, tumor tissue, inflammatory tissue, Inflamed tissue and infected tissue can be suitable for therapeutic agent delivery to the liposome size of described target tissue to realize by selection.For example, the liposome that has an average diameter of 180nm can not accumulate in solid tumor; The liposome preferably with the average diameter of 140nm is accumulated in identical solid tumor around, and the liposome preferably with the average diameter of 110nm is accumulated in surrounding and the core of this solid tumor.

In another embodiment of the invention, the Liposomal formulation that carries developer of different sizes can be used for surveying in body the size in capillary permeability and hole.This Information Availability for example, in determine the optimal granularity of the liposome of the therapeutic agent that carries the disease that is used for the treatment of particular type in a small amount of experiment (2 to 3 experiments).Because tumor is being biologically heterogeneous and even identical tumor type can show difference between different patients, thus this information for customization liposome size and for the disease that is identified for treating particular type for example the best preparation of cancer or inflammatory tissue be very useful.In another embodiment, also can be by for example, strengthening liposome to the specificity of sending of target tissue with antibody (, therapeutic agent) or other tissue marker substance markers liposomees.In another embodiment, antibody labeling can be used for realizing or strengthens in the cell of therapeutic agent and sends.

In one embodiment, the invention provides formation method, described method comprises:

A) give and have this administration compositions needing, described compositions comprises:

I) developer of sufficient quantity,

Ii) liposome that comprises bilayer, fusogenic protein matter or polypeptide and internal capacity, the amount of wherein said liposome is enough to allow by described liposome delivery to tissue, and described liposome carries developer,

B) make mammiferous imaging of tissue.

In another embodiment, for the compositions of formation method, also with therapeutic dose, comprise therapeutic agent, wherein said liposome carries described therapeutic agent.

The present invention also provides the method for drug delivery, drug delivery monitoring, tumor-killing, tumor regression, tumor growth monitoring and the drug dose administration based on sending for mammal.Described delivery method can comprise:

A) give and have this administration compositions needing, described compositions comprises:

I) have the paramagnetism chelate of paramagnetic ion, described paramagnetism chelate exists to be enough to strengthen the amount of NMR imaging,

Ii) liposome that comprises bilayer, fusogenic protein or polypeptide and internal capacity, wherein said liposome to be to be enough to the allowing amount to tissue by described liposome delivery to exist, and described liposome carries described paramagnetism chelate,

B) make described mammiferous tissue carry out MNR imaging.

In another embodiment, for the compositions of formation method, also with therapeutic dose, comprise therapeutic agent, wherein said liposome carries described therapeutic agent.

Preferably, imaging is quantitative and can assesses the amount of the described liposome that is delivered to described tissue and the amount of the medicine that calculating is sent by selectivity.These class methods and the Combination of Methods of monitoring piece of tissue can be assessed to the therapeutic efficiency of delivery method and medicine.For example, the volume that can measure tissue is with monitoring tissue volume, the reducing of display organization propagation or monitoring piece of tissue.These class methods also can be used for being determined to the best of the particular pathologies tissue of particular patient and send scheme.

In diagnostic application for example ultrasonic and CT in the situation that, by energy at least a portion that for example ultrasonic energy is applied to patient so that target tissue imaging.Then obtain the visual image of patient's interior zone, to can determine the existence of illing tissue or not exist.

Ultrasonic diagnosis and the therapeutic purposes of can be used for.In diagnostic ultrasound, available transducer ultrasound application ripple or a string ultrasonic pulse.Ultrasonic normally pulse and discrete, although it can be continuous if desired.Therefore, diagnostic ultrasound generally includes using of echo pulse, and afterwards, interim when monitoring, ultrasonic transducer receives the signal of reflection.Can use harmonic wave, ultraharmonics or subharmonic.Can use valuably second harmonic pattern, wherein receive 2x frequency, wherein x is interim frequency.The targeted contrast agent of the application of the invention, this can be used for reducing the signal and the signal strengthening from transducer, the position that described contrast agent can be expected by targeting, for example blood clot from background material.Can use similarly the method to receive other harmonic signals, for example odd harmonic signal, for example 3x or 5x.Also can receive and process subharmonic signal for example x/2 and x/3 to form image.

Except impulse method, also can use for example Power Doppler of continuous wave ultrasound.This application firm vesicle for example from polymethyl methacrylate, prepare vesicle time can be particularly useful.In this case, can make with the relative higher energy of Power Doppler vesicle resonance, thereby promote it to break.This can produce acoustic emission, and it can be within the scope of subharmonic or ultraharmonics or be identical with the supersonic frequency of using in some cases.Expection exist the acoustic signature spectrum discharge in this process and the transducer using can receive acoustic emission with test example existing as blood clot.In addition the process that, vesicle breaks can be used for momentum transfer to the surface of blood clot for example to promote dissolution of blood clot.Therefore, realize therapeutic thromboembolism in can and treating the process of ultrasonic combination in diagnosis.Also can use Spectral Doppler.Usually, from the energy level of diagnostic ultrasound, be not enough to promote breaking of vesicle and promote release and the cellular uptake of bioactivator.As noted above, diagnostic ultrasound can comprise using of one or more pings.Allow the intermittence between pulse the acoustic signals of reflection to be received and to analyze.The limited quantity that is used for the pulse of diagnostic ultrasound has limited the effective energy of the tissue that is delivered to research.

More high-octane ultrasonic, for example by the ultrasonic for the treatment of ultrasound equipment generation, conventionally can cause breaking of vesicle kind.Usually, depend on the region of the tissue of stand-by ultrasonic therapeutic, be used for the treatment of ultrasonic device and use approximately 10 to approximately 100% cycle of operation (duty cycle).Conventionally the body region that the is characterized by more substantial muscle masses for example for example heart tissue of organizing of the back of the body and thigh and height vascularization can need the cycle of operation of more growing, for example, reach approximately 100%.

In treatment is ultrasonic, continuous wave ultrasound is for sending higher energy level.For the vesicle that breaks, continuous wave ultrasound is preferred, although also can pulse acoustic energy.If use the acoustic energy of pulse, sound is conventionally with once approximately 8 to approximately 20 or the echo train length pulse of more pulses so.Preferably, echo train length is once approximately 20 pulses.The frequency of the sound using in addition, can be approximately 0.025 to approximately 100 megahertz (MHz).Generally speaking, treat ultrasonic calibration approximately 0.75 to the scope of about 3MHz, approximately 1 to about 2MHz is preferred.In addition, energy level can be at approximately 0.5 watt (W)/square centimeter (cm ²) to about 5.0W/cm ²scope in change, approximately 0.5 to about 2.5W/cm ²energy level be preferred.The energy level for the treatment of ultrasonic (it is overheated to comprise) is generally about 5W/cm ²to about 50W/cm ².For very little vesicle, for example there is the vesicle of the diameter that is less than approximately 0.5 μ m, higher acoustic frequency is normally preferred.This is because less vesicle can more effectively absorb acoustic energy under higher acoustic frequency.When using very high frequency to be for example greater than about 10MHz, acoustic energy will can only penetrate liquid and tissue conventionally to the limited degree of depth.Therefore, the external application of acoustic energy can be suitable for skin and other surface textures.Yet, for deep structure conventionally must focus supersonic energy so that it is preferentially pointed to accumulation zone (focal zone).Alternatively, can pass through interstitial probe (interstitial probe), intravascular ultrasound catheter or intraluminal catheter ultrasound application energy.Can for example in esophagus, use this type of probe or conduit with diagnosis and/or the treatment esophageal carcinoma.Except above-mentioned therapeutic use, this compositions can be used for the esophageal carcinoma or for coronary artery with treatment atherosclerosis, and the therapeutic use for describing in for example U.S. Patent No. 5,149,319 (its disclosure is complete to be by reference incorporated to herein).

Can use the treatment Vltrasonic device of two supersonic frequencies of application.First frequency can be x, and second frequency can be 2x.In a preferred form, like this design apparatus so that the focal zone of the first and second frequencies is converged to single focal zone.Then the focal zone of device can be pointed to the target composition in target tissue, for example targeting vesicle.This Vltrasonic device can provide the second harmonic therapy of the ultrasonic energy of simultaneously using x and 2x frequency.Be expected in the ultrasonic situation that relates to vesicle, this second harmonic therapy can provide breaking of the vesicle that improves of comparing with the ultrasonic energy that relates to single-frequency.Similarly, also expect that optimized frequency scope can be positioned at the basic resonant frequency of vesicle.Lower energy also can be used for this device.The Vltrasonic device that can be used for above-mentioned second harmonic therapy is described in for example Kawabata, the people such as K., and Ultrasonics Sonochemistry, the 3rd volume, pp.1-5 (1996), its disclosure is complete to be by reference incorporated to herein.

