CN102532411B - Functional oligomer used for non-viral gene vector material and application thereof - Google Patents
Functional oligomer used for non-viral gene vector material and application thereof Download PDFInfo
- Publication number
- CN102532411B CN102532411B CN2011104401623A CN201110440162A CN102532411B CN 102532411 B CN102532411 B CN 102532411B CN 2011104401623 A CN2011104401623 A CN 2011104401623A CN 201110440162 A CN201110440162 A CN 201110440162A CN 102532411 B CN102532411 B CN 102532411B
- Authority
- CN
- China
- Prior art keywords
- dna
- viral gene
- solution
- thing
- dmael
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108700005077 Viral Genes Proteins 0.000 title claims abstract description 26
- 239000000463 material Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 22
- 239000012876 carrier material Substances 0.000 claims abstract description 21
- 239000000178 monomer Substances 0.000 claims abstract description 5
- 239000003999 initiator Substances 0.000 claims abstract description 4
- 238000010526 radical polymerization reaction Methods 0.000 claims abstract description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims abstract 7
- 239000000243 solution Substances 0.000 claims description 54
- 108010039918 Polylysine Proteins 0.000 claims description 48
- 229920000656 polylysine Polymers 0.000 claims description 48
- 239000000203 mixture Substances 0.000 claims description 42
- 238000006384 oligomerization reaction Methods 0.000 claims description 34
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 238000006243 chemical reaction Methods 0.000 claims description 22
- QRUDEWIWKLJBPS-UHFFFAOYSA-N benzotriazole Chemical compound C1=CC=C2N[N][N]C2=C1 QRUDEWIWKLJBPS-UHFFFAOYSA-N 0.000 claims description 16
- 239000012964 benzotriazole Substances 0.000 claims description 16
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 11
- 239000008101 lactose Substances 0.000 claims description 11
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000000502 dialysis Methods 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 8
- -1 allyl group histidine derivative Chemical class 0.000 claims description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 claims description 6
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 6
- 235000011152 sodium sulphate Nutrition 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000004809 thin layer chromatography Methods 0.000 claims description 6
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 6
- RJUFJBKOKNCXHH-UHFFFAOYSA-N Methyl propionate Chemical compound CCC(=O)OC RJUFJBKOKNCXHH-UHFFFAOYSA-N 0.000 claims description 5
- 229930182478 glucoside Natural products 0.000 claims description 5
- 150000008131 glucosides Chemical class 0.000 claims description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 5
- 229940017219 methyl propionate Drugs 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- XRUKRHLZDVJJSX-UHFFFAOYSA-N 4-cyanopentanoic acid Chemical compound N#CC(C)CCC(O)=O XRUKRHLZDVJJSX-UHFFFAOYSA-N 0.000 claims description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- 229920001661 Chitosan Polymers 0.000 claims description 4
- 235000019270 ammonium chloride Nutrition 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- BXRMEWOQUXOLDH-LURJTMIESA-N L-Histidine methyl ester Chemical compound COC(=O)[C@@H](N)CC1=CN=CN1 BXRMEWOQUXOLDH-LURJTMIESA-N 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 238000007605 air drying Methods 0.000 claims description 3
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 3
- 238000006392 deoxygenation reaction Methods 0.000 claims description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 3
- 229960004756 ethanol Drugs 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000003818 flash chromatography Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- LRDFRRGEGBBSRN-UHFFFAOYSA-N isobutyronitrile Chemical compound CC(C)C#N LRDFRRGEGBBSRN-UHFFFAOYSA-N 0.000 claims description 3
- VLCAYQIMSMPEBW-UHFFFAOYSA-N methyl 3-hydroxy-2-methylidenebutanoate Chemical compound COC(=O)C(=C)C(C)O VLCAYQIMSMPEBW-UHFFFAOYSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000001953 recrystallisation Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000012986 chain transfer agent Substances 0.000 claims 1
- 238000001890 transfection Methods 0.000 abstract description 22
- 210000004027 cell Anatomy 0.000 abstract description 18
- 239000013612 plasmid Substances 0.000 abstract description 9
- VFXXTYGQYWRHJP-UHFFFAOYSA-N 4,4'-azobis(4-cyanopentanoic acid) Chemical compound OC(=O)CCC(C)(C#N)N=NC(C)(CCC(O)=O)C#N VFXXTYGQYWRHJP-UHFFFAOYSA-N 0.000 abstract description 5
- 101100490446 Penicillium chrysogenum PCBAB gene Proteins 0.000 abstract description 5
- 239000011246 composite particle Substances 0.000 abstract description 4
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 abstract description 3
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 210000001163 endosome Anatomy 0.000 abstract description 3
- 210000003712 lysosome Anatomy 0.000 abstract description 3
- 230000001868 lysosomic effect Effects 0.000 abstract description 3
- 238000012546 transfer Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 210000000170 cell membrane Anatomy 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 239000000969 carrier Substances 0.000 abstract 1
