CN105687165A - Tissue adhesive film loaded with protein or polypeptide drug and preparation method of tissue adhesive film - Google Patents
Tissue adhesive film loaded with protein or polypeptide drug and preparation method of tissue adhesive film Download PDFInfo
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7007—Drug-containing films, membranes or sheets
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Abstract
The invention relates to the field of biological medicine, in particular to a tissue adhesive film loaded with a protein or polypeptide drug and a preparation method of the tissue adhesive film. The tissue adhesive film comprises cationic layers and anionic layers, wherein the cationic layers and the anionic layers are stacked alternately, at least one of the cationic layers and the anionic layers is a drug layer, or at least one of the cationic layers and the anionic layers contains a charged drug which is the protein or polypeptide drug. The tissue adhesive film loaded with the protein or polypeptide drug has good tissue adhesion, good biocompatibility, degradable absorption and good stability, and physical and chemical properties of the tissue adhesive film can be adjusted by adjusting material composition.
Description
Technical field
The present invention relates to biomedicine field, particularly relating to a kind of albumen or polypeptide drugs medicine carrying tissue adhesive film and preparation method thereof, this medicine carrying tissue adhesive film can be implanted sufferer tissue site by surgical operation and carry out the local directly various disease of sustained-release and controlled release drug treatment。
Background technology
Along with developing rapidly of science and technology, the medicine of a large amount of New raxa is constantly found, but stops the biggest obstacle of a lot of novel drugs Clinical practice to be just a lack of effective medicine-feeding technology at present。In prior art, the existence of a lot of medicine-feeding technologies cannot really for focus, slow release is difficult to the problems such as control, these problems can cause that Formulations for systemic administration, dosage are big, side effect is big, targeting is poor, the drug dose of entrance cell interior is few, does not reach clinical therapeutic efficacy。So for most medicines, specifically such as albumen or polypeptide drugs, also all there is the various similar problem needing and improving in it, so for drug-supplying system, especially the research of carrier aspect increasingly comes into one's own in carrier。
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of albumen or polypeptide drugs medicine carrying tissue adhesive film and its production and use, this medicine carrying tissue adhesive film can pass through surgical operation and implant sufferer tissue site, carry out the local directly various disease of sustained-release and controlled release drug treatment, be used for solving the problems of the prior art。
For achieving the above object and other relevant purposes, first aspect present invention provides a kind of medicine carrying tissue adhesive film, more specifically albumen or polypeptide drugs medicine carrying tissue adhesive film, described medicine carrying tissue adhesive film includes cationic layer and the anion layer of alternately superposition, the at least one of which that at least one of which in described cationic layer and anion layer is in medicine layer or described cationic layer and anion layer contains charged medicine, and described medicine is albumen or polypeptide drugs。
Concrete, described cationic layer is shown generally as positive charge, and described anion layer is shown generally as negative charge。
When at least one of which in described cationic layer and anion layer is medicine layer, cationic layer and/or anion layer as medicine layer are charged medicine, cationic layer not as medicine layer can contain the carrier material with cation group, and the anion layer not as medicine layer can contain the carrier material with anionic group。
When at least one of which in described cationic layer and anion layer contains charged medicine, cationic layer can contain the carrier material with cation group, and anion layer can contain the carrier material with anionic group。
In medicine carrying tissue adhesive film provided by the present invention, the described carrier material with cation group is per se with positive charge or is being dissolved in after solvent ionizes the material with positive charge, an embodiment of the present invention can be dissolved in after water ionizes, with the material of positive charge。
The described carrier material with cation group preferably can the biocompatible materials of vivo degradation。
Concrete, the described carrier material with cation group is optional carries one or more the combination in the organic high molecular polymer of cation group, the polysaccharide with cation group, the polypeptide with cation group, albumen with cation group and cationic-liposome etc.。
Described organic high molecular polymer refers specifically to be repeated, by many identical construction units (a kind of construction unit or various structures unit), the high-molecular weight compounds that is formed by connecting by covalent bond, in an embodiment of the present invention, the concrete available organic high molecular polymer with cation group includes but not limited to: one or more the combination in polymine, tree-like daiamid macromolecule, cationic polyester (object lesson such as cation poly phosphate, poly-phosphoramide, polymethacrylates etc.), polyvinylpyridine etc.。
Described polysaccharide can generate multiple monosaccharide when referring specifically to a part hydrolysis, the more specifically sugar of more than ten monosaccharide, the described polysaccharide with cation group can be the polysaccharide with cation group own, it is also possible to be the polysaccharide with cation group by modified acquisition。Polysaccharide is modified so that it is the state of the art with the technology of cation group, can be specifically the method such as grafted amino group on side chain。In an embodiment of the present invention, the concrete available polysaccharide with cation group includes but not limited to: Chitosan-phospholipid complex (object lesson such as chitosan quaternary ammonium salt, low degree of substitution carboxymethyl chitosan, N-trimethyl chitosan TMC, containing imidazole radicals chitosan, Chitosan-Thiolated Polymers etc.), one or more combination in cationic starch and derivant (object lesson such as the cyclodextrin etc. with cation group) and other polysaccharides with cation group。
