CN103031337A - Small nucleic acid molecule delivery technology - Google Patents
Small nucleic acid molecule delivery technology Download PDFInfo
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Abstract
The invention provides a small nucleic acid molecule delivery technology. A single-chain or double-chain small molecule nucleic acid or deoxyribonucleic acid with the base number of 10-100 enters the cells through transfection by utilizing a polypeptide transfect agent, so that the small molecule nucleic acid or small molecule nucleic acid subjected to chemical modification can effectively enter the suspended or adhered cells through transfection, and the problem of the technical bottleneck of importing the gene therapy medicine in the prior art is solved.
Description
Technical field
The present invention relates to biological technical field, refer to especially a kind of small nucleic acids molecule express delivery technology.
Background technology
Gene therapy is the novel therapeutic technology that a kind of development in recent years is got up.Because the target spot of gene therapy is clear and definite, this technology is being treated major disease (such as malignant tumour, diabetes, viral hepatitis etc.) aspect demonstrates single-minded, efficient advantage, the little RNA perturbation technique that development in recent years is got up provides again a kind of new technological method for gene therapy, one of technical bottleneck that gene therapy technology is moved towards clinical application from laboratory study is the leading-in technique of nucleic acid, the transfer efficiency of nucleic acid for the shortage of correcting the expression of nucleic acid in numerous disease, relate to and excessive be necessary, nucleic acid shifts expression regulation and functional assessment and the evaluation that can also be used for the research gene, and the parsing of the molecular basis of the self of stem cell and directed differentiation and application, nowadays, people have proposed many methods and have carried out discharging in the cell of this type of genetic information, can be divided into substantially virus vector mediation and this two large class of nucleic acid leading-in technique non-viral mediation, although virus vector is having significant effect aspect the importing efficient of exogenous nucleic acid, but because they usually have immunogenicity, cytotoxicity and bio-hazard, especially the cancer that the adenovirus carrier that occurs in the clinical study causes, this long term toxicity of virus vector has produced sizable negative impact to the clinical treatment by virus vector transduction nucleic acid drug.The nucleic acid transfection technology of an other class is to use chemistry or biochemical carrier.These synthetic carriers have two major functions: compound and promote its stability increase and promote it to pass plasmalemma with the nucleic acid of wanting transfection.Cationic polymers and deae dextran or the fat transfection agents of polylysine class in the synthetic vectors of exploitation, the polymine transfection agents all is widely used.
The mechanism of action details of different synthetic polymer importing nucleic acid drugs is different, but they also exist some common features, it is electronegative that these characteristics are based on nucleic acid molecule itself, and cytolemma itself is also electronegative, nucleic acid molecule is directed to the electrostatic repulsion of the negative charge generation that must overcome nucleic acid molecule and cell surface in the intracellular process, so be widely used at present the various nucleic acid transfection agent of laboratory study all be rich in cation group with in and the negative charge of nucleic acid and cell surface, the simplest transfection agents is exactly calcium phosphate, other such as cationic-liposome, cationoid polymerisation-1B and polymine etc., yet because the cytotoxicity of above-mentioned organic polymer, the effect of missing the target of the nucleic acid drug that transfection agents causes, their widespread uses in gene therapy have been limited, simultaneously the transfection efficiency of above-mentioned transfection agents is not high enough has also limited their being widely used in gene therapy, the medicine leading-in technique of gene therapy remains current technical bottleneck, seeks new high-efficiency low-toxicity and organizes single-minded nucleic acid transfection method to remain a current very important problem.
Summary of the invention
The present invention proposes small nucleic acids molecule express delivery technology, has solved the problem of the medicine leading-in technique bottleneck of gene therapy in the prior art.
Technical scheme of the present invention is achieved in that a kind of small nucleic acids molecule express delivery technology, and utilizing polypeptide class transfection agents is that small molecules nucleic acid/picodna of 10～100 or the small molecules nucleic acid transfection of passing through chemically modified enter cell with the base number.
As preferred technical scheme, described polypeptide class transfection agents and base number are 10～100 strand or double-stranded little RNA/DNA or only are 0.02～200g polypeptide/g nucleic acid through the mass ratio of the strand of chemically modified or double-stranded little RNA/DNA.