- 230000001681 protective effect Effects 0.000 abstract 1
- 150000007970 thio esters Chemical class 0.000 abstract 1
- 239000002953 phosphate buffered saline Substances 0.000 description 20
- 239000012895 dilution Substances 0.000 description 10
- 238000010790 dilution Methods 0.000 description 10
- 239000002131 composite material Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000012637 gene transfection Methods 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
- 239000005090 green fluorescent protein Substances 0.000 description 5
- 241000700605 Viruses Species 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000001415 gene therapy Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 125000002252 acyl group Chemical group 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 231100000734 genotoxic potential Toxicity 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
一种用于非病毒基因载体材料的功能性寡聚物，由MA-His-OMe和MAEL通过以水溶性硫代酯作为分子量调节剂自由基聚合制得；其制备方法是以MA-His-OMe和MAEL为共单体，以AIBN)或ACVA为引发剂，以CPADB为连转移剂制备；将该功能性寡聚物与聚阳离子材料混合制备DNA或RNA复合物，用作非病毒基因载体材料，并进行细胞转染试验。本发明的优点：该功能性寡聚物对质粒DNA具有良好保护作用，改善了复合粒子与细胞膜表面的结合作用以及质粒DNA在细胞之内从溶酶体/内涵体中的逃离效率，从而使复合粒子对HeLa等细胞的转染效率大幅提高，毒性大幅下降，有望达到临床应用水平。
A functional oligomer for non-viral gene carrier materials, prepared by free radical polymerization of MA-His-OMe and MAEL with water-soluble thioester as a molecular weight regulator; its preparation method is based on MA-His-OMe OMe and MAEL are co-monomers, AIBN) or ACVA is used as an initiator, and CPADB is used as a transfer agent; the functional oligomer is mixed with polycation materials to prepare DNA or RNA complexes, which are used as non-viral gene carriers materials and perform cell transfection experiments. Advantages of the present invention: the functional oligomer has a good protective effect on plasmid DNA, improves the combination of composite particles and the surface of the cell membrane and the escape efficiency of plasmid DNA from lysosomes/endosomes in cells, so that The transfection efficiency of the composite particles to HeLa and other cells has been greatly improved, and the toxicity has been greatly reduced, which is expected to reach the level of clinical application.
Description
Technical field
The invention belongs to gene therapy and field of new, be specifically related to a kind of thing of the functional oligomerization for non-viral gene carrier material and application.
Background technology
Gene therapy is that a kind of molecular biology method that utilizes imports goal gene in patient body, makes it to express the goal gene product, thus the technology that disease is obtained medical treatment.Genophore is one of core technology of field of gene.Genophore is divided into virus vector and non-virus carrier usually. and as a rule, the transfection efficiency of virus vector is high, but has the problems such as genotoxic potential, immunogenicity, has limited its application in clinical treatment; Although non-virus carrier can overcome the problems referred to above, in the born of the same parents of disadvantageous bio distribution and poor efficiency, running, cause the active gene transfection efficiency low. the transfection efficiency that therefore improves non-viral gene vector is the key of current research.
Cationic polymers, as chitosan, polylysine, polymine, polymethyl acyl-oxygen ethyl-trimethyl salmiac etc. are several non-viral gene vector polymer materialss of commonly using the most.These polymer materialss as the subject matter of gene vector material are, the complex particle that itself and DNA or RNA form easily produces specific interaction and produces absorption some protein under physiological environment, thereby affects its endocytosis and the speed of escaping from endosome or lysosome affects transfection efficiency then.Overcome the way of this problem mainly by the mode of chemical modification, as introduced the PEG segment in its structure by chemical modification method, but this mode has often also consumed the amino in the polycation structure, and the ability of its compression DNA is descended simultaneously.
Summary of the invention
The objective of the invention is for above-mentioned existing problems, a kind of thing of the functional oligomerization for non-viral gene carrier material and application are provided, preparation technology is simple, easy to implement for this functional oligomerization thing; This functional oligomerization thing as non-viral gene vector, has low cytotoxicity, good antiserum(antisera) stability and high efficiency gene transfection together with other polycation material, is expected to for clinical gene therapy.
Technical scheme of the present invention:
A kind of thing of the functional oligomerization for non-viral gene carrier material P (His-co-DMAEL), by the allyl group histidine derivative, 2-methacryloyl amido-3-(4-imidazolyl) methyl propionate (MA-His-OMe) and containing lactose monomer 2-oxygen-methyl-acryloyl-oxy oxyethyl group-(2, 3, 4, 6-tetra--oxy-acetyl-β-D-semi-lactosi)-(1-4)-2, 3, by usining water-soluble monothioester, as molecular weight regulator, radical polymerization makes 6-tri--oxy-acetyl-β-D-pyranoglucose glucosides (MAEL), molecular weight is 0.5-20KDa, molecular weight distribution is 1.0-2.0, MA-His-OMe and MAEL mol ratio are 3-15: 1.