Described polypeptide with cation group or albumen can be the polypeptide per se with cation group or albumen, it is also possible to be the polypeptide with cation group by modified acquisition or albumen。In an embodiment of the present invention, concrete available polypeptide with cation group or albumen include but not limited to: one or more the combination in polylysine or poly arginine and their derivant (example of concrete derivant as: introduce PEG, galactose, lactose, folic acid, transferrins etc. on polypeptide), collagen, gelatin, serum albumin etc.。Owing to some albumen (such as collagen, gelatin, serum albumin etc.) be both possibly shown as positively charged at various ph values, it is likely to and is shown as electronegative (with positive charge under the pH value lower than isoelectric point, IP, with negative charge under the pH value higher than isoelectric point, IP), so this type of material can not only be used for cationic layer, it is possible to as anion layer。
Described cationic-liposome can be selected for the various cationic-liposome in this area, and concrete available example includes but not limited to: the cationic-liposome prepared with DOTMA analog, DOTAP, spennidine cholesterol。
In medicine carrying tissue adhesive film provided by the present invention, the described carrier material with anionic group is per se with negative charge or is being dissolved in after solvent ionizes the material with negative charge, an embodiment of the present invention can be dissolved in after water ionizes, with the material of negative charge。
The described carrier material with anionic group preferably can the biocompatible materials of vivo degradation。
Concrete, the described carrier material with anionic group is optional carries one or more the combination in the organic high molecular polymer of anionic group, the polysaccharide with anionic group, the polypeptide with anionic group, albumen with anionic group and anionic liposome etc.。
In an embodiment of the present invention, the concrete available organic high molecular polymer with anionic group includes but not limited to: the anion being made up of dicarboxylic acids provided in CN1524184A。
The described polysaccharide with anionic group can be the polysaccharide with anionic group own, it is also possible to be the polysaccharide with anionic group by modified acquisition。It is modified polysaccharide, so that it is the state of the art with the technology of anionic group, being specially such as (more specifically example such as carboxymethyl starches etc.) such as anionic starch。In an embodiment of the present invention, the concrete available polysaccharide with anionic group includes but not limited to: carboxymethyl cellulose, carboxymethyl chitosan, hyaluronic acid, alginic acid, carboxymethyl starch, chondroitin sulfate, heparin and their derivant and other are with one or more combination in the polysaccharide etc. of anionic group。
The described polypeptide with anionic group can be the polypeptide per se with anionic group, it is also possible to be the polypeptide with anionic group by modified acquisition。In an embodiment of the present invention, the concrete available polypeptide with anionic group includes but not limited to: polyglutamic acid or one or more the combination in poly-aspartate and derivant, collagen, gelatin etc.。
Described anionic liposome can be selected for the various anionic liposome in this area, and concrete available example includes but not limited to: AS-ODNs anionic liposome, DOPG/DOPE anionic liposome etc.。
In medicine carrying tissue adhesive film provided by the present invention, described charged medicine can be specifically positively charged medicine and/or electronegative medicine, as long as it is with electric charge, can be used to as medicine layer or be contained in described medicine carrying tissue adhesive film, its amount comprised is generally effective therapeutic dose。Therapeutically effective amount, corresponding to the purpose for the treatment of indication, refers specifically to a consumption after during suitable administration, it is possible to reach the effect for the treatment of indication。Treatment specifically includes preventative, healing property or the retentivity disposal of pharmacy and/or physiologic effect。This effect is preferably meant that one or more symptoms that medically can reduce indication or indication is completely eliminated, or the risk of retardance, the generation postponing indication and/or reduction indication development or deterioration。
Concrete, when at least one of which in described cationic layer and anion layer is medicine layer, described medicine layer specifically can be selected for charged medicine, specifically, when cationic layer is medicine layer, it can include positively charged medicine, and when anion layer is medicine layer, it can include electronegative medicine。