As preferred technical scheme, described polypeptide class transfection agents comprises 10～50 amino-acid residues; The transfection polypeptide of described polypeptide class transfection agents contains positively charged amino acid, and described positive charge amino acid number accounts for 20%～80% of polypeptide amino acid number; The number of described continuous distribution is 4～20; Described positive charge amino acid such as Methionin, arginine; These effects with the propylhomoserin sequence of positive electric charge are considered to form by electrostatic interaction with nucleic acid molecule the structural unit of mixture, as polypeptide-nucleic acid complexes under arginic positive charge effect with the negative charge effect of cell surface, instruct polypeptide/nucleic acid complexes to form mixture in the surface bonding of cell;
The transfection polypeptide of described polypeptide class transfection agents is a kind of or several arbitrarily in the sequence of following 9 groups of polypeptide:
Polypeptide 1:R2GSTDR1KRRRRRR3RRRR;
Polypeptide 2:AR3QAIR1IR2FQNKKRKKRKKK;
Polypeptide 3:RRRRRRRSPLMVR1GGR2GGLK;
Polypeptide 4:R2GDY1KRRRRRRRR;
Polypeptide 5:ACSR1SPSR2HCGRRRRRRR;
Polypeptide 6:AWGSR2GWSPKY1RRRRRR;
Polypeptide 7:PPLSSSTTGGGY2GGGY1HRRRKRKRRRK;
Polypeptide 8:RRRRRRRGAR2GDY1KRRRRRRRR;
Polypeptide 9:AGY2LMSTLY1RRRRRKRKR; R1 wherein, R2, R3 are any one in 20 natural amino acids or its D-type amino acid, the benefit of D-type aminoacid replacement natural amino acid is that the gained polypeptide is stronger to the tolerance of proteolytic ferment.
As preferred technical scheme, described amino-acid residue is the amino acid of D type amino acid and process chemically modified; Wherein, described chemically modified comprises that N-is terminal modified, C-is terminal modified and side chain is modified.
As preferred technical scheme, described modifier is cholesterol, cholestanol, plant sterol, the solid alkanol of plant, fluorescence molecule mark, PEG or PEI.
The other parts of polypeptide mainly are comprised of non-electric charge amino acid, improve the penetrativity of polypeptide cell membrane.Increase hydrophobic amino acid in the hydrophobic region, tryptophane especially, tyrosine, phenylalanine help to improve polypeptide to the transfection ability of nucleic acid molecule.A plurality of continuous non-electric charge amino acid regions also help to improve polypeptide to the transfection ability of nucleic acid such as the existence of a plurality of Serines; The ligand sequence that has cell-membrane receptor in some polypeptide as; The RGS sequence helps the integrin v III combination on polypeptide and the cytolemma, help to improve nucleic acid complexes at the cell that is rich in integrin v III, such as the transfection efficiency in the HeLa cell, amino acid (such as arginine and the Methionin) sequence of the positive electric charge of one section continuous band the 2nd or/and the 3rd will be to be easy to discharge nucleic acid molecule after Serine can make mixture enter cytoplasm with the amino-acid substitution of positive electric charge, thereby so that nucleic acid molecule can be sought its action site, bring into play its biological function, in addition, because cell contains proteolytic enzyme, proteolytic enzyme can further help the release of nucleic acid molecule from polypeptide-nucleic acid molecule mixture to the hydrolytic action of polypeptide, for the polypeptide that guarantees to synthesize have good water-soluble, the also suitable a small amount of charge residue of design in the non-positive charge amino acid region of polypeptide.
The modification of polypeptide comprises that also PEG modifies, and steroid is modified, stearic acid-base, and the bay acidic group, polyethylene imine beautify, trimethyl-glycine is modified.The position of modifying comprises any position except Methionin/arginine bunch, comprises that N-is terminal modified, and C-is terminal modified, sulfydryl modification etc.
The cell that this peptide species class transfection agents can be used for little RNA/DNA imports, thereby can be in clinical gene therapy and cell therapy.