A kind of preparation method of the described thing of the functional oligomerization for non-viral gene carrier material, take MA-His-OMe and MAEL as being total to monomer, with 2,2 '-azo two (2-methyl propionitrile) (AIBN) or 4,4 '-azo (4-cyanopentanoic acid) is (ACVA) initiator, take 4-cyanopentanoic acid-dithio phenyl ester (CPADB) as connecting the transfer agent preparation, and step is as follows:
1) take Histidine methyl esters and allyl group benzo triazole as synthetic 2-methacryloyl amido-3-(4-imidazolyl) methyl propionate (MA-His-OMe) of raw material:
The 4.8g benzotriazole is dissolved in the 25ml methylene dichloride, add the 1.2g thionyl chloride, after stirring 30min, add the 0.86g methacrylic acid, continue reaction 2h, the sodium hydroxide solution extraction that organic phase is 2 mol/L by concentration three times, organic phase dried over sodium sulfate 12h, revolve to steam and remove methylene dichloride, product flash chromatography column purification, obtain allyl group benzo triazole.1.87g allyl group benzo triazole is dissolved in the 50ml dioxane, 1.69g Histidine methyl esters is dissolved in the 10ml deionized water, under the magnetic agitation condition, regulating its pH value is 8.0, then will be dissolved in allyl group benzo triazole in dioxane slowly drops in above-mentioned Histidine methyl ester solution and is reacted, and by thin-layer chromatography monitoring reaction process, treat that thin-layer chromatography shows that allyl group benzo triazole runs out of full-time, stop stirring, the dilute hydrochloric acid regulator solution pH value that is 10% with mass percent concentration is to neutral, revolve to steam and remove dioxane, remove by filter insolubles and revolve to steam and remove water, product is dissolved in dehydrated alcohol and filters after lyophilize, again filtrate is spin-dried for and vacuum-drying, obtain MA-His-OMe,
2) 2-oxygen-methyl-acryloyl-oxy oxyethyl group-(2,3,4,6-, tetra--oxy-acetyl-β-D-semi-lactosi)-(1-4)-2,3, the preparation of 6-tri--oxy-acetyl-β-D-pyranoglucose glucosides (MAEL):
20g lactose, 10g sodium acetate, anhydrous are added in the 120ml acetic anhydride, mixed being incorporated under reflux temperature reacted 2h, and reaction is poured product in frozen water into and stirred after finishing, and filters and wash with water solid three times, be placed in air drying, then with ethyl alcohol recrystallization, obtain the octoacetyl lactose; In the 20ml methylene dichloride, add 5g eight acyl group lactose and 4.8ml hydroxyethyl methylacrylate and 2mL Eorontrifluoride etherate, react 2h in ice-water bath, then room temperature reaction 12h, reaction adds the 20ml deionized water after finishing, and after separation, water, saturated sodium bicarbonate solution and water washing methylene dichloride, mutually to neutral, are then used dried over sodium sulfate successively, revolve to steam and remove methylene dichloride and separate by chromatographic column, obtain MAEL;
3) for the preparation of the functional oligomerization thing of non-viral gene carrier material:
0.96g MA-His-OMe, 0.5g MAEL, 0.0075g ACVA and 0.023g CPADB are dissolved in 10ml N, in N '-dimethyl formamide, freezing deoxygenation seals afterwards for three times and is placed in 70 ℃ of water-baths reactions 48 hours, after reaction finishes, the dialysis tubing that is first 3500 with molecular weight cut-off in deionized water, dialyse 48h lyophilize, then product is dissolved in dehydrated alcohol, add the methanol solution that the 1ml mass percent concentration is 30% sodium methylate and react 2 hours, the dialysis tubing that is 3500 with molecular weight cut-off again is in deionized water dialysis 48 hours lyophilize, obtain functional oligomerization thing P (His-co-DMAEL).
A kind of application of the described thing of the functional oligomerization for non-viral gene carrier material, prepare DNA or RNA mixture by this functional oligomerization thing and polycation material mixing, as non-viral gene carrier material, and carries out the cell transfecting test.
Described polycation material comprises chitosan (being designated hereinafter simply as CS), polylysine (PLL), polymine (PEI) and polymethyl acyl-oxygen ethyl-trimethyl salmiac (PDMMC).
The preparation method of described DNA or RNA mixture is first the functional oligomerization thing of 0.1-2.0 μ g/ μ l to be mixed and places 15 minutes with under DNA or RNA room temperature; And then the polycation material solution of 0.1-2.0 μ g/ μ l is added dropwise in above-mentioned DNA mixed solution, vibration is stirred and solution was mixed in 10 seconds and place 15 minutes, can make N/P=0.25: 1-40: the DNA of 1 ratio or RNA mixture.
The mass ratio of described functional oligomerization thing and DNA or RNA is 0-200: 1.
The mass ratio of described functional oligomerization thing and polycation material is 0-40: 1.
Major advantage of the present invention:
Some polycation materials commonly used are during separately as gene vector material, often or because toxicity is large or transfection efficiency is low, are difficult to clinical application.Method of the present invention is by introducing a kind of polyfunctional nonionic oligomer, both obtained that plasmid DNA was had to the good protection effect, the composite nanoparticle that stability is high, improved again the keying action of composite particles and surface of cell membrane and plasmid DNA simultaneously and fled from efficiency from lysosome/endosome within cell, thereby make composite particles to HeLa, HepG2, the more unmodified polylysine of the isocellular transfection efficiency of NIH3T3, chitosan, polymine, the polycations such as polymethyl acyl-oxygen ethyl-trimethyl salmiac significantly improve, be expected to reach the clinical application level, polymkeric substance through the inventive method modification also declines to a great extent aspect cytotoxicity than unmodified polymer simultaneously.