More specifically, for positively charged medicine, when at least one of which of cationic layer is medicine layer, positively charged medicine as medicine layer, can form electrostatic interaction with the anion layer closed on；When it is contained in cationic layer and/or anion layer as pharmaceutical pack, it can be located in cationic layer and forms electrostatic interaction with the anion layer closed on, thus being stably contained in described medicine carrying tissue adhesive film, or can be located in anion layer, positively charged medicine with after the carrier material effect of anionic group, this layer still appears as negative charge on the whole, forms electrostatic interaction with the carrier material with cation group, thus being stably contained in described medicine carrying tissue adhesive film。And for electronegative medicine, when at least one of which of anion layer is medicine layer, electronegative medicine as medicine layer, can form electrostatic interaction with the cationic layer closed on；When it is contained in cationic layer and/or anion layer as pharmaceutical pack, it can be located in anion layer and forms electrostatic interaction with the cationic layer closed on, thus being stably contained in described medicine carrying tissue adhesive film, or can be located at band cation group material and rush in layer, electronegative medicine with after the carrier material effect of cation group, this layer still appears as positive charge on the whole, electrostatic interaction is formed, thus being stably contained in described medicine carrying tissue adhesive film with the carrier material with anionic group。
Described charged medicine, it is specially charged albumen or polypeptide drugs, can be some itself namely with the albumen of electric charge or polypeptide drugs, it is also possible to be some albumen or the polypeptide drugs drug composite with electric charge through being combined with electrically charged (positive charge or negative charge) material or material or be wrapped to form。The method of the drug composite with electric charge that medicine is combined with electrically charged (positive charge or negative charge) material or material or is wrapped to form is the state of the art, and concrete drug composite includes but not limited to the polymer etc. that polymer, medicine and cation group material that medicinal liposome, medicine phospholipid, medicine and anionic group material are formed are formed。The form of drug composite can be microsphere, microcapsule, microgranule, micelle, micelle etc.。
Concrete, described medicine is albumen or polypeptide drugs, described polypeptide drugs include polypeptide or derivatives thereof, be specifically including but not limited to cell-targeting polypeptide (concrete as containing-arginine-glycine-aspartic acid, valine-glycine-val-ala-proline-glycine, Isoleucine-lysine-valine-alanine-valine fragment polypeptide etc.), antibacterial peptide etc.；Described protein drug includes albumen or derivatives thereof, is specifically including but not limited to cytokine, antibody, part, glycoprotein, thrombin, interferon etc.。
In medicine carrying tissue adhesive film provided by the present invention, cationic layer and anion layer replace superposition, and can be combined by electrostatic attraction, form the tissue adhesive film for medicine carrying, described cationic layer is shown generally as positive charge, and described anion layer is shown generally as negative charge。Can be specifically cationic layer material and the method carrying out alternating deposit by electrostatic attraction with anion layer material, such as polyelectrolyte self-assembly method layer by layer etc.。Its number of times replacing superposition for cationic layer and anion layer is not particularly limited, those skilled in the art can according to actual needs (as, drug loading, degradation time etc.) adjust tissue adhesive film gross thickness, the gross thickness of described tissue adhesive film can≤1000 μm, more specifically can≤600 μm, more specifically can≤400 μm, more specifically can≤200 μm, be specifically as follows 0.05 μm-1000 μm further。For each layer of cationic layer and/or anion layer, those skilled in the art can according to the kind of material and be actually needed (as, drug loading, degradation time etc.) adjust the thickness of each cationic layer and/or anion layer, and may further determine that medicine, carrier material with cation group and the carrier material with anionic group make consumption, its thickness can be 0.0005 μm--100 μm, more specifically can 0.001 μm--50 μm, it can be more specifically Nano grade, and cationic layer and anion layer when preparing the consumption that makes of material be 1:0.005--200。
The preparation method that second aspect present invention provides described medicine carrying tissue adhesive film, comprises the steps: alternating deposit cationic layer and anion layer on substrate, prepares tissue adhesive film。
Medicine carrying tissue adhesive film provided by the present invention can intactly be taken off from substrate after preparation completes, and stably can exist independent of other attachment material (such as substrate, backing etc.)。
Concrete, when at least one of which in described cationic layer and anion layer is medicine layer, described preparation method can be polyelectrolyte self-assembly method layer by layer, specifically may include steps of:
Alternating deposit cationic layer and anion layer on substrate, prepare tissue adhesive film, when deposition is as the material layer of medicine layer, uses charged medicine to be deposited。
Concrete, on substrate, alternating deposit cationic layer and anion layer method specifically include following steps: when depositing non-drug layer, the solution of the solution of substrate alternate immersion or the spraying carrier material with cation group and the carrier material with anionic group, when deposition of medicament layer, then soak or spray the solution of charged medicine, wash (be specifically as follows and wash with water) every time after deposition, dry, take off film after completing alternating deposit, obtain described tissue adhesive film。
More specifically, the described solution with cation group material, the solution with anionic group material, charged medicine solution can be all aqueous solution, also can add suitable salt ion in aqueous and stretch situation with the space changing molecule, the kind being particularly used in the salt ion that situation is stretched in the space changing molecule is well known in the prior art, as added NaCl, KCl etc. in the solution。
The concentration of solution of used material, pH value, salt ionic concentration etc. when those skilled in the art can deposit according to the kind adjustment of material, in an embodiment of the present invention, in the solution used during deposition, salt ionic concentration is specifically as follows≤10mol/L, can being more specifically 0.05-10mol/L, pH value can be 3-11。