Table 1 has been listed and has been met the sequence that the present invention protects the little peptide with nucleic acid transfection function of claim.These sequences only are the constructional features with polypeptide transfection agents of high-efficiency transfection nucleic acid molecule of the present invention for convenience of explanation, and not only are confined to the cited polypeptide of table 1, as change the sequence that positive amino acid whose number and permutation and combination can form thousands of meters
| Sequence number | The sequence of polypeptide |
| 1 | X 1GSTDX 1KXXXXXXX 1 |
| 2 | AX 1QAIXIXFQNKKXKKXKKK |
| 3 | X1XXXXXXSPLMVX 1GGXGGLK |
| 4 | X 1GDX 1KXXXXXXXX |
| 5 | ACSX
1SPSX
1 |
| 6 | AWGSX 1GWSPKYXXXXXX |
| 7 | PPLSSSTTGGGYGGGYHXXXKXKXXXK |
| 8 | X 1XXXXXXGAXGDYKXXXXXXXX |
| 9 | AGYLMSTLYXXXXXKXKX |
| 10 | X 1GDYKXXXXXXXX-FITC |
| 11 | GALFLGFLGAAGSTMGAWSQPKKKXXXXK |
| 12 | GALFLGFLGAAGSTMGAWPKXKXKXKX |
| 13 | GALFLGFLGAAGSTMGAWSQPKSKXKXKSKX |
| 14 | KXKXKXKXGDCDGXKXKXKXXXXKXKXGDCGXXX |
| 15 | GALFLGFLGAAGSTMGAGKSKXKXXXV |
| 16 | GALFLGFLGAAGSTMGAGKSXXXXXXS |
| 17 | KETWWETWWTEWSQPKKKXXXX |
| 18 | KWFETWFTEWPKSKKKKKKSK |
| 19 | KETWFETWFTEWSQPKXKXKXKX |
| 20 | KWFETWFTEWSQPKXKXKXK |
| 21 | KWFETWFTEWPKXKXKXKX |
| 22 | KWFETWFTEWPXSXXXXXX |
| 23 | KWFETWFTEWPKKKXKKKSK |
| 24 | YAXAAAXQAXAXXXXXXX |
| 25 | PLSSIFSXIGDPKSKXKXKSKX |
| 26 | GCXKKXXQXXXKKWPXXSXXXCA |
| 27 | XQIKIWFQNXXMKWKKXXXX |
| 28 | GIGAVLKVLTTGLPALISWIKXKXXXXX |
| 29 | ACSSSPSKHCGGKKKKKKSX |
| 30 | ACSSSPSKHCGGKSKKKKKX |
| 31 | ACSSSPSKHCGGXSXKXKXX |
| 32 | ACSSSPSKHCGGKKKKXXXX |
| 33 | WEAKLAKALAKALAKHLAKALAKALKACEAXSXXXXX |
| 34 | YTIWMPENPXPGTPCDIFTNSXGKXASNGKSKKKKKK |
| 35 | YTIWMPENPXPGTPCDIFTNSXGKXASNGGKKSKXXXXX |
| 36 | KFTIVFPHNQKGNWKNVPSNYHYCPXXXXXXXXX |
| 37 | KFTIVFPHNQKGNWKNVPSNYHYCPKKKKKKKK |
| 38 | GLFEALLELLESLWELLLLEAXXXXXXXX |
| 39 | LIXLWSHLIHIWFQNXXLKWXXXXX |
| 40 | AGYLLGKINLKALAALAKKILXXXXXXX |
| 41 | GWTLNSAGYLLGKINLKALAALAKKXXXXKK |
| 42 | YAXAAAXQAXAXSXXXXXK |
| 43 | YAXAAAXQAXAXXXXXSXK |
| 44 | LLIILXXXXIXKQAHAHSKXXX |
| 45 | MVXXFLVTLXIXXACGPPXVXVXXX |
| 46 | LIXLWSHLIHIWFQNXXLKWKKKXXX |
| 47 | TPWWXLWTKWHHKXXDLPXKPEKXXXX |
| 48 | GLWXALWXLLXSLWXLLWXAXXXKKK |
| 49 | DSHAKXHHGYKXKFHEKHHSHXGYXXX |
| 50 | GCG(AXKKAAKXXCA)3 |
| 51 | MANLGYWLLALFVTMWTDVGLCKKXPKPKKXXX |
| 52 | ALWKTLLKKVLKAPKKKXKV-amide |
| 53 | PKKKXKVALWKTLLKKVLKA-amide |
Table 1
Wherein, R1 can be any in 20 kinds of natural amino acids, also can be corresponding D-type amino acid.
The method of polypeptide transfection nucleic acid and liposome, PEI class transfection agents are similar.But can make the high transfection efficiency of various kinds of cell (comprising the stem cell in former generation, the hemocyte of suspension and adherent tumour cell) acquisition and show low toxicity with this transfection agents.The nucleic acid molecule of institute's transfection can be brought into play function in cell, what the present invention can be used for nucleic acid molecule in addition organizes transfection such as skin and other tissue.