The accompanying drawing explanation
Fig. 1 is poly-[2-methacryloyl amido-3-(4-imidazolyl) methyl propionate-co-2-oxygen-methyl-acryloyl-oxy oxyethyl group-(2; 3; 4; 6-tetra--oxy-acetyl-β-D-semi-lactosi)-(1-4)-2; 3,6-, tri--oxy-acetyl-β-D-pyranoglucose glucosides] the 400M nuclear magnetic spectrogram.
Fig. 2 mixture that to be different polycations form with DNA identical N/P than the time cell in vitro transfection green fluorescence figure and corresponding light field figure.
Fig. 3 mixture that to be identical polycation form with DNA different N/P than the time cell in vitro transfection green fluorescence figure and corresponding light field figure.
PLL/DNA/P (His-co-DMAEL) mixture that Fig. 4 is different P (His-co-DMAEL) content identical N/P than the time cell in vitro transfection green fluorescence figure and corresponding light field figure.
Fig. 5 is that PLL/DNA, PLL/DNA/P (His-co-DMAEL) mixture particle diameter in 0.5% bovine serum albumin (BSA) solution is schemed over time, mixture DNA consumption is 3 μ g, N/P=15: 1, the mass ratio of functional oligomerization thing and polycation material is 4: 1, and the cultivation time is followed successively by 0,6,24h.
Embodiment
Below in conjunction with specific embodiment, the present invention program is described further, but this explanation does not limit the scope of the invention.
Embodiment:
A kind of preparation method of the thing of the functional oligomerization for non-viral gene carrier material, take MA-His-OMe and MAEL as reactant, with 2,2 '-azo two (2-methyl propionitrile) (being designated hereinafter simply as AIBN) or 4,4 '-azo (4-cyanopentanoic acid) is (ACVA) initiator, take 4-cyanopentanoic acid-dithio phenyl ester (CPADB) as connecting the transfer agent preparation, and step is as follows:
1) preparation of MA-His-OMe
4.8g benzotriazole is dissolved in the 25ml methylene dichloride, adds the 1.2g thionyl chloride, after stirring 30min, adds the 0.86g methacrylic acid, continues reaction 2h.After reaction finishes, organic phase extracts three times with the sodium hydroxide solution of 2 moles every liter, uses dried over sodium sulfate 12h.Revolve to steam and remove methylene dichloride, product flash chromatography column purification, obtain allyl group benzo triazole.
1.69g Histidine methyl esters is dissolved in the 10ml deionized water, under the magnetic agitation condition, regulate its pH value to 8.0 left and right, 1.87g allyl group benzo triazole is dissolved in the 50ml dioxane, then slowly drop in the Histidine methyl ester solution and react, and by thin-layer chromatography monitoring reaction process, treat that thin-layer chromatography shows that the consumption of allyl group benzo triazole is complete, stop stirring, extremely neutral by dilute hydrochloric acid regulator solution pH value, revolve to steam and remove dioxane, remove by filter insolubles and revolve to steam and remove water.Be dissolved in dehydrated alcohol after the product lyophilize and filter, filtrate is spin-dried for and vacuum-drying, obtaining 2-methacryloyl amido-3-(4-imidazolyl) methyl propionate.
2) preparation of MAEL;
20g lactose, 10g sodium acetate, anhydrous are added in the 120ml acetic anhydride, and mixed being incorporated under reflux temperature reacted 2h.Reaction is poured product in frozen water into and is stirred after finishing, and filters and wash with water solid three times, is placed in air drying, then with ethyl alcohol recrystallization, obtains the octoacetyl lactose.
In the 20ml methylene dichloride, add the Eorontrifluoride etherate of 5g eight acyl group lactose and 4.8ml hydroxyethyl methylacrylate and 2mL, react 2h in ice-water bath, then room temperature reaction 12h.Reaction adds the 20ml deionized water after finishing and water, saturated sodium bicarbonate solution and water washing methylene dichloride are extremely neutral mutually successively.Then use dried over sodium sulfate, revolve to steam and remove methylene dichloride, and separate by chromatographic column, obtain MAEL.
3) preparation of functional oligomerization thing P (His-co-DMAEL).
0.96g MA-His-OMe, 0.5g MAEL, 0.023g CPADB, 0.0075g ACVA are dissolved in 10mlN, in N '-dimethyl formamide, freezing deoxygenation seals afterwards for three times and is placed in 70 ℃ of water-baths reactions 48 hours.After reaction finishes, the dialysis tubing that is first 3500 with molecular weight cut-off in deionized water, dialyse 48h lyophilize, then product is dissolved in dehydrated alcohol, add sodium methylate/methanol solution of 1ml 30% and react 2 hours, the dialysis tubing that is 3500 with molecular weight cut-off again, in deionized water dialysis 48 hours lyophilize, obtains P (His-co-DMAEL).
A kind of application of the described thing of the functional oligomerization for non-viral gene carrier material, prepare DNA or RNA mixture by this functional oligomerization thing and polycation material mixing, as non-viral gene carrier material, and carries out the cell transfecting test.