When at least one of which in described cationic layer and anion layer contains charged medicine, described preparation method can be polyelectrolyte self-assembly method layer by layer, specifically may include steps of:
Alternating deposit cationic layer and anion layer on substrate, prepare tissue adhesive film；And in the process preparing tissue adhesive film, medicine is implanted in described tissue adhesive film。
Concrete, on substrate, alternating deposit cationic layer and anion layer method specifically include following steps: the solution of the solution of substrate alternate immersion or the spraying carrier material with cation group and the carrier material with anionic group is (when deposition includes the material layer of medicine, medicine is then may further include) for depositing in the solution of this material layer, wash (be specifically as follows and wash with water) every time after deposition, dry, take off film after completing alternating deposit, obtain described tissue adhesive film。
Those skilled in the art can be selected for suitable method, medicine is implanted in described tissue adhesive film, specifically may is that when deposition contains the cationic layer of charged medicine, medicine is mixed with the carrier material with cation group and is deposited (can be specifically the mixed solution soaking or spraying the carrier material with cation group and medicine)；When deposition contains the anion layer of charged medicine, medicine is mixed with the carrier material with anionic group and is deposited (can be specifically the mixed solution soaking or spraying the carrier material with anionic group and medicine)；In addition it is also possible to after preparing tissue adhesive film, re-use the solution that the instantaneous spraying of high pressure contains medicine, and carry out further washing, drying。
Third aspect present invention provides a kind of tissue adhesive film purposes in preparing drug carrier material。
Described medicine is specially charged medicine, charged albumen more specifically as above or polypeptide drugs。
Concrete, described tissue adhesive film includes cationic layer and the anion layer of alternately superposition。
More specifically, at least one of which in described cationic layer and anion layer is that at least one of which in medicine layer or described cationic layer and anion layer is for comprising charged medicine。
Medicine carrying tissue adhesive film provided by the present invention has good tissue adhesion, good biocompatibility and degradable absorbability, good stability, and its physics, chemical property can regulate by regulating the composition of material。In addition, described medicine carrying tissue adhesive membrane preparation method is simple, drug loading can be measured as required and be adjusted, carrier material can protect the medicine (as making RNA avoid being degraded) being carried, and the medicine being carried can be transported in target cell expeditiously, specifically such as: medicine carrying tissue adhesive film directly can be pasted onto lesions position (namely treating various diseases by surgical operation implanted sustained-release administration) by described medicine carrying tissue adhesive film by surgical operation, administration is directly, targeting is good, medicine local concentration height therapeutic effect is good, side effect is little, the genotoxic potential of organism is low, there is significantly high biological safety。
Accompanying drawing explanation
Fig. 1 is shown as the gel electrophoresis experiment schematic diagram of embodiment 12siRNA。
Fig. 2 is shown as embodiment 13 cell transfection assay schematic diagram。
Fig. 3 is shown as embodiment 13 relative intensity of fluorescence schematic diagram。
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art the content disclosed by this specification can understand other advantages and effect of the present invention easily。The present invention can also be carried out by additionally different detailed description of the invention or apply, and the every details in this specification based on different viewpoints and application, can also carry out various modification or change under the spirit without departing from the present invention。
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, rather than in order to limit the scope of the invention；In specification and claims of the present invention, unless additionally explicitly pointed out in literary composition, singulative " ", " one " and " this " include plural form。
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, between two end points and two end points of each numerical range, any one numerical value all can be selected for。Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique are generally understood that。Except the concrete grammar used in embodiment, equipment, material, record according to those skilled in the art's grasp to prior art and the present invention, it is also possible to use similar with the method described in the embodiment of the present invention, equipment, material or that be equal to any method of prior art, equipment and material to realize the present invention。
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all adopt the conventional molecular biology of the art, biochemistry, chromatin Structure and analysis, analytical chemistry, cell are cultivated, the routine techniques of recombinant DNA technology and association area。These technology existing improving in existing document illustrates, specifically can referring to the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001；Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, john wiley & sons, NewYork, 1987andperiodicupdates；TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego；Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998；METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999；And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc.。
Embodiment 1