About relating to the method for ultra sonic imaging, particularly in relating to the embodiment of vesicle, diagnostic ultrasound imaging can carry out with hyperacoustic the using simultaneously for the treatment of, with the vesicle that breaks, thereby for example increases cavitation (cavitation) or discharges with the targeting of the bioactivator of vesicle combination.Described method comprises step: (i) to patient, use a certain amount of vesicle; (ii) utilize the treatment ultrasound wave cause frequency that vesicle breaks and energy to make the vesicle in patient's region accept ul-trasonic irradiation; (iii) receive from accepting the ultrasound emission of hyperacoustic vesicle and produce the image in described region according to the ultrasound emission receiving treating hyperacoustic harmonic frequency simultaneously.Imaging simultaneously allows operator to monitor in real time breaking of vesicle.

As those skilled in the art recognize that, after the instruction in arms with present disclosure, the vesicle of the amount of broad range can be used for the practice of method described herein.As used herein, term " amount of vesicle " is intended to comprise all such amounts.

Diagnosing image means to make patient's inside body region visual.Diagnosing image comprises for example ultrasonic (US), nuclear magnetic resonance (MRI), nuclear magnetic resonance, NMR (NMR), computerized tomography (CT), electron spin resonance (ESR); Nuclear medicine (when contrast agent comprises active material); And optical imagery, particularly utilize the optical imagery of fluorescent contrast agent.Diagnosing image also comprises breaking by method promotion vesicle of the present invention.For example, can be by ultrasonic for making the visual and checking vesicle of vesicle in the position of a certain tissue.In addition, the target that arrives expectation at vesicle comprises that, after tissue and/or receptor destination, ultrasonic also can be used for promotes breaking of vesicle, thus release bioactive agent and/or diagnostic agent.

According to the present invention, provide for carry out the method for general imaging to patient, and/or especially for diagnosing the method for existence of patient's illing tissue.Can carry out formation method of the present invention by following: to patient, use contrast agent of the present invention, then use for example ultrasonic, computerized tomography and/or MRI scan patient, to obtain patient's interior zone and/or the visual image of any illing tissue in this region.Patient's region means whole patient or patient's specific region or position.

When using contrast agent, preferably they be suspended in aqueous solution and use aseptic technique preparation contrast agent.(for example use less liposome, size is for 200nm and following) and the favourable aspect of the lipid of micelle or emulsifying and the simple suspension of paramagnetic ion and fat-soluble compound be, can, to be about to use (for example, by intravenous injection) front or as the whole step of the preparation of contrast agent, contrast agent be filtered to remove any potential pyrogen by 0.22 micro wire filter (line filter).

For this type of contrast agent is mixed with to stable preparation, can use other additives.For example, when preparation is used for the contrast agent of intravenous injection, can in preparation, comprise parenteral additive.Examples of such additives comprises tension adjustment additive for example glucose and sodium chloride, and it oozes contrast agent for preparing etc.Conventionally so that this type of tension force additive to be provided on a small quantity, for example, calculate by weight total preparation of approximately 0.1% to approximately 0.5%.In addition, can in whole preparation, comprise microbicidal additives to avoid bacterial growth.This type of microbicidal additives (common acceptable amount) can include but not limited to benzalkonium chloride (conventionally calculating by weight total preparation of 0.01%), benzylalcohol (conventionally calculating by weight 1-2%), chlorobutanol (conventionally calculating by weight 0.25-0.5%), metacresol (conventionally calculating by weight 0.1-0.3%), butyl p-hydroxybenzoate (conventionally calculating by weight 0.015%), methyl parahydroxybenzoate (conventionally calculating by weight 0.1-0.2%), propyl p-hydroxybenzoate (conventionally calculating by weight 0.2%), phenol (conventionally calculating by weight 0.25-0.5%) and thimerosal (conventionally calculating by weight 0.01%).In addition, can in preparation, comprise antioxidant, it is particularly useful when contrast agent comprises unsaturated lipids.This type of antioxidant (it is useful amount conventionally) comprises ascorbic acid (conventionally calculating by weight 0.01-0.5%), cysteine (conventionally calculating by weight 0.1-0.5%), monothioglycerol (monothioglycerol) (conventionally calculating by weight 0.1-1.0%), sodium sulfite (conventionally calculating by weight 0.1-1.0%), sodium metabisulfite (conventionally calculating by weight 0.1-1.0%) and tocopherol (conventionally calculating by weight 0.05-0.5%).As those skilled in the art recognize that, can contrast agent of the present invention be formulated as with several different methods and be particularly suitable for endovascular delivery, to any endoceliac send or other send target.

Other reagent

It is also a part of the present invention that the compositions of the material of use except above-mentioned biocompatibility lipid and polymer is prepared microsphere, as long as prepared microsphere stability and the other standards shown in meeting herein.

Can add propylene glycol with the dispersion by promotion lipid granule and dissolve and remove muddiness.Propylene glycol also can be used as thickening agent, and it is promoted the formation of microsphere and stablized by the surface tension increasing on microsphere film or shell.Propylene glycol also can be used as the film of coated microsphere or thereby the extra layer of shell provides extra stability., there is spendable conventional surfactants in the example as this type of other basic or auxiliary stable compound, for example United States Patent (USP) 4,684, and 479 and 5,215,680.

Auxiliary and basicly stable compound in addition comprises that such reagent is as Oleum Arachidis hypogaeae semen, canola oil (canola oil), olive oil, safflower oil, Semen Maydis oil or known absorbable, any other oil of being suitable as stable compound according to the requirement as shown in this description and explanation conventionally.

In addition, compound for the preparation of mixed micelles system can be suitable as basic or auxiliary stable compound, and this compounds includes but not limited to: lauryl trimethylammonium bromide (dodecyl-), cetyl trimethylammonium bromide (cetyl-), myristyl trimethylammonium bromide (myristyl-), zephiran (alkyl=C ₁₂, C ₁₄, C ₁₆), benzyl dimethyl dodecyl bromination ammonium/benzyl dimethyl lauryl ammonium chloride, benzyl dimethyl cetyl ammonium bromide/benzyl dimethyl cetyl chloride ammonium, benzyl dimethyl Tetra-n-decylammonium bromide/benzyl dimethyl tetradecyl ammonium chloride, cetyl-dimethyl ethyl ammonium bromide/cetyl-dimethyl ethyl ammonium chloride or cetyl pyridinium bromide/hexadecylpyridinium chloride.

Find, can, according to size, dissolubility and heat stability by selecting in the various other or auxiliary stabilizer of describing in this article, control for liposome of the present invention.This type of reagent not only can be by they and lipid coating Physical interaction but also these parameters that can affect microsphere by viscosity and the capillary ability on their modified liposome surfaces.Therefore, for liposome of the present invention, can advantageously modify and further stable, for example, by adding following one or more to carry out: (a) viscous regulator, includes but not limited to saccharide and their phosphorylation and sulfonated derivant, and polyethers, preferably it has the molecular weight between 400 and 100,000, two and the polymer of trihydroxy alkane and they, preferably it has the molecular weight between 200 and 50,000, (b) emulsifying agent and/or solubilizing agent also can be used to obtain the modification of expectation and further stable with lipid binding, this type of reagent includes but not limited to arabic gum, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohol, lecithin, monoglyceride and diester, monoethanolamine, oleic acid, oleyl alcohol, poloxamer (for example, PLURONICS F87, poloxamer 184 and poloxamer 181), polyoxyethylene 50 stearate, polyoxyethylene (35) Oleum Ricini, polyoxyethylene 10-oil ether, polyoxyethylene (20) cetyl-octadecyl ether, polyoxyethylene (40) stearate, polysorbate20, polysorbate40, polysorbate60, polysorbate80, propylene-glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan mono-laurate, sorbitan monooleate, anhydrous sorbitol monopalmitate, sorbitan monostearate, stearic acid, triethanolamine and emulsifing wax, (c) suspending agent and/or the viscosifier that can use together with lipid include but not limited to arabic gum, agar, alginic acid, aluminum monostearate, bentonite, magma, carbomer940, carboxymethyl cellulose, calcium and sodium and sodium 12, carrageenan, cellulose, glucosan, gelatin, guar gum, locust bean gum, aluminium silicate magnesium salt, hydroxyethyl-cellulose, hydroxypropyl emthylcellulose, aluminium-magnesium silicate, methylcellulose, pectin, polyethylene glycol oxide, polyvidone, alginate propylene glycol, silicon dioxide, sodium alginate, Tragacanth, xanthan gum, alpha-d-glucose acid lactone, glycerol and mannitol, (d) also can use synthetic suspending agent for example Polyethylene Glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polypropylene glycol and polysorbate, (e) can comprise tension force improving agent (tonicity raising agent), this type of reagent includes but not limited to sorbitol, propylene glycol and glycerol.