1) preparation of PLL/DNA/P (His-co-DMAEL), PLL/DNA cytogene transfection composite:
P (His-co-DMAEL) is dissolved in to the phosphate buffered saline buffer (hereinafter to be referred as PBS) of the 0.01M of pH=7.4, be mixed with P (His-co-DMAEL) solution of 1mg/ml, the plasmid DNA solution through Green Fluorescent Protein that is 0.2 μ g/ μ L by the concentration configured, with equal-volume after the PBS dilution, join in P (His-co-DMAEL) solution, the DNA consumption is 1 μ g, the standing 15min of room temperature after mixing.Then according to N/P=5: 1 or 15: 1, the PLL solution of the 1mg/ml that calculates is added with equal-volume after the PBS dilution, P (His-co-DMAEL) is 4: 1 with the PLL mass ratio, mixes and the standing 15min of room temperature, obtains PLL/DNA/P (His-co-DMAEL) mixture.Replace P (His-co-DMAEL) solution with isopyknic PBS solution, by method same as described above, prepare the PLL/DNA mixture.
2) preparation of PEI/DNA/P (His-co-DMAEL), PEI/DNA cytogene transfection composite:
P (His-co-DMAEL) is dissolved in to the PBS of the 0.01M of pH=7.4, be mixed with P (His-co-DMAEL) solution of 1mg/ml, the plasmid DNA solution through Green Fluorescent Protein that is 0.2 μ g/ μ L by the concentration configured, with equal-volume after the PBS dilution, join in P (His-co-DMAEL) solution, the DNA consumption is 1 μ g, the standing 15min of room temperature after mixing.Then according to N/P=15: 1, the PEI solution of the 1mg/ml that calculates is added with equal-volume after the PBS dilution, P (His-co-DMAEL) is 4: 1 with the PEI mass ratio, mixes and the standing 15min of room temperature, obtains PEI/DNA/P (His-co-DMAEL) mixture.Replace P (His-co-DMAEL) solution with isopyknic PBS solution, by method same as described above, prepare the PEI/DNA mixture.
3) preparation of PDMMC/DNA/P (His-co-DMAEL), PDMMC/DNA cytogene transfection composite:
P (His-co-DMAEL) is dissolved in to the PBS solution of the 0.01M of pH=7.4, be mixed with P (His-co-DMAEL) solution of 1mg/ml, the plasmid DNA solution through Green Fluorescent Protein that is 0.2 μ g/ μ L by the concentration configured, with equal-volume after the PBS dilution, join in P (His-co-DMAEL) solution, the DNA consumption is 1 μ g, the standing 15min of room temperature after mixing.Then according to N/P=15: 1, the PDMMC solution of the 1mg/ml that calculates is added with equal-volume after the PBS dilution, P (His-co-DMAEL) is 4: 1 with the PDMMC mass ratio, mixes and the standing 15min of room temperature, obtains PDMMC/DNA/P (His-co-DMAEL) mixture.Replace P (His-co-DMAEL) solution with isopyknic PBS solution, by method same as described above, prepare the PDMMC/DNA mixture.
4) preparation of CS/DNA/P (His-co-DMAEL), CS/DNA cytogene transfection composite:
P (His-co-DMAEL) is dissolved in to the PBS solution of the 0.01M of pH=7.4, be mixed with P (His-co-DMAEL) solution of 1mg/ml, the plasmid DNA solution through Green Fluorescent Protein that is 0.2 μ g/ μ L by the concentration configured, with equal-volume after the PBS dilution, join in P (His-co-DMAEL) solution, the DNA consumption is 1 μ g, the standing 15min of room temperature after mixing.Then according to N/P=15: 1, by the CS solution (CH of the 0.01M of pH=5.5 of the 1mg/ml that calculates
3COONa/CH
3COOH buffered soln) use the CH of the 0.01M of pH=5.5
3COONa/CH
3After the dilution of COOH buffered soln, equal-volume adds, and P (His-co-DMAEL) is 4: 1 with the CS mass ratio, mixes and the standing 15min of room temperature, obtains CS/DNA/P (His-co-DMAEL) mixture.Replace P (His-co-DMAEL) solution with isopyknic PBS solution, by method same as described above, prepare the CS/DNA mixture.
5) preparation of the PLL/DNA/P of different functionalities oligomer content (His-co-DMAEL) cytogene transfection composite:
P (His-co-DMAEL) is dissolved in to the PBS solution of the 0.01M of pH=7.4, be mixed with P (His-co-DMAEL) solution of 1mg/ml, the plasmid DNA solution through Green Fluorescent Protein that is 0.2 μ g/ μ L by the concentration configured, with equal-volume after the PBS dilution, join in P (His-co-DMAEL) solution, the DNA consumption is 1 μ g, the standing 15min of room temperature after mixing.Then according to N/P=15: 1, the PLL solution of the 1mg/ml that calculates is added with equal-volume after the PBS dilution, mix and the standing 15min of room temperature, obtain P (His-co-DMAEL) and be respectively 1: 1 with the PLL mass ratio, 2: 1,4: 1, the mixture of 8: 1, mean respectively PLL/DNA/P (His-co-DMAEL)-1, PLL/DNA/P (His-co-DMAEL)-2, PLL/DNA/P (His-co-DMAEL)-4, PLL/DNA/P (His-co-DMAEL)-8.