In aseptic working platform, prepare one 12 cm diameter culture dishs, a built-in same diameter silicon chip film (scrubbed, sterilizing and depyrogenation process), dry；The preparation solution A (1mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) with cation group material and the B liquid (1mg/ml carboxymethyl chitosan, 0.15mol/LNaCl, pH=6) with anionic group material respectively；Take part B liquid add small RNA medicine (eGFP-siRNA (and sense:5 '-GGCACAAGCUGGAGUACAAUU-3 '；Antisense:5 '-UUGUACUCCAGCUUGUGCCUU-3 ', 20ug/mL) and the cell-targeting factor (hyaluronic acid, molecular weight is less than 20,000 dalton, 10ug/mL) and target polypeptide (1 × 10-4Mg/ml) being configured to C liquid, target polypeptide amino acid sequence is: valine-glycine-val-ala-proline-glycine；Above-mentioned solution is filled in the instantaneous spraying equipment of high pressure respectively, first toward the instantaneous spraying C liquid of culture dish inner high voltage, drying and forming-film；Then the instantaneous spraying A liquid of high pressure, rinses with water for injection；Then the instantaneous spraying B liquid of high pressure, rinses with water for injection, such alternating spray A liquid, B liquid, flushing, repetitive operation；Then the instantaneous spraying A liquid of high pressure again, rinses with water for injection；The instantaneous spraying C liquid of last high pressure, rinses with water for injection, dry, takes off film, and veneer water for injection rinses, and dried must carry small RNA medicine implanted tissue adhesive film。
Embodiment 2
In aseptic working platform, prepare a macromolecular material flat board (scrubbed, sterilizing and depyrogenation process), dry；Preparation solution A (2mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/LNaCl with cation group material respectively, pH=4) the B liquid (alginic acid of 2mg/ml with anionic group material, 6 to 8 ten thousand molecular weight, 0.15mol/LNaCl, pH=6) it is called for short B liquid；B liquid adds small RNA medicine (with embodiment 1,20ug/mL) and the cell-targeting factor (hyaluronic acid, molecular weight is less than 20,000 dalton, 10ug/mL), A liquid adds target polypeptide (1 × 10-4Mg/ml), target polypeptide amino acid sequence is: Isoleucine-lysine-valine-alanine-valine；Flat board is submerged initially in solution A 20 minutes, takes out into cleanout fluid washing dry；Again flat board is immersed in B solution 30 minutes, take out into cleanout fluid washing dry, so hocket 160 times, finally rinse with water for injection, dry, take off film, small RNA medicine implanted tissue adhesive film must be carried。
Embodiment 3
In aseptic working platform, prepare a macromolecular material flat board (scrubbed, sterilizing and depyrogenation process), dry；Preparation solution A (the 2mg/ml chitosan with cation group material respectively, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) and the B liquid (hyaluronic acid of 2mg/ml with anionic group material, 0.15mol/LNaCl, pH=6) it is called for short B liquid；(cation lipid preparation method is with reference to " gene transfection of anionic liposome-cationic liposome complex mediation " to add small RNA cationic-liposome in A liquid, pharmacy practice magazine, 2011 (29): 4, small RNA is with embodiment 1, small RNA cationic-liposome addition is 0.5mg/ml, small nucleic acids drug loading 21.7%) stir and target polypeptide (1 × 10-4Mg/ml), target polypeptide amino acid sequence is: arginine-glycine-aspartic acid；Flat board is submerged initially in solution A 20 minutes, takes out into cleanout fluid washing dry；Again flat board is immersed in B solution 30 minutes, take out into cleanout fluid washing dry, so hocket 180 times, finally rinse with water for injection, dry, take off film, small RNA medicine implanted tissue adhesive film must be carried。
Embodiment 4
In aseptic working platform, prepare a macromolecular material flat board (scrubbed, sterilizing and depyrogenation process), dry；Preparation solution A (the 2mg/ml chitosan with cation group material respectively, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) and the B liquid (hyaluronic acid of 2mg/ml with anionic group material, 0.15mol/LNaCl, pH=6) it is called for short B liquid；(preparation method is with reference to " gene transfection of anionic liposome-cationic liposome complex mediation " to add small RNA anionic liposome in B liquid, pharmacy practice magazine, 2011 (29): 4, small RNA is with embodiment 1, small RNA anionic liposome addition is 0.5mg/ml, small nucleic acids drug loading 12.4%) stir and target polypeptide (1 × 10-4Mg/ml), target polypeptide amino acid sequence is: valine-glycine-val-ala-proline-glycine, is submerged initially in solution A by flat board 20 minutes, takes out into cleanout fluid washing dry；Again flat board is immersed in B solution 30 minutes, take out into cleanout fluid washing dry, so hocket 180 times, finally rinse with water for injection, dry, take off film, small RNA medicine implanted tissue adhesive film must be carried。
Embodiment 5
In aseptic working platform, preparing one 12 cm diameter culture dishs, a built-in same diameter polymer material film (scrubbed and sterilizing depyrogenation processes), water for injection rinses dry；The preparation A liquid (1mg/ml collagen, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=3.5) with cation group material and the B liquid (1mg/ml hyaluronic acid, 0.15mol/LNaCl, pH=6) with anionic group material respectively；A liquid adds small RNA cationic-liposome (preparation method is embodiment 3 such as, and concentration is 0.5mg/ml) and target polypeptide (1 × 10-4Mg/ml) stirring, target polypeptide amino acid sequence is: arginine-glycine-aspartic acid；Above-mentioned solution is filled in the instantaneous spraying equipment of high pressure respectively, first toward the instantaneous spraying A liquid of culture dish inner high voltage, dry, the then instantaneous spraying B liquid of high pressure, rinses with water for injection, such alternating spray A liquid, B liquid, flushing, repetitive operation 200 times；The last instantaneous spraying A liquid of high pressure again, rinses with water for injection, dry, takes off film, and veneer water for injection rinses, and dried must carry small RNA medicine implanted tissue adhesive film。