The diluent that can be used for producing aqueous environments includes but not limited to water, water of deionized water or the salt that contains multiple dissolving etc., its not the microsphere of interference stability generation and maintain or they are as the purposes of MRI contrast agent; With conventional saline and normal saline.

Although described the present invention in conjunction with its most preferred embodiment, other embodiments are in claimed scope of invention and spirit.Preferred embodiment of the present invention is only intended to illustrate the present invention, but is not intended to limit scope of the present invention, and scope of the present invention defines in claims.

Experimental embodiment

Embodiment 1: Saposin C and Liposomal formulation and in vitro and in vivo are sent

Material-following material is from commercial source: mice laminin，LN, P/S, hyclone and DMEM (Gibco BRL, Gaithersborg, MD); The Neurobasal culture medium (Life Technologies) with B27 fill-in; Restriction endonuclease (New England Biolabs, Beverly, MA); PET21a (+) DNA vector, escherichia coli (E.Coli) host strain [BL21 (DE3)] and His ● Bind resin (Novagen, Medison, WI); Be conjugated with the monoclonal anti His antibody (QIAGEN, Valencia, CA) of Alexa Fluor488; Be conjugated with the goat anti-rabbit antibodies and the continuous goat anti-mouse antibody (ICN/CAPPEL, Aurora, OH) that is conjugated with rhodamine of fluorescein; The anti-ageing reagent (Ventana Medical Systems, Tucson, AZ) that moves back; C ₄reversed-phase HPLC post (Alltech Association Inc., Deerfield, IL); DOPS and 1; 2-dioleoyl-sn-glycerol-3-phosphate-Serine-N-(7-nitro-2-l; 3-Ben Bing oxadiazole-4-yl) (NBD-DOPS) (as the stock solution in chloroform) (Avanti Polar Lipids, Alabaster, AL); Polymine and papain (Sigma, St.Louis, MO).Anion lipid is sodium salt.Every other chemical drugs is SILVER REAGENT or better.

Fibroblast cell cultures-people and mouse primary fibroblast are for all experiments were and and utilize standard method to set up at this laboratory. ¹⁵mice Prosaposin defect fibroblast is from PSAP-/-mice.By all cells at 37 ℃ with monolayer culture in DMEM/FBS (10%) culture medium to use after treating.

Primary cortical neuron culture-as described by people such as Whitmarsh, cortical neuron is cultivated in the serum-free Neuroblasal culture medium that contains B27 fill-in. ⁴⁴take out E16 mice embryonic, cut head and by they be placed in ice-cold have papain (1mg/ml) not containing the Hank Balance Salt Solution (HBSS) of Ca/Mg.Dissect and take out brain, dissecting knife is placed along the dorsal part center line (dorsal midline) between two cerebral hemispheres, but when it cuts, be partial to a little side.This will produce clean cerebral cortex.Peel off gently meninges (meninge) and do not touch interior honest and clean (the medial side) of the residing cortex of Hippocampus.With the refining surgery shears of bending, cut off cortex, they are collected in ice-cold HBSS.Again cortex tissue is placed in to papain HBSS solution, at room temperature carries out 15 to 20 minutes with softening tissue.At room temperature they are transferred in antipain solution, carry out other 5 minutes, finally return in the HBSS that 2ml is ice-cold.With Fisherbran12-546 (18CIR-2) coverslip in coated 12 orifice plates of the PEI that contains laminin，LN, spend the night.Utilize Neurobasal/B27 culture medium by separated cortex tissue culture on the coated coverslip of the PEI in plate.By adding medicine to carry out kainate processing in culture medium.

The preparation of Saposin C and liposome

In our laboratory, use the pET system convention ground of the IPTG-induction in escherichia coli to produce restructuring Saposin C. ¹¹the protein of all expression comprises His-label, and carries out purification on nickel post and by C4 reversed-phase HPLC chromatograph (using linearity (0-100%) gradient of the acetonitrile in 0.1% trifluoroacetic acid).Collect main protein peak and by its lyophilization.As the mensuration protein concentration of being described by people such as Qi before. ¹¹

At N ₂with the DOPS lipid (16.2 μ g) in dry chloroform under vacuum to form lipid film.Saposin C (79 μ g) is added in lipid film, is then suspended in 0.1M citric acid/0.2M phosphate (pH4.7) of 50 μ l.Add other medium or PBS.By bathing the unilamellar vesicle that ultrasonic preparation is large (LUV). ¹⁴use N4+ submicron particle size analyser (Coulter, Miami, FL) to utilize photon correlation spectroscopy to measure liposome size.Use the size of N4+ submicron particle size analyser assessment LUV colony, disperseed, average diameter is 250 ± 100nm.

The in vitro and in vivo of Saposin C-DOPS proteoliposome is sent

By cell (10 ⁵) in thering are 8 vestibule chambers cultivation microscope slides (Lab-Tek II, Nalge Nunc International) of coverslip, in DMEM culture medium, cultivate 48 hours.In cell culture, be added in the Saposin C-DOPS complex in culture medium.At 37 ℃ of incubations, after 48 hours, use PBS washed cell 2 times, fixing to carry out immunofluorescence assay with 2% paraformaldehyde.In order to carry out studying in body, the proteoliposome in PBS is entered to mice by tail vein injection.Within 48 hours after administration of protein-lipid complex, collect mouse brain tissue, to carry out immunofluorescence assay.

Histopathology and immunofluorescence

Before processing fixedly mouse brain tissue and by its quick freezing in 10% formalin.With h and E (H & E), paraffin section is dyeed, under optical microscope, analyze.

(there is little improvement) as described, carry out immunofluorescence dyeing. ¹⁵cultured cell (1x10 with PBS washing in thering is the culture dish of coverslip ⁵), then at room temperature with 2% paraformaldehyde, fix 10 minutes.After the Triton X-100 in PBS with 0.1% processes, that at 37 ℃, sample is puted together with the first antiserum (carrying out 2 hours) separately and fluorescence two resists incubation together with (carrying out 1 hour).Primary antibodie and two anti-dilution factors are respectively 1:30 and 1:60.By the incubation together with the lock solution that contains 5% mice serum of the mouse brain tissue slice in 4% paraformaldehyde, then add the first anti-His antibody.The anti-mouse antibodies that rhodamine is puted together is as two anti-detections.In section, add and anti-ageingly move back agent (Antifade) to prevent fluorescent quenching.Utilize Laser Scanning Confocal Microscope (LSM510, Zeiss) or fluorescence microscope (Zeiss Axioskop) to detect fluorescence signal.

Embodiment 2: use the synthetic of liposome that acid long-chain lipid, neutral long-chain lipid and neutral short chain lipid carry out

Materials and methods

All phospholipid DOPS, DPPC and DHPC are used purchased from Avanti polar lipid and in the situation that nothing is further purified with powder type.In order to carry out dynamic light scattering (DLS), measure, in mixture, DOPS is approximately 10 to approximately 1 to the mol ratio of DPPC, for whole samples ([DPPC]+[DOPS])/DHPC=approximately 4.Use the combination of vortex and temperature cycles (between 50 and 4 ℃) lipid mixture to be dissolved in to the ultrapure H of filtration with the TL concentration of 10wt.% ₂in O (Millipore EASYpure UV).Then with the H filtering ₂o is diluted to 5,2,1,0.5 and 0.1wt.% progressively by the 10wt.% solution of homogenize.