6) mixture that different polycations and DNA form identical N/P than the time the cell in vitro transfection
By the HeLa cell with every hole 5 * 10
4Cell several enter 24 well culture plates and cultivate 24 hours, then the substratum containing 10% bovine serum albumin FBS that contains the different genes transfection composite with 0.5ml is replaced former substratum and is continued and cultivates, the consumption of DNA is the 1 every hole of μ g, N/P=15: 1, P (His-co-DMAEL) is 4: 1 with the mass ratio of polycation, after 24 hours, with 0.5ml, containing the substratum of 10%FBS, replace the substratum that contains the gene transfection mixture and continue to cultivate, observing with the fluorescence inverted microscope after 24h.Result as shown in Figure 2.
Fig. 2 shows, identical N/P than the time, PLL/DNA/P (His-co-DMAEL), PEI/DNA/P (His-co-DMAEL), PDMMC/DNA/P (His-co-DMAEL), PLL/DNA, PEI/DNA, PDMMC/DNA, CS/DNA mixture that CS/DNA/P (His-co-DMAEL) mixture is more corresponding are significantly increased to the transfection efficiency of HeLa cell.
7) mixture that identical polycation and DNA form different N/P than the time the cell in vitro transfection
By the HeLa cell with every hole 5 * 10
4Cell several enter 24 well culture plates and cultivate 24 hours, then the substratum containing 10%FBS that contains the gene transfection mixture with 0.5ml is replaced former substratum and is continued and cultivates, the consumption of DNA is the 1 every hole of μ g, N/P=5: 1 or 15: 1, P (His-co-DMAEL) is 4: 1 with the mass ratio of PLL, after 24 hours, with 0.5ml, containing the substratum of 10%FBS, replace the substratum that contains the gene transfection mixture and continue to cultivate, observing with the fluorescence inverted microscope after 24h.Result as shown in Figure 3.Fig. 3 shows, PLL/DNA/P (His-co-DMAEL) mixture is at N/P=5: 1 and 15: 1 o'clock, and more corresponding PLL/DNA mixture is significantly increased to the transfection efficiency of HeLa cell.
8) PLL/DNA/P (His-co-DMAEL) mixture of different P (His-co-DMAEL) content identical N/P than the time the cell in vitro transfection
By the HeLa cell with every hole 5 * 10
4cell several enter 24 well culture plates and cultivate 24 hours, then contain PLL/DNA/P (His-co-DMAEL)-1 with 0.5ml, PLL/DNA/P (His-co-DMAEL)-2, PLL/DNA/P (His-co-DMAEL)-4, the substratum containing 10%FBS of PLL/DNA/P (His-co-DMAEL)-8 or PLL/DNA mixture is replaced former substratum and is continued and cultivates, the consumption of DNA is the 1 every hole of μ g, N/P=15: 1, after 24 hours, replace the substratum that contains the gene transfection mixture and continue to cultivate containing the substratum of 10%FBS with 0.5ml, after 24h, with the fluorescence inverted microscope, observe.Result as shown in Figure 4.
Fig. 4 shows, PLL/DNA/P (His-co-DMAEL)-1, PLL/DNA/P (His-co-DMAEL)-2, PLL/DNA/P (His-co-DMAEL)-4, PLL/DNA/P (His-co-DMAEL)-8 mixture is at N/P=15: 1 o'clock, more corresponding PLL/DNA mixture was significantly increased to the transfection efficiency of HeLa cell.
This stable composite test:
Prepare PLL/DNA/P (His-co-DMAEL)-1, PLL/DNA/P (His-co-DMAEL)-2, PLL/DNA/P (His-co-DMAEL)-4, PLL/DNA/P (His-co-DMAEL)-8 and PLL/DNA mixture in the PBS of 0.01M solution, every kind of mixture DNA consumption is 3 μ g, N/P=15: 1, then add bovine serum albumin BSA solution in mixture solution, to make the BSA ultimate density be 0.5% and mix.Cultivate respectively 0,6,24h under 37 ℃ of conditions, then with dynamic light scattering DLS, measure the mixture particle diameter.Result as shown in Figure 5.
Fig. 5 shows, in protein soln, PLL/DNA/P (His-co-DMAEL)-1, PLL/DNA/P (His-co-DMAEL)-2, PLL/DNA/P (His-co-DMAEL)-4, PLL/DNA/P (His-co-DMAEL)-8 mixture are significantly increased than the PLL/DNA stable composite.