Nucleic acid molecule among the present invention should be understood to strand or double-stranded thymus nucleic acid and Yeast Nucleic Acid.May relate to natural or artificial sequence, particularly such as the sequence of genome, complementary DNA (cDNA), messenger mrna, transfer RNA (tRNA) (tRNA), ribosome-RNA(rRNA) (rRNA), heterozygosis sequence or synthetic, semisynthetic sequence, oligonucleotide such as the miRNA of modification or unmodified, siRNA, piRNA, shRNA and antisense nucleotide.These nucleic acid can be from people, animal, plant, bacterium, virus etc.They can adopt any technology well known by persons skilled in the art to obtain, and they can be modified with chemical process.
The polypeptide transfection agents prepared according to the present invention is extremely low to the toxicity of cell, is 10: 1 to 30: 1 at best transfection agents weight range such as polypeptide/nucleic acid ratio; Classify 1: 1 to 60: 1 as at suboptimum transfection agents weight range such as polypeptide/nucleic acid ratio; Classify 0.1: 1 to 90: 1 as at general transfection agents weight range such as polypeptide/nucleic acid ratio; The polypeptide transfection agents does not all have observable cytotoxicity to the propagation of cell.
Owing to having adopted technique scheme, a kind of small nucleic acids molecule express delivery technology, utilizing polypeptide class transfection agents is that 10～100 small molecules nucleic acid or the efficiently transfection of small molecules nucleic acid of passing through chemically modified enter suspension or adherent cell with the base number, can be effectively enter cell with small molecules nucleic acid or through the small molecules nucleic acid transfection of chemically modified, solved the problem of the medicine leading-in technique bottleneck of gene therapy in the prior art.
Further specify the present invention below in conjunction with specific examples:
Example 1: transfection polypeptide synthetic
All polypeptide all adopt 9050 solid phase peptide synthesizers of Milipore company, are that stationary phase synthesizes with the AEDI-expansin resin.Resin activates with diisopropylethylamine, and transfection PEPC end first is amino acid whose amino with after the protection of uncle's fourth oxygen, and take DIC as condensing agent, carboxyl is namely to be covalently bound on the resin.Dissociate behind the amino acid protecting group with anhydrous trifluoroacetic acid, it is synthetic that peptide C-second amino acid of end repeats the above-mentioned steps circulation, makes peptide chain sequentially extend synthetic polypeptide according to the aminoacid sequence of above-mentioned transfection peptide.When polypeptide is synthesized to final step, Resin Suspension in anhydrous trifluoroacetic acid, is passed into dry HBr, polypeptide is disintegrated down from resin, some blocking groups also are removed simultaneously, and the product that obtains is transfection peptide crude product.It is a band that the polypeptide crude product carries out purifying with half preparative high-performance liquid chromatographic, and structure verification carries out the mass-to-charge ratio analysis with electrospray ionization mass spectrum, is 444.7 (M5+/5) such as the mass-to-charge ratio to polypeptide 10 (R1=F).
Example 2: enter in the adherent tumour cell with the fluorescently-labeled single stranded DNA of polypeptide transfection
Transfection polypeptide 4 (R1=F, R2=R) is made into the storage liquid of 10mg/ml with sterilized water, with the synthetic 3 ' strand with the red fluorescence mark of TAMRA mark
DNA (5 '-GTTCCGAGGTTGTTGTTGAAG-3 ') is configured to the 400g/ml storage liquid.Get 1l polypeptide and 1l nucleic acid join contain in 100 1 serum free mediums concussion and mix after, hatched 30 minutes for 37 ℃.
Other gets 24 porocyte culture plates, and before 12 hours, every hole inoculation prostate cancer tumour cell Dul45 (available from ATCC, the U.S.) 200,000 is uniformly dispersed cell.(substratum was the DMEM substratum that contains 10% foetal calf serum through 12 hours cultivations, culture condition is 37 ℃, 5% carbonic acid gas) after, change fresh serum free medium 500l into, to hatch polypeptide after 30 minutes-nucleic acid substratum is added drop-wise in the culture hole of 24 well culture plates uniformly again, under the light shaking culture plate 5-10, cell put back in the cell culture incubator continue to cultivate 6 hours.At this moment the DMEM substratum that contains 10% foetal calf serum that more renews continued culturing cell 12 hours.
With 18 hours Du145 Tissue Culture Plate after the transfection at inverted fluorescence microscope (Olympus IX-71, OLYMPUS, Japan) the lower observation, excite with green light, in red channels, observe fluorescent rna (as shown in Figure 1), as negative control, in only adding the cell cultures hole of fluorescently-labeled little RNA, almost do not observe the nucleic acid molecule of mark, show that independent nucleic acid molecule can not enter into cell, and under the effect of transfection polypeptide, can observe a large amount of red fluorescence spots, these spots all are positioned at cell, show that the polypeptide transfection agents can be transfected into nucleic acid molecule in the attached cell.