Before DLS, by 5,50 and 200 times of lipid sample stock solution dilutions, use N4 ⁺particle Size Analyzer (Coulter, Miami, FL) is analyzed.Through determining, dilution system is on not impact of size measurement.In the situation that SANS tests, except using D ₂o (99.9%, Chalk River Laboratories, Chalk River, ON) substitutes H ₂o, to obtain outside the sample of the TL concentration with 0.5wt.%, is used for identical sample preparation scheme in the sample of [DOPS]/[DPPC]=10.Then use the acidic buffer comprise isopyknic 0.1N sodium acetate (NaAc) and 0.1N acetic acid (HAc) that 0.5wt.% solution is further diluted to 0.1 and the mixture of 0.05wt.%.The solution of gained is at D ₂in O, there is 4.78 ± 0.02 pH value, and the pH of buffer is being used D ₂o is stable when dilution surpasses 12 times.

In the situation that SANS tests, except using D ₂o (99.9%, Chalk River Lab.) substitutes the H filtering ₂o, until TL concentration is outside 0.5wt.%, is used for identical sample preparation methods in the sample of [DOPS]/[DPPC]=10.Then use the acidic buffer of the equal-volume mixture that comprises 0.1N sodium acetate (NaAc) and 0.1N acetic acid (HAc) solution by 0.5wt.% solution dilution to 0.1 and 0.05wt.%, at D ₂in O, produce the pH value of (4.78 ± 0.02).The pH value of buffer is utilizing D ₂o is stable when dilution surpasses 12 times.

By using the pET system (26) of IPTG-induction, in Bacillus coli cells, cross expression SapC.From nickel, note the protein that eluting has the expression of His-label.After dialysis, by HPLC chromatography, be further purified protein as follows: with 0.1% trifluoroacetic acid (TFA) balance C4 reversed-phase column 10 minutes, elute protein more than 60 minutes in linearity (0-100%) gradient of 0.1%TFA (in acetonitrile) then.Collect main protein peak and by its lyophilization.Measure as described above protein concentration.The people such as Qi, 1994.

By the synthetic Hl (YCEVCEFLVKEVTKLID) of SynPep Corp. (California, USA) and H2 (EKEILDAFDKMCSKLPK) peptide, the concentration by it with 1.5mg/mL is dissolved in D ₂in O.Then to 0.1wt.% lipid soln ([DOPS]/[DPPC]=10 and ([DPPC]+[DOPS])/DHPC=4)) in add respectively two kinds of peptide solutions (1.5mg/mL) (with the volume ratio of about 12:1) and SapC solution (with the volume ratio of about 12:1) to produce whole peptide (or SapC) concentration of 62.5 μ M, this concentration is greater than induces the required SapC concentration of film stabilization removal.(Wang, waits people, 2003).

Be positioned at National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR, Gaithersburg, Maryland, USA) one of 30m SANS instrument NG7 on carry out SANS experiment.Will

wavelength X and long sample to the detector distance (SDD) of neutron condenser lens and 15.3m be used from the less Scattering of Vector value of acquisition, q=4 π/λ sin (θ/2), wherein θ is angle of scattering.Also by 5 and other two SDD of 1m for cover from 0.002 to

whole q scope.Then with regard to the transmission of detector sensitivity, background, empty chamber scattering (empty cell scattering) and sample, proofread and correct original 2-D data, then described data are circulated to average (circularly averaged) to produce 1-D data around beam center.According to the flux of direct beam, 1-D data are placed on absolute coordinate.Intensity by average last 10 to 20 data points is measured discontinuous platform, and the data of deduction reduction.

(14,15) as described, are used N4+ submicron particle size analyser (Coulter, Miami, FL) to utilize photon correlation spectroscopy to measure liposome size.The size of using N4+ submicron particle size analyser assessment LUV colony, it is polydisperse, has 20 to 800nm average diameter.With 90 ° of angles, obtain the data of liposome size assessment, with medium auto-correlation function (fair autocorrelation function), use size distribution to process (size distribution process, SDP) analyzing and processing data.By SDP, measure the size of the vesicle that provides main fraction.Utilize ANOVA analysis and evaluation statistical significance.Error bar represents standard deviation.

Utilize Hitachi TEM (H-7600, HITACHI, Japan) to take transmission electron microscope (TEM) image.The microdroplet of each sample is placed on nickel screen with the covering of the magnificent film in the side of supporting (200 orders, thickness range is 30 to 75nm, Electron Microscopy Sciences, PA).At room temperature nickel screen is placed on filter paper and carries out 2 hours, then carry out tem analysis.Under the accelerating potential of 80kV, operate TEM.Optimal imaging background under high-amplification-factor, and in object region, the lower location of low amplification (50-1,000X).Use the high amplification to 50,000X to focus on single vesicle.Manual adjustment contrast and brightness are until obtain " clearly " image, optimal imaging background under high-amplification-factor.Use has two AMT CCD digital cameras (2Kx2K, 16bit) of suitable image capture software and takes TEM microphotograph.

About using the embodiment of short chain lipid, the dynamic (dynamical) generally accepted model that forms low polydispersity ULV is as described below:

At first, plate-like micelle precursor starts to form, and short chain lipid coating is at edge, and long-chain lipid is on the plane bilayer surface of dish, so that the curvature energy minimization at edge.The rising of dilution factor or temperature causes that edge short chain lipid is to the loss of bilayer or solution, and this causes the increase of line tension and with the coalescence between hub disk, thereby forms larger dish.When the increase of line tension overwhelms the coalescence of contiguous plate-like micelle, the stretched length at edge has to reduce, thereby causes that bilayer is folded into the spherical shell with opening, and its edge is covered by short chain lipid.Finally, opening can be closed, disappears, thereby cause the morphology of vesicle around the short chain lipid at edge.

Embodiment 3: the MR of the tumor cell with USPIO labelling that use DOPS liposome carries out detects

In order preparing, to comprise for example liposome of USPIO of MR detectable label, to use following method.In having the aqueous solution of DOPS, the coated USPIO granule of supersound process glucosan fails to produce sufficient encapsulation in liposome.In order to increase the content of USPIO in liposome, use has little improved by people such as Bogdanov, Trapping of dextran-coated colloids in liposomes by transient binding to aminophospholipid:preparation of ferrosomes.Biochim Biophys Acta, 1994.1193 (1): the chemical coupling method of p.212-8 describing.In brief, oxidation is coated on glucosan on USPIO granule to produce aldehyde radical.Aldehyde radical forms covalency Schiff key (Schiff bond) with the amine of DOPS under high pH.The liposome obtaining has the mean size of 150nm, as passes through N4 ⁺particle Size Analyzer (Beckman Coulter, CA) analysis confirmation.Liposome solutions is dialysed to low pH solution, so that be bonded to the outer field USPIO of liposome, depart from.Use Con-ASepharose4B post (Amersham Biosciences Corp., NJ) to remove the not USPIO of encapsulation by affinity chromatograph.By conventional electrical microscopy, confirm USPIO-DOPS liposome structure.To use the standard R2 relaxation curve of the free USPIO of known quantity and the generation of the mixture of DOPS liposome for assessment of the concentration of iron in DOPS liposome.Use 1mM DOPS concentration to obtain the maximum level of 32 μ g Fe/ml.Prepare 4 neuroblastoma cell samples, every group of approximately 10,000 cells.By the first and second samples respectively with l00uM incubation together with 300 μ MUSPIO-DOPS liposomees in culture medium.The 3rd sample comprises cell but without USPIO or liposome.At incubation, after 36 hours, washed cell 4 times by trypsin treatment, is then fixed on it in mixture of 0.5% agarose solution and growth medium (1:1) in 4ml vial.

Use, with regard to the gtadient echo method of T2 Weighted optimal, utilizes 7T Bruker Biospec scanner to carry out the high-resolution MR imaging of cell.By the 3D FLASH imaging sequence with the TR/TE/0 of 200ms/35ms/10 ° and 320X320X64 matrix for 3.2cm X3.2cm X0.64cm FOV, thereby produce isotropism l00um resolution.

The demonstration of MR image, USPIO granule is by the cellular uptake in sample 1 and 2, and corresponding to higher USPIO-DOPS liposome concentration, sample 2 shows the picked-up increasing.In the sample that comprises cell and the liposome-USPIO by ultrasonic preparation, the much lower cell of quantity detected.After processing in use IDL, algorithm obtains the estimated value of the number of the cell detecting in each bottle.Compare with bottle 1, the quantity of the cell detecting in bottle 2 is about 1.4 times.Gel and represent that the average contrast-noise between the hypo-intense region of cell is 20.15, SD11 than (CNR).