Claims (6)
1. the thing of the functional oligomerization for a non-viral gene carrier material P (His-co-DMAEL), it is characterized in that: by the allyl group histidine derivative, 2-methacryloyl amido-3-(4-imidazolyl) methyl propionate (MA-His-OMe) and containing lactose monomer 2-oxygen-methyl-acryloyl-oxy oxyethyl group-(2, 3, 4, 6-tetra--oxy-acetyl-β-D-semi-lactosi)-(1-4)-2, 3, by usining water-soluble monothioester, as molecular weight regulator, radical polymerization makes 6-tri--oxy-acetyl-β-D-pyranoglucose glucosides (MAEL), molecular weight is 0.5-20KDa, molecular weight distribution is 1.0-2.0, MA-His-OMe and MAEL mol ratio are 3-15:1,
The preparation method of the described thing of the functional oligomerization for non-viral gene carrier material, take MA-His-OMe and MAEL as being total to monomer, with 2,2'-azo two (2-methyl propionitrile) or 4,4'-azo (4-cyanopentanoic acid) is initiator, take 4-cyanopentanoic acid-dithio phenyl ester as the chain-transfer agent preparation, and step is as follows:
1) take Histidine methyl esters and allyl group benzo triazole as synthetic 2-methacryloyl amido-3-(4-imidazolyl) methyl propionate (MA-His-OMe) of raw material:
The 4.8g benzotriazole is dissolved in the 25ml methylene dichloride, add the 1.2g thionyl chloride, after stirring 30min, add the 0.86g methacrylic acid, continue reaction 2h, the sodium hydroxide solution extraction that organic phase is 2 mol/L by concentration three times, organic phase dried over sodium sulfate 12h, revolve to steam and remove methylene dichloride, product flash chromatography column purification, obtain allyl group benzo triazole, 1.87g allyl group benzo triazole is dissolved in the 50ml dioxane, 1.69g Histidine methyl esters is dissolved in the 10ml deionized water, under the magnetic agitation condition, regulating its pH value is 8.0, then will be dissolved in allyl group benzo triazole in dioxane slowly drops in above-mentioned Histidine methyl ester solution and is reacted, and by thin-layer chromatography monitoring reaction process, treat that thin-layer chromatography shows that allyl group benzo triazole runs out of full-time, stop stirring, the dilute hydrochloric acid regulator solution pH value that is 10% with mass percent concentration is to neutral, revolve to steam and remove dioxane, remove by filter insolubles and revolve to steam and remove water, product is dissolved in dehydrated alcohol and filters after lyophilize, again filtrate is spin-dried for and vacuum-drying, obtain MA-His-OMe,
2) 2-oxygen-methyl-acryloyl-oxy oxyethyl group-(2,3,4,6-, tetra--oxy-acetyl-β-D-semi-lactosi)-(1-4)-2,3, the preparation of 6-tri--oxy-acetyl-β-D-pyranoglucose glucosides (MAEL):
20g lactose, 10g sodium acetate, anhydrous are added in the 120ml acetic anhydride, mixed being incorporated under reflux temperature reacted 2h, and reaction is poured product in frozen water into and stirred after finishing, and filters and wash with water solid three times, be placed in air drying, then with ethyl alcohol recrystallization, obtain the octoacetyl lactose; In the 20ml methylene dichloride, add 5g octoacetyl lactose and 4.8ml hydroxyethyl methylacrylate and 2mL Eorontrifluoride etherate, react 2h in ice-water bath, then room temperature reaction 12h, add the 20ml deionized water after the reaction end, and after separation, water, saturated sodium bicarbonate solution and water washing methylene dichloride are extremely neutral mutually successively, then use dried over sodium sulfate, revolve to steam and remove methylene dichloride, separate by chromatographic column, obtain MAEL;
3) for the preparation of the functional oligomerization thing of non-viral gene carrier material:
0.96g MA-His-OMe, 0.5g MAEL, 0.0075g4, 4'-azo (4-cyanopentanoic acid) and 0.023g4-cyanopentanoic acid-dithio phenyl ester are dissolved in 10ml N, in the N'-dimethyl formamide, freezing deoxygenation seals afterwards for three times and is placed in 70 ℃ of water-baths reactions 48 hours, after reaction finishes, the dialysis tubing that is first 3500 with molecular weight cut-off in deionized water, dialyse 48h lyophilize, then product is dissolved in dehydrated alcohol, add the methanol solution that the 1ml mass percent concentration is 30% sodium methylate and react 2 hours, the dialysis tubing that is 3500 with molecular weight cut-off again is in deionized water dialysis 48 hours lyophilize, obtain functional oligomerization thing P (His-co-DMAEL).
2. one kind as claimed in claim 1 for the application of the functional oligomerization thing of non-viral gene carrier material, it is characterized in that: this functional oligomerization thing and polycation material mixing are prepared to DNA or RNA mixture, as non-viral gene carrier material, and carry out the cell transfecting test.
3. according to claim 2 for the application of the functional oligomerization thing of non-viral gene carrier material, it is characterized in that: described polycation material comprises chitosan (being designated hereinafter simply as CS), polylysine (PLL), polymine (PEI) and polymethyl acyl-oxygen ethyl-trimethyl salmiac (PDMMC).
4. according to claim 2 for the application of the functional oligomerization thing of non-viral gene carrier material, it is characterized in that: the preparation method of described DNA or RNA mixture is, first the functional oligomerization thing of 0.1-2.0 μ g/ μ l obtained to mixed solution in 15 minutes with under DNA or RNA room temperature, mixing and place; And then the polycation material solution of 0.1-2.0 μ g/ μ l is added dropwise in above-mentioned mixed solution, vibration is stirred and solution was mixed in 10 seconds and place 15 minutes, can make DNA or the RNA mixture of N/P=0.25:1-40:1 ratio.