Polypeptide has obvious dose-effect relationship to this transfection effect of small nucleic acids molecule.Fig. 2 has shown that the mixing transfection polypeptide P1-4x transfection of various dose is for the double-strandednucleic acid siRNA (positive-sense strand: 5 '-FAM-GCAUUUAUCGUUAACCUAAdTdT-3 ' of the green fluorescence mark of GSK gene; The antisense strand sequence is the variation that 5 '-UUAGGUUAACGAUAAAUGCdTdT-3 ' (Shanghai JiMa pharmacy Technology Co., Ltd is synthetic) enters the transfection degree of Adenocarcinoma of lung cell line A549.Inoculating cell is 50000 in 24 porocyte culture plates, and the DMEM substratum that the rear usefulness of spending the night does not contain serum changes liquid three times to cell.Other gets the sample hose without the RNA enzyme, adds the DMEM serum free medium of 50 microlitres in every pipe, adds the GSK siRNA of 400ng again, in the other sample hose that same DMEM substratum is housed, add respectively 3ug, 6ug, 12ug, the mixed polypeptide of 24ug, behind above-mentioned sample concussion mixing, the substratum that polypeptide is housed is transferred to respectively in the substratum that contains siRNA mixed 37 ℃ of insulations 20 minutes that are incorporated in of concussion, form the polypeptide metering and be followed successively by 3, the polypeptide of 6,12,24ug, RNA transfection mixture.Above-mentioned transfection mixture evenly is added drop-wise to respectively in the cell cultures hole, behind the serum-free culture 5h, adds the DMEM substratum that contains foetal calf serum, making the serum final concentration in the culture hole is 5%.After cell cultures is spent the night, the luminous situation of green fluorescence under fluorescent microscope in the observation of cell, can see under the condition in lowest dose level (3ug/ hole), the A549 cell only has the transfected of only a few, send absinthe-green fluorescence, along with the rising of transfection polypeptide dosage, transfected cell count obviously increases, transfected mark fluorescent intensity also significantly strengthens, and illustrates that the amount that increases the transfection polypeptide can improve polypeptide to the transfection ability of cell.When polypeptide dosage is 24ug, the transfection ability of cell is reached approximately more than 80%, and the growth of cell do not had the restraining effect that can examine, demonstrate polypeptide to the good transfection ability of small nucleic acids.
Example 3: the polypeptide transfection agents can be transferred to the double-stranded RNA nucleic acid molecule of chemically modified in the suspension cell
Transfection polypeptide 5 (R1=S) is made into the storage liquid of 10mg/ml with sterilized water, with the double-stranded little RNA of synthetic green fluorescence mark
(positive-sense strand:
5 '-AUCCUCCCACCUCAGCCUCCCAAGUAGCUGGGACUACAGG-3 '; The antisense strand sequence is
5 '-CCUGUAGUCCCAGCUACUUGGGAGGCUGAGGUGGGAGGAU-3 ', nucleic acid molecule 5 ' end is with gfp molecule FAM covalent labeling, entrusts the Shanghai JiMa pharmacy Technology Co., Ltd synthetic) be configured to the 400g/ml storage liquid.Get 1l polypeptide and 1l nucleic acid join contain in the 100l serum free medium concussion and mix after, hatched 30 minutes for 37 ℃.
K562 cell (available from ATCC, the U.S.) is inoculated in the 24 porocyte culture plates, and the inoculation number in every hole is 50,000.Polypeptide-the nucleic acid complexes of hatching is added drop-wise in the DMEM cell culture medium that contains 10% foetal calf serum, and vibrations make it even.Under inverted fluorescence microscope, observe the distribution of green fluorescence after 20 hours as shown in Figure 3.Can see under the condition that does not add the polypeptide transfection agents, fluorescent rna can not enter in the cell, and behind the adding polypeptide transfection agents, cell sends green fluorescence.The little RNA of indication fluorescence is directed in the cell.