The preparation of embodiment 4:SapC-DOPS proteoliposome

SapC's is protonated for promoting the combination of SapC and DOPS film.First, by SapC being dissolved in the decile (pH5,20 μ l) of acidic buffer, carry out protonated SapC, then with PBS or neutral buffered liquid (pH7), be diluted to 1ml final volume.Alternatively, can be by bronsted acid (such as TFE, chloroform, methanol etc.) for dissolving SapC and DOPS lipid.To have reported this type of bronsted acid be good hydrogen bond donor and protein is had to Protonation effect (1).Can under nitrogen atmosphere or vacuum system, evaporate this kind solvent to be dried.Add suitable plecable (for example PBS) to form SapC-DOPS proteoliposome.The method merges the DOPS liposome of avoiding being induced by SapC under acid pH.The proteoliposome of preparing by the method exists with single discrete form, and mean size is 200nm.

The temperature of embodiment 5:SapC-DOPS proteoliposome is controlled seepage

Utilize thermally sensitive various lipid composition design SapC-DOPS proteoliposome, with seepage, be packaged into the content in liposome.

Embodiment 6: the sign of liposome/Saposin C

For time and the steric interaction of sphingolipid activator protein and liposome membrane are described, inventor has been devoted in the exploitation of inside (Trp) and/or outside (NBD, pyrene etc.) fluorimetry.These class methods comprise flow analysis (flow-analysis) and the fluorescence microscopy of emission maximum spectral displacement, FRET (fluorescence resonance energy transfer), fluorescence stop-flow analysis (stopped-flow analysis), fluorescence beadlet-sphingolipid activator protein-liposome complex.In addition, circular dichroism (CD) is for assessment of never containing lipid, the relative secondary structure to the sphingolipid activator protein of lipid binding changes.The analysis of initial results is evolved into the hypothesis of proposition.

What summarize below is the relevant research of fluorescence analysis of merging to expression, purification, functional analysis, mutation and sphingolipid activator protein-phospholipid interaction and film.

I. purification and the sign of natural and restructuring sphingolipid activator protein

A) from the expression of the sphingolipid activator protein of prokaryotic system

Although separated and characterized natural sphingolipid activator protein, set up recombinant expression system and think that it is very important that the research of mentioning provides the obtained source of the sphingolipid activator protein of a large amount of normal, sudden changes and Trp-labelling.Based on following aspect developing prokaryotic system: 1) sphingolipid activator protein has the N-glycosylation site that at least one is occupied, but for sphingolipid activator protein B and C, occupying of this type of site is not that function is necessary.2) in eukaryotic system protein expression be effort and resource-intensive and slowly.By contrast, prokaryotic system is fast and produces the wild type of high yield and the protein of sudden change.And 3) available Trp residue is as inner fluorescent probe labelled protein, because wild type A is the unique sphingolipid activator protein that comprises natural Trp (37W).

B) generation of active sphingolipid activator protein in escherichia coli

Use pET21a serial carrier in BL21 (DE3) cell, to cross expressive function sphingolipid activator protein.Be to carry out after IPTG induction at 37 ℃ or 30 ℃, in the soluble fraction of the cell breaking, find to contain in a large number the sphingolipid activator protein of His label.Can load on the pillar of nickel easily this type of protein purification to electrophoresis homogeneity.Alternatively, by introduce termination codon after protein coding region, produce the sphingolipid activator protein without His label, then utilize T7-taq monoclonal antibody, use immune affinity column to carry out purification.The restructuring Saposin C of purification shows activation and the other biological character of good acid β-glucosyl enzym.Circular dichroism spectra, light scattering and ES-MS analyze the physical property for assessment of the sphingolipid activator protein of purification, for example coherent condition and molecular weight.Also produce Trp-sphingolipid activator protein without His label for essential control experiment.In liposome reconstructing system and neural axon growth mensuration, use the functional completeness of the acid β-glucosyl enzym mensuration restructuring Saposin C of defat and homogeneity.With sulfatide in conjunction with the function of measuring restructuring sphingolipid activator protein B.The external function of restructuring sphingolipid activator protein B and C is similar to natural or deglycosylated sphingolipid activator protein.

The functional conformation of the sphingolipid activator protein of II. being induced by phospholipid

In order to determine the interactional specificity of Saposin C-phospholipid, use CD, fluorescent emission displacement and Fluorimetric Quenching Method exploitation liposome system.The Saposin C of the sudden change that comprises single Trp (W) producing is called Saposin C (0W), (S37W) and (81W).The Saposin C of this type of Trp labelling is as follows: Saposin C (0W) is at first NH of ripe Saposin C ₂before-end amino acid, have Trp, Saposin C (S37W) has Trp at residue 37 (that is, in centre), and Saposin C (81W) in the end has Trp after a COOH-end amino acid.This type of displacement is on the activation character of Saposin C or the not impact of CD spectrum.

A) CD spectrum

By using CD spectrographic method, the relative secondary structure by film zygotic induction restructuring sphingolipid activator protein changes.Available from the relative secondary structure of the sphingolipid activator protein of the unsaturated Phosphatidylserine of acidity (PS)/Saposin C complex and neutral phosphatidylcholine (PC)/sphingolipid activator protein B complex, change similar and cause the minimizing of β chain content and the increase (table 4) of α helical content.

Table 4

Table 4.

The circular dichroism (195-250nm) with the sphingolipid activator protein of different phospholipid is analyzed

For sphingolipid activator protein A and B in PS (18:1,1) complex, do not observe change.These results show that sphingolipid activator protein A has the membrane interaction different from Saposin C with B.On Jasco710 instrument, gather CD data, and the method for use Yang (referring to Chang, C.T., Wu, C.S. and Yang, J.T.Anal.Biochem (1978) 91,13-31) deconvolute.

B) fluorescence emission spectrum

When tryptophanyl environment change polarity chron protein emission spectra is subjected to displacement.Adding the fluorescence spectrum of sphingolipid activator protein A (0W), the A (37W), A (81W), C (0W) and the C (81W) that obtain after cephalin acyl serine (BPS) liposome to show blue shift (table 5).

Table 5

Table 5.

The fluorescent emission maximum of Trp-sphingolipid activator protein in the situation that cephalin acyl serine (BPS) does not exist and exists

Experiment condition: pH4.7, protein: lipid=1:20 to 40.At 22 or 37 ℃, do not observe difference.

Blue shift is implying the interaction of sphingolipid activator protein and lipid in complex forming process.Yet Saposin C (S37W) does not show displacement in the situation that BPS exists.This means the NH of Saposin C ₂-(0W) and COOH-(81W) end enter film, yet the stage casing of sequence does not enter film.For sphingolipid activator protein A, situation is just in time contrary, and the stage casing of sequence (37W) is in film.This means that sphingolipid activator protein A-film associates completely different from the film association of Saposin C.These results are analyzed consistent with CD.In the situation that neutral EPC exists, for sphingolipid activator protein A or C, do not observe maximum emission wavelength variation, while using the PS that comprises satisfied fatty acid chain, do not observe maximum emission wavelength yet and change.

1. the time of sphingolipid activator protein and immobilized artificial membrane and steric interaction

In order to study time and the steric interaction of sphingolipid activator protein and immobilized artificial membrane, the inside fluorescent probe using Trp as sphingolipid activator protein, is used fluorescence to arrhea and cancellation method.This type of experiment allows to identify the regional interaction between sphingolipid activator protein and lipid bilayer, also can identify the kinetics of their combination.

Time interphase interaction

Under acid pH, at Saposin C (0W) and synthetic Phosphatidylserine [PS (18:1,1)] vesicle, in conjunction with rear fluorescence intensity, significantly increase.The change of this zygotic induction is that lipid concentration is dependent and need at least one unsaturated fatty acid chain.In order to assess this interactional kinetics, arrhea experiment, the variation of monitoring fluorescence in Saposin C/liposome complex forming process.When Saposin C (0W) and PS (18:1,1) or BPS vesicle are mixed, the fluorescence of Trp increases, but the time course of this variation can not detect because of the restriction of the ability of machine.Obviously, Saposin C occurs at least 10 milliseconds with the interaction of the film that comprises unsaturated PS.