5. according to claim 2 for the application of the functional oligomerization thing of non-viral gene carrier material, it is characterized in that: the mass ratio of described functional oligomerization thing and DNA or RNA is 0-200:1, and described functional oligomerization thing is not 0.
6. according to claim 2 for the application of the functional oligomerization thing of non-viral gene carrier material, it is characterized in that: the mass ratio of described functional oligomerization thing and polycation material is 0-40:1, and described functional oligomerization thing is not 0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104401623A CN102532411B (en) | 2011-12-26 | 2011-12-26 | Functional oligomer used for non-viral gene vector material and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104401623A CN102532411B (en) | 2011-12-26 | 2011-12-26 | Functional oligomer used for non-viral gene vector material and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102532411A CN102532411A (en) | 2012-07-04 |
| CN102532411B true CN102532411B (en) | 2013-12-04 |
Family
ID=46340555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011104401623A Expired - Fee Related CN102532411B (en) | 2011-12-26 | 2011-12-26 | Functional oligomer used for non-viral gene vector material and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102532411B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8703197B2 (en) * | 2012-09-13 | 2014-04-22 | International Business Machines Corporation | Branched polyamines for delivery of biologically active materials |
| CN102943086B (en) * | 2012-10-22 | 2014-10-22 | 山东大学 | Application of cationic imidazole Gemini surfactant [Cn-s-Cnim]Br2 in gene transfection |
| CN114874146B (en) * | 2022-03-25 | 2023-09-12 | 圣诺生物医药技术(苏州)有限公司 | Histidine lipid, histidine lipid nanoparticle, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845451A (en) * | 2010-04-30 | 2010-09-29 | 天津大学 | PEI (Polyetherimide)-chitosan triply compound gene vector with low molecular weight and preparation method and application thereof |
| CN102140171A (en) * | 2010-12-22 | 2011-08-03 | 南开大学 | Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof |
-
2011
- 2011-12-26 CN CN2011104401623A patent/CN102532411B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845451A (en) * | 2010-04-30 | 2010-09-29 | 天津大学 | PEI (Polyetherimide)-chitosan triply compound gene vector with low molecular weight and preparation method and application thereof |
| CN102140171A (en) * | 2010-12-22 | 2011-08-03 | 南开大学 | Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102532411A (en) | 2012-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yi et al. | Nanoscale zeolitic imidazolate framework-8 for ratiometric fluorescence imaging of microRNA in living cells | |
| Shandilya et al. | Small interfering RNA in milk exosomes is resistant to digestion and crosses the intestinal barrier in vitro | |
| Yang et al. | Theranostic niosomes for efficient siRNA/MicroRNA delivery and activatable near-infrared fluorescent tracking of stem cells | |
| CN105503833B (en) | Indolepiperazine-dione, its preparation and therapeutic effect | |
| CN102145173B (en) | Human serum albumin complex hydrophobically modified pullulan nanoparticles and preparation method thereof | |
| CN102241790B (en) | Amphipathic chitosan derivative and preparation method and application thereof | |
| Zhang et al. | CRISPR/Cas9 Delivery Mediated with Hydroxyl‐Rich Nanosystems for Gene Editing in Aorta | |
| Wu et al. | A functional DNA nanosensor for highly sensitive and selective imaging of ClO− in atherosclerotic plaques | |
| Guo et al. | NIR-responsive spatiotemporally controlled cyanobacteria micro-nanodevice for intensity-modulated chemotherapeutics in rheumatoid arthritis | |
| CN110746599A (en) | UV light-responsive hyperbranched poly-β-amino ester with high-efficiency gene delivery ability, and preparation method and application thereof | |
| Su et al. | Metal–Organic Framework-Loaded Engineering DNAzyme for the Self-Powered Amplified Detection of MicroRNA | |
| Zhao et al. | Synthesis of amphiphilic poly (β-amino ester) for efficiently minicircle DNA delivery in vivo | |
| CN106834355B (en) | A kind of cationic composite lipid nanodisk and its preparation method and use | |
| CN102532411B (en) | Functional oligomer used for non-viral gene vector material and application thereof | |
| CN102140171B (en) | Glutathione-modified chitosan copolymer serving as non-viral gene carrier material and preparation and application thereof | |
| CN111116904A (en) | Phenylboronic acid modified fluorine-containing high polymer material and application thereof in intracellular delivery of protein | |
| CN104910252A (en) | PH response type lipid based on dendrimers as well as preparation method and application of pH response type lipid | |
| Liu et al. | Fullerene-based nanocomplex assists pulmonary delivery of siRNA for treating metastatic lung cancer | |
| Ding et al. | A cyclodextrin‐based ph‐responsive microRNA delivery platform targeting polarization of M1 to M2 macrophages for sepsis therapy | |
| Tang et al. | Soybean Oil‐Derived Lipids for Efficient mRNA Delivery | |
| CN116803995A (en) | Amino amide lipoid compound and preparation method and application thereof | |
| CN103721269B (en) | Nm of gold genophore of liposome protection and preparation method thereof | |
| CN102731775A (en) | Poly spermine cations, construction method thereof, and preparation method of nano-grade particles | |
| CN106750273B (en) | A kind of block polymer tumor radiotherapy sensitive-increasing agent and preparation method thereof | |
| CN118027409A (en) | A polymer and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131204 |