Embodiment 6
In aseptic working platform, prepare a macromolecular material flat board (scrubbed, sterilizing and depyrogenation process), dry；Preparation solution A (2mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/LNaCl with cation group material respectively, pH=4) the B liquid (alginic acid of 2mg/ml with anionic group material, 6 to 8 ten thousand molecular weight, 0.15mol/LNaCl, pH=6) it is called for short B liquid；B liquid adds Sorafenib hyaluronate microspheres (concentration 1.5mg/ml, microsphere diameter 10 μm to 16 μm；Drug loading 7.58%), A liquid adds target polypeptide (1 × 10-4Mg/ml), target polypeptide amino acid sequence is: Isoleucine-lysine-valine-alanine-valine；Flat board is submerged initially in solution A 20 minutes, takes out into cleanout fluid washing dry；Again flat board is immersed in B solution 30 minutes, take out into cleanout fluid washing dry, so hocket 30 times, finally rinse with water for injection, dry, take off film, Sorafenib medicine implanted tissue adhesive film must be carried。
Embodiment 7
In aseptic working platform, preparing one 12 cm diameter culture dishs, a built-in same diameter polymer material film (scrubbed and sterilizing depyrogenation processes), water for injection rinses dry；The preparation A liquid (1mg/ml collagen, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=3.5) with cation group material and the B liquid (1mg/ml hyaluronic acid, 0.15mol/LNaCl, pH=6) with anionic group material respectively；B liquid adds Sutent sodium alginate micro ball (concentration 1.0mg/ml, microsphere diameter 10 μm to 15 μm；Drug loading 6.28%), A liquid adds target polypeptide (1 × 10-4Mg/ml) stirring, target polypeptide amino acid sequence is: arginine-glycine-aspartic acid；Above-mentioned solution is filled in the instantaneous spraying equipment of high pressure respectively, first toward the instantaneous spraying A liquid of culture dish inner high voltage, dry, the then instantaneous spraying B liquid of high pressure, rinses with water for injection, such alternating spray A liquid, B liquid, flushing, repetitive operation 50 times；The last instantaneous spraying A liquid of high pressure again, rinses with water for injection, dry, takes off film, and veneer water for injection rinses, and dried must carry Sutent medicine implanted tissue adhesive film。
Embodiment 8
In aseptic working platform, prepare a macromolecular material flat board (scrubbed, sterilizing and depyrogenation process), dry；Preparation solution A (the 2mg/ml chitosan with cation group material respectively, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) and the B liquid (hyaluronic acid of 2mg/ml with anionic group material, 0.15mol/LNaCl, pH=6) it is called for short B liquid；B liquid adds ganglioside sodium alginate micro ball (concentration 1.0mg/ml, microsphere diameter 0.15 μm to 0.26 μm；Drug loading 7.3%) stir and target polypeptide (1 × 10-4Mg/ml), target polypeptide amino acid sequence is: valine-glycine-val-ala-proline-glycine, is submerged initially in solution A by flat board 20 minutes, takes out into cleanout fluid washing dry；Again flat board is immersed in B solution 30 minutes, take out into cleanout fluid washing dry, so hocket 30 times, finally rinse with water for injection, dry, take off film, ganglioside medicine implanted tissue adhesive film must be carried。
Embodiment 9
In aseptic working platform, preparing one 12 cm diameter culture dishs, a built-in same diameter polymer material film (scrubbed and sterilizing depyrogenation processes), water for injection rinses dry；The preparation A liquid (1mg/ml collagen, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=3.5) with cation group material and the B liquid (1mg/ml hyaluronic acid, 0.15mol/LNaCl, pH=6) with anionic group material respectively；B liquid adds antibacterial peptide (1.5mg/ml)；Above-mentioned solution is filled in the instantaneous spraying equipment of high pressure respectively, first toward the instantaneous spraying A liquid of culture dish inner high voltage, dry, the then instantaneous spraying B liquid of high pressure, rinses with water for injection, such alternating spray A liquid, B liquid, flushing, repetitive operation 80 times；The last instantaneous spraying A liquid of high pressure again, rinses with water for injection, dry, takes off film, and veneer water for injection rinses, and dried obtains antimicrobial peptide medicine implanted tissue adhesive film。
Embodiment 10
In aseptic working platform, preparing one 12 cm diameter culture dishs, a built-in same diameter polymer material film (scrubbed and sterilizing depyrogenation processes), water for injection rinses dry；The preparation A liquid (1mg/ml collagen, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=3.5) with cation group material and the B liquid (1mg/ml hyaluronic acid, 0.15mol/LNaCl, pH=6) with anionic group material respectively；B liquid adds interleukin-2 (0.5mg/ml)；Above-mentioned solution is filled in the instantaneous spraying equipment of high pressure respectively, first toward the instantaneous spraying A liquid of culture dish inner high voltage, dry, the then instantaneous spraying B liquid of high pressure, rinses with water for injection, such alternating spray A liquid, B liquid, flushing, repetitive operation 50 times；The last instantaneous spraying A liquid of high pressure again, rinses with water for injection, dry, takes off film, and veneer water for injection rinses, and dried must carry interleukin-2 medicine implanted tissue adhesive film。
Embodiment 11
The medicine carrying tissue adhesive membrane drug delivery system prepared by embodiment 1-10 carries out evaluation of its biocompatibility detection, possesses excellent biocompatibility, specific as follows:
1. cell toxicity test:
Reference/technical standard: GB/T16886.5-2003
Cell strain: L-929 cell (l cell)
Culture fluid: containing the DMEM of 10% (V/V) calf serum
Blank: with batch cell culture fluid
Negative control: high density polyethylene (HDPE) (see GB/T16886 cell toxicity test standard)
Positive control: 5g/L phenol solution
Lixiviate medium: with the DMEM criticized without calf serum
Extraction time: 24 hours
Test sample lixiviate ratio: 1g/5ml