According to CD and emission spectra data, the unsaturated phospholipid of Saposin C combined belt negative charge.This shows to have electrostatic interaction between positively charged residue in Saposin C and electronegative film surface.After this initial phase mutual effect, protein passes through hydrophobic interaction embedding to film.Mixture for Saposin C (0W) with PS (18:0,0), does not observe the variation of the glimmering light intensity of Trp and the displacement of transmitting.

Steric interaction

In order to measure sphingolipid activator protein, insert the degree of depth of BPS liposome, with ever-increasing molar percentage (0-50%), the phosphatidylcholine of spin labeling (SLPC) is mixed to BPS liposome.SLPC, hydrophobicity fluorescence quenching, comprises doxyl group, and its different carbon (n) that are positioned at acyl chain are upper: SLPC5 (n=5), SLPCl0 (n=10) and SLPC16 (n=16).After adding Trp-sphingolipid activator protein, by protein-liposome mixture (protein: lipid=1:20) incubation 30 minutes at room temperature, the variation of then recording fluorescence intensity.For the Trp-sphingolipid activator protein that is displayed in Table 2 blue shift, use BPS/SLPC5 liposome to observe significant quenching effect (30-60%).Cancellation efficiency depends on doxyl group on the SLPC position in acyl chain.Doxyl group is darker in film, and cancellation efficiency is so just lower.For BPS/SLPC10, the tryptophanyl fluorescence of Saposin C (0W) is quenched 30%.

2. the film of Saposin C-induction merges

Saposin C is the polyfunctional molecule with lysosomal enzyme activation and neural axon growth active (neuritogenic activity).Detailed functions/the structure organization of Saposin C is shown in Fig. 3.

Amino acid residue 51-67 is that its best enzymatic mobilizing function is necessary, but non-sufficient.After lipid binding, the disulfide structure of Saposin C and conformational change are also that this activity is necessary.By three methods for this research: (1) arrheas the minimizing of the self-cancellation that monitoring causes because of the vesicle that comprises fluorescent probe of Saposin C induction and the fusion of non-fluorescence vesicle; (2) variation of monitoring lipid vesicle size add Saposin C in vesicle after, the distribution of size, as used N4+ submicron particle size analyser (Coulter Co.) to measure; (3) variation of monitoring inside fluorescence of Trp-Saposin C in liposome fusion process.These results have been determined the short fusion activity region in αhelix territory on the amino of Saposin C and c-terminus, and the kinetics (vide infra) that merges of the liposome of Saposin C induction.

The liposome of Saposin C induction merges

Fluorescent probe has been widely used in measuring film and has merged, and for example fluorescence takes off cancellation method and FRET (fluorescence resonance energy transfer) (FET), and can be used for quantitative and dynamic analysis.De-cancellation method is for studying the short fusion activity of Saposin C.Octadecyl rhodamine B (Rl8) is selected as fluorescent probe and by with BPS or PS (18:1,1) together altogether in the ultrasonic inside aqueous compartment that is captured in liposome vesicle.Rl8 shows self-cancellation under high concentration.

When Rl8 concentration reduces, the fluorescence of R18 increases (de-cancellation effect).After the Vesicle fusion of cold vesicle and labelling, the concentration of Rl8 is diluted, thereby causes the increase of fluorescence intensity.The vesicle of R18 labelling (lipid: R18=96:4, mol:mol) is mixed with the identical lipid vesicle without fluorescent probe.Arrhea and measure with rapidly by these vesicles and Saposin C or Ca ²⁺ar ion mixing.Generation time-tracking (Time-trace) curve is to carry out dynamic analysis.Unsaturated PS (18:1, the 1) film of Saposin C induction merges demonstration and uses Ca ²⁺the same kinetics of dynamical phase producing.When reaction temperature is during higher than the phase transition temperature (Tc) of phospholipid, merge widely and occur.The Tc of synthetic PS (18:1) is approximately-11 ℃, however the Tc of PS (18:0) very high (68 ℃).Therefore, the lipid bilayer phase of PS at 24 ℃ (18:1,1) and BPS's (18:0 and 18:1) is different.Result shows, the kinetics that the film of Saposin C induction merges can be measured by the physical state of bilayer lipid.

3. the mensuration of size variation

Electron microscopy (EM) can be used for Vesicle fusion analysis, because the size of the vesicle merging is greater than nonfused size.N4+ submicron particle size is analyzed for assessment of the granularity within the scope of 3nm to 3 μ m, because most of liposome is within the scope of this.Use the monodispersed BPS-liposome of the big or small about 200nm of supersound process condition generation of cup ultrasonic disruption instrument (cup sonicator).After adding Saposin C, this type of vesicle becomes larger size (reaching 2-3 μ m).The increase of size is relevant to Vesicle fusion, as shown by above-mentioned de-cancellation experiment.Saposin C makes vesicle expanded in size within the time of 10 minutes under pH4.7, but under pH7.4, does not expand vesicle size (referring to Fig. 4).

These data show the short fusion activity of the pH sensitivity of Saposin C.Saposin C promotes size variation in about 50nM concentration.In order determine to be responsible for the region of this Fusogenic properties, test only contains 50% NH of Saposin C ₂the peptide of end half part or 50% COOH end half part.Two kinds of peptides all show fusion activity.These data show that the linear order of mediates fusion is positioned on the two ends of Saposin C.

4. the mechanism merging

Protein conformation variation is considered to play an important role in the film of protein mediation merges.Use Saposin C dependency film to merge this syncretizing mechanism of assessment.First, form Saposin C-PS (18:1,1) liposome complex.In the film of this Saposin C-grappling, protein conformation is changed.This complex is at pH3 to 10 time and be stable in the SDS of low concentration solution.This shows, Saposin C is extremely slow from the dissociation rate of PS vesicle.

Because the Trp in Saposin C (0W) is embedded in the inside of lipid bilayer, so the variation of its signal indicating, and the surrounding of Trp changes.After approximately 20 to 30 milliseconds, Trp fluorescence signal is down to base level.This shows that Saposin C and other PS-vesicles in complex interact.Shortly after that, signal falls and is back to base level, shows that fusion process finishes.These data show, even if Saposin C still keeps short fusion activity when lipid film is combined at it.Therefore, when lipid binding, the conformation change of Saposin C is not that its short fusion activity is necessary.This result is enough to induce the conclusion that film merges to be consistent with linear order.

XI. the gene optimization of Saposin C is with synthetic

Consider mRNA secondary structure and afterwards for the elimination of the restriction site of sub-clone, the DNA sequence of Saposin C is carried out codon optimized to express in escherichia coli.At the 5' of gene and 3' end, add restriction site NdeI and SalI respectively, and add two termination codon with the correct termination of the protein guaranteeing to express at the end of the coded sequence of Saposin C.By DNA2.0, carry out gene and synthesize, and the gene of optimizing by sequence verification, provide it to the VTI in cloning vehicle " pJ2 ".This vector construction body is called pJ2-SapCg; The Saposin C box gene of the optimization that restriction site NdeI and SalI be border of take is called SapCg.

Be cloned into pET24a

In the following manner SapCg is cloned into pET24a.Use restriction site NdeI and SalI that SapCg is cut out from pJ2-SapCg, then connect into the expression vector pET24a (Novagen) also having cut on identical site.The construct of this connection is transformed into escherichia coli cloning strain TOP10 (Invitrogen).Use kanamycin (50mg/L) to select.Carry out bacterium colony PCR to determine the bacterium colony of the conversion of carrying the carrier with SapCg insert.From test those positive bacterium colonies, select a bacterium colony for further operation.Utilize Plasmid Miniprep test kit (Qiagen) from this clone's preparation plasmid DNA, by the existence of restrictive diges-tion and sequence analysis checking SapCg insert DNA.This plasmid construction body is called pET24a-SapCg.

Shaking flask induction

Expression construct pET24a-SapCg is transformed into competence escherichia coli expression bacterial strain BL21 (DE3) (Novagen).Select 3 bacterium colonies (clone), with regard to the initial testing of Saposin C, express and test.For BL21 (DE3) clone, use the LB culture medium with kanamycin (50mg/L) in 125ml shaking flask, to carry out small-scale expression.When cell OD600 reaches approximately 0.6 by adding the final concentration of IPTG to 1mM to realize induction.Before being about to induction and in latter 4 hours collected specimens of induction.For whole 3 BL21 (DE3) clone, the protein expression of correct size is similar.The work glycerol seed stock solution of preparing whole 3 clones.Random selection 1 clone further develop.