Example 4 can effectively reticent its target gene with polypeptide transfection agents transfection activity siRNA
Transfection polypeptide 5 (R1=K) is made into the storage liquid of 10mg/ml with sterilized water, with the double-stranded siRNA for people GSK gene of synthetic green fluorescence mark
(positive-sense strand: 5 '-FAM-GCAUUUAUCGUUAACCUAAdTdT-3 '; The antisense strand sequence is that 5 '-UUAGGUUAACGAUAAAUGCdTdT-3 ' (entrusting Shanghai JiMa pharmacy Technology Co., Ltd to synthesize) is configured to the 400g/ml storage liquid.Get 1l polypeptide and 1l nucleic acid join contain in the 100l serum free medium concussion and mix after, hatched 30 minutes for 37 ℃.HeLa cell (available from ATCC) is inoculated in the 24 porocyte culture plates, and the inoculation number in every hole is 50,000, after the incubated overnight, changes serum free medium into.Polypeptide-the nucleic acid complexes of hatching is added drop-wise in the DMEM cell culture medium that does not contain serum, and vibrations make it even.After the serum-free culture 5 hours, change the DMEM substratum that contains 10% foetal calf serum into, overnight incubation.Use simultaneously the double-stranded siRNA of negative control
(positive-sense strand: 5 '-UUCUCCGAACGUGUCACGUTT-3 ', antisense strand: 5 '-ACGUGACACGUUCGGAGAATT-3 ') make parallel control.
Cell extracts total RNA with the Trizol 1ml of Invitrogen company according to the specification sheets that reagent uses, the OD260/OD280 of total RNA is higher than 1.8, get total RNA of 2ug, reverse transcription test kit with Promega company, operational condition is to specifications carried out reverse transcription, after responsive transcription carries out 50min, transcribe the enzyme in the sample 95C5min deactivation sample.The PCR reaction adopts day quantitative PCR premixed liquid (containing PCR fluorescent probe molecule SYBR Green I) of root biotech company to carry out according to the operation of product description, the premixed liquid that in the reaction system of 20ul, adds 10ul, RNA reverse transcription product, forward and the inverse PCR primer of 1ul
(5’-AATTACGGGACCCAAATGTC-3’；5’-TCCACCTCTGGCTACCATCC-3’)
Each 1ul, the 7ul sterilized water mixes, and take-Actin (primer 5 '-agcgagcatcccccaaagtt-3 ' and 5 '-gggcacgaaggctcatcatt-3 ') gene as contrast, carries out quantitative pcr amplification.Each template is established 3 groups of parallel sample in the experiment.Amplification program is 95C10min, then carries out 40 (60C15s, 72C30s, 95C20s) stopped reaction of PCR circulation, and response curve is after normalized, and the PCR reaction cycle when acquisition fluorescent strength of signal occurs 5% is counted the Ct value, sees Table 3
Table 3: the reaction cycle during the quantitative PCR threshold values is counted the Ct value
The amount of its template is calculated in calculating according to amplification efficiency 100%, calculate the siRNA for the GSK gene with the polypeptide transfection
Expression that can reticent GSK, silence efficiency is 75%, the result of this silence efficiency and bibliographical information approaches, and illustrates that the peptide sequence that the present invention protects has the effect that imports to functional small nucleic acids in the cell and bring into play function.
Example 5: the polypeptide transfection agents can enter in the histocyte by skin
Transfection polypeptide 10 (R1=R) is made into the storage liquid of 10mg/ml with sterilized water, the little RNA of synthetic red fluorescence mark is configured to the 400g/ml storage liquid.Get 50l polypeptide and 50l nucleic acid join contain in the 100l serum free medium concussion and mix after, hatched 30 minutes for 37 ℃, add 10l glycerine.
With the male guinea pig (available from laboratory animal institute of the Chinese Academy of Medical Sciences) in 8 ages in week with the U.S. good quick-acting depilatory creams of poem (manufacturer: slough the hair of guinea pig back the flower makeup company limited in southern Guangdong, Guangzhou), behind warm water cleaning, get the transfection composite 20l that mixes, spread upon the guinea pig skin surface, area is 1cm2,1.5 after hour, cavy is put to death, behind skin peeling, make freezing microtome section (MEV freezing-microtome along the plumb cut of epidermis, SLEE, Germany), slice thickness is 5m.Section is in the lower observation of laser confocal microscope (Nikon, Japan), such as Fig. 4.Can see little RNA (Fig. 4-I) and green fluorescence labeling polypeptide 10 (Fig. 4-H) can enter in the tegumental cell of red fluorescence mark, ruddiness and green glow are excited simultaneously and send sodium yellow (Fig. 4-J) has verified that further single strand dna is that transfection effect by polypeptide enters the cavy skin.Only give single stranded DNA on the contrary in control group and during to polypeptide, DNA can not see through cavy skin (Fig. 4-A-E).