Test method: lixiviating solution test (mtt assay)
At 27 DEG C, 5%CO2Blank, negative control, positive control contact the cell of adherent growth with test sample lixiviating solution, after cultivating 72h, add MTT liquid, hatch 4h。After absorption, add DMSO, by spectrophotometer, each group is carried out absorbance measurement under wavelength 630nm, and calculate the relative rate of increase of cell。
Result: the cytotoxicity of test sample: 0 grade
Conclusion: with reference to GB/T16886.5-2003, this test sample no cytotoxicity。
2. picosecond laser pulse
Reference/technical standard: GB/T16886.10-2005
Experimental animal: healthy new zealand rabbit
Lixiviate medium: 0.9% sodium chloride injection
Test sample: material lixiviating solution
Negative control: with batch lixiviate medium
Route of exposure: intradermal injection
Judging quota: observe 24h, 48h, 72h erythema, the edema extent of reaction
Result: after injection, 24h, 48h, 72h injection site and surrounding skin tissue are all without erythema, edema reaction, and test side dermoreaction is not more than control sides dermoreaction。
Conclusion: with reference to GB/T16886.10-2005, this test sample is without Intradermal irritant reaction。
3. acute systemic toxicity
Reference/technical standard: GB/T16886.11-1997/ASTMF750
Experimental animal: healthy mice
Lixiviate medium: 0.9% sodium chloride injection
Test sample: material lixiviating solution
Negative control: with batch lixiviate medium
Route of exposure: tail vein injection
Judging quota: observe 4h, 24h, 48h, 72h animal general state, toxicity performance and dead animal number。
Result: in the 72h observation period, the reaction of test sample treated animal is not more than negative control treated animal。
Conclusion: with reference to GB/T16886.11-1997/ASTMF750, this test sample acute systemic toxicity result meets the requirements。
4. hemolytic test
Reference/technical standard: GB/T16886.4-2003/GB/T16175-1996
Experimental animal: healthy new zealand rabbit
Prepared by dilution anticoagulant Sanguis Leporis seu oryctolagi: fresh anticoagulant Sanguis Leporis seu oryctolagi+0.9% sodium chloride injection
Negative control: 0.9% sodium chloride injection
Positive control: distilled water
Route of exposure: tail vein injection
Test method: immersed by a certain percentage by test sample in 0.9% sodium chloride injection, after 37 DEG C of water bath heat preservation 30min, adds 0.2ml and dilutes fresh anticoagulant Sanguis Leporis seu oryctolagi, 37 DEG C of water bath heat preservation 60min。Take supernatant after the centrifugal 5min of 2500rpm, measure its absorbance with ultraviolet spectrophotometer at 545nm place, calculate hemolysis rate。
Result: the hemolysis rate of this test sample is 0.2%。
Conclusion: with reference to GB/T16886.4-2003, the hemolytic test result of this test sample meets the requirement of medical material。
Embodiment 12
Stability and integrity test:
In aseptic working platform, prepare a macromolecular material flat board (scrubbed, sterilizing and depyrogenation process), dry；Preparation solution A (the 2mg/ml chitosan with cation group material respectively, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) and the B liquid (hyaluronic acid of 2mg/ml with anionic group material, 0.15mol/LNaCl, pH=6) it is called for short B liquid；Take part B liquid addition small RNA medicine (such as embodiment 1,10ug/mL) and be configured to C liquid, above-mentioned solution is filled in the instantaneous spraying equipment of high pressure respectively, first toward the instantaneous spraying A liquid of culture dish inner high voltage, drying and forming-film；Then the instantaneous spray coating liquor B of high pressure, rinses with water for injection；Then the instantaneous spray coating liquor A of high pressure, rinses with water for injection, such alternating spray A liquid, B liquid, flushing, repetitive operation 4 times altogether；Then the instantaneous spraying A liquid of high pressure again, rinses with water for injection；The instantaneous spraying C liquid of high pressure again, rinses with water for injection, dry, such alternating spray A liquid, C liquid, flushing, and film is taken off in repetitive operation 7 times altogether, and veneer water for injection rinses, and dried must carry small RNA medicine implanted tissue adhesive film。
The above-mentioned plural layers prepared are placed in 1MNaCl solution, hatch certain time, the sustained-release liquid of gained is first passed through the ultra-filtration centrifuge tube filtration treatment that molecular cut off is 30KDa, remove macromolecular substances。It is then passed through the ultra-filtration centrifuge tube that molecular cut off is 3KDa and refilters process, remove the salt ion in sustained-release liquid, i.e. desalting processing。Finally the sustained-release liquid processed, run glue as sample liquid at electrophoresis tank and process。Adopt polyacrylamide gel electrophoresis method (PAGE) herein, verify genomic integrity and the Dependent Stability experiment of plural layers release siRNA。
The stability of the gel electrophoresis experimental verification slow release siRNA sequence of Fig. 1 and integrity。The tissue adhesive film of load siRNA is hatched in 1MNaCl solution, and film breaks dissolving gradually, after 6 days, has been difficult to observe by article shaped。Testing from Fig. 1 gel electrophoresis, it can be seen that, not it is observed that the siRNA dissociated after lysate desalination in 6 days, and the siRNA dissociated can be it can be clearly seen that after lysate desalination in 10 days, thus it is presumed that, film is breaking in course of dissolution gradually, leachable is siRNA and anions and canons group coalition, along with the degraded of anions and canons group, siRNA dissociates out gradually, and maintains stability and the integrity of siRNA sequence。
Embodiment 13
Cell transfection assays:
In aseptic working platform, prepare a macromolecular material flat board (scrubbed, sterilizing and depyrogenation process), dry；Preparation solution A (the 2mg/ml chitosan with cation group material respectively, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) and the B liquid (hyaluronic acid of 2mg/ml with anionic group material, 0.15mol/LNaCl, pH=6) it is called for short B liquid；Take part B liquid addition small RNA medicine (such as embodiment 1,10ug/mL) and be configured to C liquid, above-mentioned solution is filled in the instantaneous spraying equipment of high pressure respectively, first toward the instantaneous spraying A liquid of culture dish inner high voltage, drying and forming-film；Then the instantaneous spray coating liquor B of high pressure, rinses with water for injection；Then the instantaneous spray coating liquor A of high pressure, rinses with water for injection, such alternating spray A liquid, B liquid, flushing, repetitive operation 4 times altogether；Then the instantaneous spraying A liquid of high pressure again, rinses with water for injection；The instantaneous spraying C liquid of high pressure again, rinses with water for injection, dry, such alternating spray A liquid, C liquid, flushing, and film is taken off in repetitive operation 2 times altogether, and veneer water for injection rinses, and dried must carry small RNA medicine implanted tissue adhesive film。
Using the above-mentioned tissue adhesive film prepared as experimental group, namely experimental group is be loaded with eGFP-siRNA to replace polyelectrolyte and react the film of 2 times, negative control group is be loaded with common siRNA to replace polyelectrolyte and react the film (the same experimental group of preparation method, only eGFP-siRNA replaces with common siRNA) of 2 times。Experimental group and negative control group are placed in 6 orifice plates respectively, respectively eGFP-HEK293T cell is inoculated in 6 orifice plates thereon (additionally using the hole direct inoculation cell of not placing adhesive film as blank group), observing its fluorescence intensity change situation respectively at 1 day, 2 days and 3 days, result shows Fig. 2。In Fig. 3, list the relative intensity of fluorescence of experimental group, negative control group and blank group, it can be seen that blank group and negative control group fluorescence intensity in 3 days have almost no change, and experimental group increased fluorescence intensity in 3 days over time and significantly reduces。The tissue adhesive film being loaded with eGFPsiRNA by secondary checking can make the fluorescence intensity of eGFP-HEK293T cell weaken, and illustrates that eGFPsiRNA has transfected into cell induction target gene reticent。
In sum, present invention effect overcomes various shortcoming of the prior art and has high industrial utilization。
Above-described embodiment is illustrative principles of the invention and effect thereof only, not for the restriction present invention。Above-described embodiment all under the spirit and category of the present invention, can be modified or change by any those skilled in the art。Therefore, art has usually intellectual such as modifying without departing from all equivalences completed under disclosed spirit and technological thought or change, must be contained by the claim of the present invention。
Claims (10)
1. a medicine carrying tissue adhesive film, described medicine carrying tissue adhesive film includes cationic layer and the anion layer of alternately superposition, the at least one of which that at least one of which in described cationic layer and anion layer is in medicine layer or described cationic layer and anion layer contains charged medicine, and described medicine is albumen or polypeptide drugs。
2. a kind of medicine carrying tissue adhesive film as claimed in claim 1, it is characterized in that, when at least one of which in described cationic layer and anion layer is medicine layer, cationic layer and/or anion layer as medicine layer are charged medicine, cationic layer not as medicine layer contains the carrier material with cation group, and the anion layer not as medicine layer contains the carrier material with anionic group；When at least one of which in described cationic layer and anion layer contains charged medicine, cationic layer contains the carrier material with cation group, and anion layer contains the carrier material with anionic group。
3. a kind of medicine carrying tissue adhesive film as claimed in claim 2, it is characterized in that, one or more the combination in the organic high molecular polymer with cation group, the polysaccharide with cation group, the polypeptide with cation group, the albumen with cation group and cationic-liposome of the described carrier material with cation group。
4. a kind of medicine carrying tissue adhesive film as claimed in claim 2, it is characterized in that, one or more the combination in the organic high molecular polymer with anionic group, the polysaccharide with anionic group, the polypeptide with anionic group, the albumen with anionic group and anionic liposome of the described carrier material with anionic group。
5. a kind of medicine carrying tissue adhesive film as claimed in claim 2, it is characterised in that the described carrier material with cation group and the carrier material with anionic group select biocompatible materials。
6. a kind of medicine carrying tissue adhesive film as claimed in claim 1, it is characterised in that described cationic layer is shown generally as positive charge, and described anion layer is shown generally as negative charge。
7. a kind of medicine carrying tissue adhesive film as claimed in claim 1, it is characterised in that described charged medicine is drug composite。
8. the preparation method of the medicine carrying tissue adhesive film as described in claim 1-7 any claim, comprises the steps: alternating deposit cationic layer and anion layer on substrate, prepares tissue adhesive film。
9. tissue adhesive film purposes in preparing drug carrier material, described tissue adhesive film includes cationic layer and the anion layer of alternately superposition。
10. purposes as claimed in claim 9, it is characterised in that at least one of which in described cationic layer and anion layer is that at least one of which in medicine layer or described cationic layer and anion layer is for comprising charged medicine。
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