Fermentation condition

For the clone of fermenting, be the pET24a-SapCg (above-mentioned) transforming at escherichia coli expression bacterial strain BL21 (DE3).Use 8 liters of NBSC BioFlow3000 fermentation tanks to ferment, described fermentation is by using the fed-batch fermentation of DO-Stat feeding strategy to form.Initial incubation volume is 5 liters.The composition of batch culture base (Batch media) and supplemented medium (feeding media) is shown in table 1 and 2.Fermentation temperature before induction is 30 ℃.By adding the final concentration of IPTG to 1mM to realize induction in late logarithmic (log) phase, meanwhile by temperature transition to 37 ℃.Whole fermentation continues 22 hours, and induction is carried out 10 hours.

Table 1-batch culture base

	Composition	Amt/L	Unit
				1	(NH 4) 2HPO 4	4	g
2	KH 2PO 4	13.3	g
				3	MgSO 4.7H 2O	1.2	g
4	Citric acid	1.7	g
				5	Yeast extract	2	g
6	Trace metal solution	10	ml
				7	Glucose .H 2O	22	g
8	With I type water, volume is adjusted to 5L, pH6.8	QS	ml

Table 2-supplemented medium

	Composition	Amt/	Unit
				1	D-Glucose-monohydrate	660	g
2	Yeast extract	60	g
				3	MgSO 4.7H 2O	20	g
4	I type water	QS	ml

The preparation of inclusion body

The preparation of inclusion body is carried out in use from the pastel of above-mentioned fermentation.About 20g pastel is resuspended to altogether in the lysis buffer of 200ml (50mM Tris pH8,1mM EDTA, 100mM NaCl).After resuspension and complete homogenate, use Micro Fluid (microfluidization) smudge cells.By at 4 ℃ with the insoluble part of centrifugal 60 minutes sedimentation cell lysates of 16,000X g.The precipitation that homogenizes in 800ml lysis buffer+l%triton X-100 altogether, at room temperature mixes 45 minutes.At 4 ℃ with 16,000X g centrifugal 60 minutes.The lysis buffer that use has 1%triton X-100 carries out 2 washings again, then uses the lysis buffer that does not contain triton X-100 to wash 1 time.Then by pellet resuspended in 600ml6M carbamide pH8.5 altogether (by 20mM Tris buffering), at room temperature stir 3 hours.At 4 ℃ with 16,000X g centrifugal 60 minutes, to clarify sample.After using the existence of Saposin C specific antibody confirmation Saposin C, the supernatant of gained is used for being further purified.

The chromatography of SapC and refolding

Following chromatography, refolding and concentration step are not all carrying out containing under endotoxic condition.Use Q-sepharose Fast Flow resin (GE Amersham) to carry out the purification from the Saposin C of inclusion body by ion-exchange chromatography.Level pad (buffer A) is 6M carbamide/0.02M Tris, pH8.5.Elution buffer (buffer B) is 6M carbamide/1MNaCl/0.02M Tris, pH8.5.By discontinuous gradient, carry out eluting at first, by 5% buffer B/95% buffer A, carry out 10 column volumes, then use 10% buffer B/90% buffer A to carry out 10 column volumes, then use the linear gradient of from 10% to 100% buffer B to carry out eluting, surpass 10 column volumes.Collect and retain whole fraction.Existence with regard to Saposin C utilizes SDS-PAGE to carry out the analysis of fraction, selects the fraction that comprises most of Saposin C to carry out refolding.

By dialysing, to McIlvaine buffer, (0.05M citric acid/0.1M phosphate carries out refolding in pH4.7).Then Saposin C albumen is concentrated into about 0.2mg/ml.By the visual inspection of SDS-PAGE, determine that said preparation is approximately 90% purity.

Sequence table

Sequence table

<100> general information:

The number of <160>SEQ ID NO: 6

<200> sequence signature:

<210>SEQ ID NO1

<211> length: 40

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 1

Ser Asp Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro

<200> sequence signature:

<210>SEQ ID NO2

<211> length: 38

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 2

Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro

<200> sequence signature:

<210>SEQ ID NO3

<211> length: 38

<212> type: PRT

<213> is biological: homo sapiens

<220> feature:

<221> title/key word: MISC_FEATURE

<222> position: (1) .. (1)

Other information of <223>: when being positioned at aminoacid on 1 and being hydrophobic amino acid, comprise Val, Leu, Ile, Met, Pro, Phe and Ala

<400> sequence: 3

Xaa Xaa Cys Glu Xaa Cys Glu Xaa Xaa Xaa Lys Glu Xaa Xaa Lys Xaa Xaa Asp Asn Asn Lys Xaa Glu Lys Glu Xaa Xaa Asp Xaa Xaa Asp Lys Xaa Cys Xaa Lys Xaa Xaa

<200> sequence signature:

<210>SEQ ID NO 4

<211> length: 39

<212> type: PRT

<213> is biological: homo sapiens

<220> feature:

<221> title/key word: MISC_FEATURE

<222> position: (1) .. (2)

Other information of <223>: when being positioned at aminoacid on 1 and 2 and being hydrophobic amino acid, comprise Val, Leu, Ile, Met, Pro, Phe and Ala

<400> sequence: 4

Xaa Xaa Xaa Cys Glu Xaa Cys Glu Xaa Xaa Xaa Lys Glu Xaa Xaa Lys Xaa Xaa Asp Asn Asn Lys Xaa Glu Lys Glu Xaa Xaa Asp Xaa Xaa Asp Lys Xaa Cys Xaa Lys Xaa Xaa

<200> sequence signature:

<210>SEQ ID NO 5

<211> length: 38

<212> type: PRT

<213> is biological: homo sapiens

<220> feature:

<221> title/key word: MISC_FEATURE

<222> position: (1) .. (1)

Other information of <223>: when being positioned at aminoacid on 1 and being hydrophobic amino acid, comprise Val, Leu, Ile, Met, Pro, Phe and Ala

<400> sequence: 5

Xaa Xaa Cys Glu Xaa Cys Glu Xaa Xaa Xaa Lys Glu Xaa Xaa Lys Xaa Xaa Asp Asn Asn Lys Xaa Glu Lys Glu Xaa Xaa Asp Xaa Xaa Asp Lys Xaa Cys Xaa Lys Xaa Xaa

<200> sequence signature:

<210>SEQ ID NO6

<211> length: 38

<212> type: PRT

<213> is biological: homo sapiens

<220> feature:

<221> title/key word: MISC_FEATURE

<222> position: (1) .. (1)

Other information of <223>: when being positioned at aminoacid on 1 and being hydrophobic amino acid, comprise Val, Leu, Ile, Met, Pro, Phe and Ala

<400> sequence: 6

Xaa Xaa Cys Glu Xaa Cys Glu Xaa Xaa Xaa Lys Glu Xaa Xaa Lys Xaa Xaa Asp Asn Asn Lys Xaa Glu Lys Glu Xaa Xaa Asp Xaa Xaa Asp Lys Xaa Cys Xaa Lys Xaa Xaa

<200> sequence signature:

<210>SEQ ID NO7

<211> length: 22

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 7

CysGlnPheValMetAsnLysPheSerGluLeuIleValAsnAsnAlaThrGluGluLeuLeuTyr

<200> sequence signature:

<210>SEQ ID NO8

<211> length: 21

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 8

CysGlnLeuValAsnArgLysLeuSerGluLeuIleIleAsnAsnAlaThrGluGluLeuLeu

<200> sequence signature:

<210>SEQ ID NO9

<211> length: 22

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 9

CysGluTyrValValLysLysValMetLeuLeuIleAspAsnAsnArgThrGluGluLysIleIle

<200> sequence signature:

<210>SEQ ID NO10

<211> length: 22

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 10

CysGluPheValValLysGluValAlaLysLeuIleAspAsnAsnArgThrGluGluGluIleLeu

<200> sequence signature:

<210>SEQ ID NO11

<211> length: 22

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 11

(B) position: 21...21 (D) other information: Xaa=I or L

CysXaaXaaXaaValXaaXaaXaaXaaXaaLeuIleXaaAsnAsnXaaThrGluXaaXaaXaaXaa

<200> sequence signature:

<210>SEQ ID NO12

<211> length: 22

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 12

CysLysAspValValThrAlaAlaGlyAspMetLeuLysAspAsnAlaThrGluGluGluIleLeu 20

<200> sequence signature:

<210>SEQ ID NO13

<211> length: 523

<212> type: PRT

<213> is biological: homo sapiens

<400> sequence: 13

MetTyrAlaLeuPheLeuLeuAlaSerLeuLeuGlyAlaAlaLeuAlaGlyProValLeuGlyLeuLysGluCysThr ArgGlySerAlaValTrpCysGlnAsnValLysThrAlaSerAspCysGlyAlaValLysHisCysLeuGlnThrValT rpAsnLysProThrValLysSerLeuProCysAspIleCysLysAspValValThrAlaAlaGlyAspMetLeuLysAs pAsnAlaThrGluGluGluIleLeuValTyrLeuGluLysThrCysAspTrpLeuProLysProAsnMetSerAlaSer CysLysGluIleValAspSerTyrLeuProValIleLeuAspIleIleLysGlyGluMetSerArgProGlyGluValCysS erAlaLeuAsnLeuCysGluSerLeuGlnLysHisLeuAlaGluLeuAsnHisGlnLysGlnLeuGluSerAsnLysIle ProGluLeuAspMetThrGluValValAlaProPheMetAlaAsnIleProLeuLeuLeuTyrProGlnAspGlyProA rgSerLysProGlnProLysAspGlyAspValCysGlnAspCysIleGlnMetValThrAspIleGlnThrAlaValArg ThrAsnSerThrPheValGlnAlaLeuValGluHisValLysGluGluCysAspArgLeuGlyProGlyMetAlaAspI leCysLysAsnTyrIleSerGlnTyrSerGluIleAlaIleGlnMetMetMetHisMetGlnProLysGluIleCysAlaLeu ValGlyPheCysAspGluValLysGluMetProMetGlnThrLeuValProAlaLysValAlaSerLysAsnValIlePr oAlaLeuAspLeuValAspProIleLysLysHisGluValProAlaLysSerAspValTyrCysGluValCysGluPheL euValLysGluValThrLysLeuIleAspAsnAsnLysThrGluLysGluIleLeuAspAlaPheAspLysMetCysSe rLysLeuProLysSerLeuSerGluGluCysGlnGluValValAspThrTyrGlySerSerIleLeuSerIleLeuLeuGlu GluValSerProGluLeuValCysSerMetLeuHisLeuCysSerGlyThrArgLeuProAlaLeuThrValHisValTh rGlnProLysAspGlyGlyPheCysGluValCysLysLysLeuValGlyThrLeuAspArgAsnLeuGluLysAsnSe rThrLysGlnGluIleLeuAlaAlaLeuGluLysGlyCysSerPheLeuProAspProTyrGlnLysGlnCysAspGlnP heValAlaGluTyrGluProValLeuIleGluIleLeuValGluValMetAspProSerPheValCysLeuLysIleGlyAl aCysProSerAlaHisLysProLeuLeuGlyThrGluLysCysIleTrpGlyProSerTyrTrpCysGlnAsnThrGluTh rAlaAlaGlnCysAsnAlaValGluHisCysLysArgHisValTrpAsn。

Claims (18)

1. for reagent being delivered to the nano-capsule bubble of biomembranous target biology film, described nano-capsule bubble comprises:

A. be selected from one or more phospholipid of long-chain phospholipid, short chain lipid and its mixture;

B. the developer of safe and effective amount or therapeutic agent;

C. be derived from the peptide of going back to the nest of Saposin C; With

D. the carrier of pharmaceutically accepting,

Wherein said nano-capsule bubble can with by the biomembrane of targeting, merged.

2. the compositions of claim 1, the wherein said peptide of going back to the nest has such sequence, and described sequence comprises approximately 9 to approximately 20 continuous amino acids that are selected from following formula:

EKEILDAFDKMCSKLPK

EKEILDAFDKMCSKLP

FDKMCSKLPK

FDKMCSKLP

EVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLP

KSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSG

SPELVCSMLHLCSG。

3. the compositions of claim 1, the wherein said peptide of going back to the nest has approximately 9 sequences to approximately 20 continuous amino acids of following formula:

AsnLysThrGluLysGluIleLeuAspAlaPheAspLysMetCysSerLysLeuProLys。

4. the compositions of claim 1, the wherein said peptide of going back to the nest has approximately 9 sequences to approximately 20 continuous amino acids of following formula:

XaaXaaXaaXaaXaaXaaXaaPheAspLysMetCysSerLysLeuProXaa

Wherein the Xaa on position 1 is Glu or does not exist; Wherein the Xaa on position 2 is Lys or does not exist; Wherein the Xaa on position 3 is Glu or does not exist; Wherein the Xaa on position 4 is Ile or does not exist; Wherein the Xaa on position 5 is Leu or does not exist; Wherein the Xaa on position 6 is Asp or does not exist; Wherein the Xaa on position 7 is Ala or does not exist; And wherein the Xaa on position 17 is Lys or does not exist.

5. the compositions of claim 1, the wherein said peptide of going back to the nest has the sequence that is selected from following formula:

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 17

phe-asp-lys-met-cys-ser-lys-leu-pro-lys 10

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 16

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 15

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 14

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 13

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 12

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro 11

ala-phe-asp-lys-met-cys-ser-lys-leu-pro 10

phe-asp-lys-met-cys-ser-lys-leu-pro 9

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 16

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 15

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 14

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 13

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 12

ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 11

phe-asp-lys-met-cys-ser-lys-leu-pro-lys 10

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 17

glu-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 17

lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 16

glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 15

ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 14

leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 13

asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 12

ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 11

phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 10

Xaa-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 17

Xaa-Xaa-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 16

Xaa-Xaa-Xaa-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 15

Xaa-Xaa-Xaa-Xaa-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 14

Xaa-Xaa-Xaa-Xaa-Xaa-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 13

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 12

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-phe-asp-lys-met-cys-ser-lys-leu-pro-lys 11

phe-asp-lys-met-cys-ser-lys-leu-pro-lys 10

Xaa-lys-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 17

Xaa-Xaa-glu-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 16

Xaa-Xaa-Xaa-ile-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 15

Xaa-Xaa-Xaa-Xaa-leu-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 14

Xaa-Xaa-Xaa-Xaa-Xaa-asp-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 13

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-ala-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 12

Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 11

phe-asp-lys-met-cys-ser-lys-leu-pro-Xaa 10

Wherein Xaa is selected from val, leu, ile, met, pro, the hydrophobic amino acid of phe and ala; Be selected from thr, ser, tyr, gly, uncharged polar amino acid of gln and ash; Or do not exist.

6. the compositions of claim 1, wherein said phospholipid is phosphatidylcholine.

7. the compositions of claim 1, wherein said phosphatidylcholine is selected from DOPC, dimyristoyl phosphatidyl choline, two pentadecanoyl phosphatidylcholine, DLPC, dipalmitoyl phosphatidyl choline and distearoyl phosphatidylcholine.

8. the compositions of claim 1, wherein said phospholipid is selected from DOPS, dipalmitoyl phosphatidyl choline and hexanoyl phosphatidylcholine.

9. the compositions of claim 1, wherein said reagent is therapeutic agent or developer.

10. the compositions of claim 1, wherein said reagent is developer.

The compositions of 11. claim 1, the nanometer that wherein said developer is selected from radionuclide, biotin, fluorogen, antibody, horseradish peroxidase, alkali phosphatase, nano-particle, quantum dot, detectable anticarcinogen is dripped, liposome medicament and cytokine.

The compositions of 12. claim 1, wherein said developer is conjugated to the peptide of going back to the nest.

13. for by the method for the film of reagent targeted cells or tissue, and wherein said method comprises the compositions of using claim 1 to described film, and wherein the concentration of peptide is to be enough to described reagent to send the amount by film.

The method of 14. claim 13, wherein said reagent is developer.

The method of 15. claim 13, wherein said developer is one or more reagent that are selected from magnetic resonance, fluorescence or CT/PET detectable label.

The method of 16. claim 13, wherein said developer has two or more imaging character.

The method of 17. claim 13, wherein said developer is the PTIR dyestuff that comprises fluorogen and Gd (III) part, it can detect by nuclear magnetic resonance (MRI) or confocal fluorescent microscopy.

The method of 18. claim 13, wherein described developer is sent to the film through blood brain barrier, the concentration of wherein said nano-capsule bubble is to be enough to send the pharmaceutical agent of safe and effective amount by the amount of described film, and wherein said phospholipid formation overall charge is negative liposome.

Images