Example 6: the polypeptide transfection agents does not have obvious restraining effect to the propagation of cell
Mtt assay is a kind of effective ways that characterize the impact of medicine on cell proliferation, uses very wide in the research of cancer therapy drug.We have characterized the cytotoxicity of polypeptide transfection agents to tumour cell by mtt assay.Cell is in inoculation the day before yesterday of administration, and inoculum size is 5000/hole, and cell was cultivated 48 hours in the 5%CO2 incubator after to the transfection polypeptide of various dose.Add according to the every hole of the method for document to continue again to cultivate in the incubator behind the MTT sterile solution of 20 15mg/ml and stop after 4 hours cultivating.The careful nutrient solution of drawing in the hole is abandoned or adopted supernatant liquor and is added 150l dimethyl sulfoxide (DMSO) (Sigma, the U.S.), and concussion 10min makes the Viola crystallina dissolving, and on enzyme-linked immunosorbent assay instrument, 490nm measures each hole absorbance value.Fig. 5 A and Fig. 5 B have provided polypeptide transfection agents 2 propagation impact on prostate cancer Du145 cell strain and esophageal cancer cell strain CaES-17 under different polypeptide dosage, and to set the cell sample that does not add the transfection polypeptide be 100%.The result of Fig. 5 shows the cytotoxicity that the polypeptide transfection agents can not be observed cell, even the DU145 cell is also had slight stimulating growth effect.
Description of drawings
Fig. 1 wherein, A) only adds the little RNA of strand for the transfection design sketch with the fluorescently-labeled single stranded DNA of polypeptide transfection agents P2 transfection, and the cell image of observing under white light; B) at A) in the visual field under, excite the cell image of observing at the red fluorescence passage with green fluorescence; C) add the little RNA of strand and polypeptide transfection agents, and the cell image of under white light, observing; D) at C) the visual field under, use green fluorescence instead and excite, the cell image of observing at the red fluorescence passage;
Fig. 2 be the polypeptide transfection agents to the dose-effect relationship of little RNA transfection cell strain A549, wherein scheme A, B, what C and D showed is under identical exposure condition, transfection degree and transfection efficiency during the transfection polypeptide transfectional cell A549 of various dose.Wherein the dosage of fluorescently-labeled little RNA is every hole 400ng, and the dosage of transfection polypeptide is followed successively by 3,6,12 and the 24ug/ hole, figure E, F, G and H be respectively cell A549 at A, B, the cellular form figure of the gained of under white light, taking pictures under C and the D figure respective conditions;
Fig. 3 is that polypeptide 5 will dye the transfection effect that the fluorescence double-stranded RNA is transfected into Leukemia K562 cell, wherein, A) behind the double-stranded little RNA of adding and the polypeptide transfection agents, excite with blue-fluorescence, the cell image of observing at the green fluorescence passage, B) cell figure A) is under the same visual field, with the cell image of observing under the white light;
Fig. 4 is the laser co-focusing photo that polypeptide transfection agents P2 transshipment note chain DNA sees through guinea pig skin, wherein, and an A-E) administration single stranded DNA; F-J) percutaneous drug delivery after transfection polypeptide and single stranded DNA mix, A and F) white light copolymerization Jiao's frozen section photo; B) and G) the fluorescence excitation photo of DAPI dyeing, blue color spot point shows subcutaneous nuclear position, C) and H) be the polypeptide transfection green fluorescence photo of mark, the epidermal area of the corresponding cavy skin of green area that outermost layer is continuous, D) and I) be the photo of the single-chain nucleic acid red fluorescence of mark, E) and J) be blue-fluorescence, the stacking diagram of green fluorescence and red fluorescence, the zone of corresponding white, nuclear position in the indication dermis, the common zone that exists of yellow area correspondence markings polypeptide and labeling nucleic acid, these zones comprise the epidermal area of no cell structure and the nuclear exterior domain of skin corium cell, and used each parameter of fluorescence imaging is all identical in above-mentioned each figure;
Fig. 5 is to the cytotoxicity figure of cell strain Du145 and CaES-17 behind the transfection polypeptide effect 48h of different concns; Wherein the cell toxicant analysis adopts mtt assay to detect, and take the cell sample of not administration in the absorbancy at 490nm place as 100%.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the invention, the technical scheme in the embodiment of the invention is clearly and completely described, obviously, described embodiment only is the present invention's part embodiment, rather than whole embodiment.Based on the embodiment among the present invention, those of ordinary skills belong to the scope of protection of the invention not making the every other embodiment that obtains under the creative work prerequisite.
A kind of small nucleic acids molecule express delivery technology is characterized in that: utilizing polypeptide class transfection agents is that 10～100 small molecules nucleic acid or the small molecules nucleic acid transfection of passing through chemically modified enter cell with the base number.
Described polypeptide class transfection agents and base number are that 10～100 small molecules nucleic acid or the mass ratio that passes through the small molecules nucleic acid of chemically modified only are 0.02～200g polypeptide/g nucleic acid.
Described polypeptide class transfection agents comprises 10～50 amino-acid residues; The transfection polypeptide of described polypeptide class transfection agents contains positively charged amino acid, and described positive charge amino acid number accounts for 20%～80% of polypeptide amino acid number; The number of described positive charge amino acid continuous distribution is 4～20;
The transfection polypeptide of described polypeptide class transfection agents is a kind of or several arbitrarily in the sequence of following 9 groups of polypeptide:
Polypeptide 1:R2GSTDR1KRRRRRR3RRRR;
Polypeptide 2:AR3QAIR1IR2FQNKKRKKRKKK;
Polypeptide 3:RRRRRRRSPLMVR1GGR2GGLK;
Polypeptide 4:R2GDY1KRRRRRRRR;
Polypeptide 5:ACSR1SPSR2HCGRRRRRRR;
Polypeptide 6:AWGSR2GWSPKY1RRRRRR;
Polypeptide 7:PPLSSSTTGGGY2GGGY1HRRRKRKRRRK;
Polypeptide 8:RRRRRRRGAR2GDY1KRRRRRRRR;
Polypeptide 9:AGY2LMSTLY1RRRRRKRKR; R1 wherein, R2, R3 are any one in 20 natural amino acids or its D-type amino acid.
Described amino-acid residue is the amino acid of D type amino acid and process chemically modified; Wherein, described chemically modified comprises that N-is terminal modified, C-is terminal modified and side chain is modified.
Described modifier is cholesterol, cholestanol, plant sterol, the solid alkanol of plant, fluorescence molecule mark, PEG or PEI.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. small nucleic acids molecule express delivery technology is characterized in that: to utilize polypeptide class transfection agents be small molecules nucleic acid/picodna of 10～100 with the base number or enter cell through the small molecules nucleic acid transfection of chemically modified.
2. a kind of small nucleic acids molecule express delivery technology as claimed in claim 1 is characterized in that: described polypeptide class transfection agents and base number are 10～100 strand or double-chain small molecule RNA/DNA or only are 0.02～200g polypeptide/g nucleic acid through the strand of chemically modified or the mass ratio of double-chain small molecule RNA/DNA.
3. a kind of small nucleic acids molecule express delivery technology as claimed in claim 1, it is characterized in that: described polypeptide class transfection agents comprises 10～50 amino-acid residues; The transfection polypeptide of described polypeptide class transfection agents contains positively charged amino acid, and described positive charge amino acid number accounts for 20%～80% of polypeptide amino acid number; The number of described positive charge amino acid continuous distribution is 4～20;
The transfection polypeptide of described polypeptide class transfection agents is a kind of or several arbitrarily in the sequence of following 9 groups of polypeptide:
Polypeptide 1:R2GSTDR1KRRRRRR3RRRR;
Polypeptide 2:AR3QAIR1IR2FQNKKRKKRKKK;
Polypeptide 3:RRRRRRRSPLMVR1GGR2GGLK;
Polypeptide 4:R2GDY1KRRRRRRRR;
Polypeptide 5:ACSR1SPSR2HCGRRRRRRR;
Polypeptide 6:AWGSR2GWSPKY1RRRRRR;
Polypeptide 7:PPLSSSTTGGGY2GGGY1HRRRKRKRRRK;
Polypeptide 8:RRRRRRRGAR2GDY1KRRRRRRRR;
Polypeptide 9:AGY2LMSTLY1RRRRRKRKR; R1 wherein, R2, R3 are any one in 20 natural amino acids or its D-type amino acid.
4. a kind of small nucleic acids molecule express delivery technology as claimed in claim 3 is characterized in that: described amino-acid residue is D type amino acid and through the amino acid of chemically modified; Wherein, described chemically modified comprises that N-is terminal modified, C-is terminal modified and side chain is modified.
5. a kind of small nucleic acids molecule express delivery technology as claimed in claim 4 is characterized in that: described modifier is cholesterol, cholestanol, plant sterol, the solid alkanol of plant, fluorescence molecule mark, PEG or PEI.
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