CN114478301B - Gene delivery vector, preparation method and application thereof - Google Patents
Gene delivery vector, preparation method and application thereof Download PDFInfo
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- CN114478301B CN114478301B CN202210243938.0A CN202210243938A CN114478301B CN 114478301 B CN114478301 B CN 114478301B CN 202210243938 A CN202210243938 A CN 202210243938A CN 114478301 B CN114478301 B CN 114478301B
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- 238000001476 gene delivery Methods 0.000 title claims abstract description 36
- 239000013598 vector Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 238000001415 gene therapy Methods 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims description 79
- 229920002873 Polyethylenimine Polymers 0.000 claims description 58
- 239000002253 acid Substances 0.000 claims description 28
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 11
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims description 10
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 8
- 239000003054 catalyst Substances 0.000 claims description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 8
- 235000011090 malic acid Nutrition 0.000 claims description 8
- 239000013067 intermediate product Substances 0.000 claims description 6
- 239000001630 malic acid Substances 0.000 claims description 6
- NPXOKRUENSOPAO-UHFFFAOYSA-N Raney nickel Chemical group [Al].[Ni] NPXOKRUENSOPAO-UHFFFAOYSA-N 0.000 claims description 5
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 5
- 238000009833 condensation Methods 0.000 claims description 5
- 230000005494 condensation Effects 0.000 claims description 5
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- 239000003814 drug Substances 0.000 claims description 5
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- 238000004519 manufacturing process Methods 0.000 claims description 5
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- 125000003277 amino group Chemical group 0.000 claims description 4
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- 229910000564 Raney nickel Inorganic materials 0.000 claims description 3
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- 239000002994 raw material Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
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- 231100000135 cytotoxicity Toxicity 0.000 abstract description 8
- 230000003013 cytotoxicity Effects 0.000 abstract description 8
- 238000003153 stable transfection Methods 0.000 abstract description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 33
- 238000005481 NMR spectroscopy Methods 0.000 description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- 239000012074 organic phase Substances 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
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- 231100000419 toxicity Toxicity 0.000 description 8
- 230000001988 toxicity Effects 0.000 description 8
- 239000013603 viral vector Substances 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- PBMIETCUUSQZCG-UHFFFAOYSA-N n'-cyclohexylmethanediimine Chemical compound N=C=NC1CCCCC1 PBMIETCUUSQZCG-UHFFFAOYSA-N 0.000 description 7
- 239000000741 silica gel Substances 0.000 description 7
- 229910002027 silica gel Inorganic materials 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 230000008020 evaporation Effects 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 238000002390 rotary evaporation Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 229940099690 malic acid Drugs 0.000 description 4
- KJIFKLIQANRMOU-UHFFFAOYSA-N oxidanium;4-methylbenzenesulfonate Chemical compound O.CC1=CC=C(S(O)(=O)=O)C=C1 KJIFKLIQANRMOU-UHFFFAOYSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- MHYGQXWCZAYSLJ-UHFFFAOYSA-N tert-butyl-chloro-diphenylsilane Chemical compound C=1C=CC=CC=1[Si](Cl)(C(C)(C)C)C1=CC=CC=C1 MHYGQXWCZAYSLJ-UHFFFAOYSA-N 0.000 description 4
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 4
- HEWZVZIVELJPQZ-UHFFFAOYSA-N 2,2-dimethoxypropane Chemical compound COC(C)(C)OC HEWZVZIVELJPQZ-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 238000012637 gene transfection Methods 0.000 description 3
- -1 hexafluorophosphate Chemical compound 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 3
- 230000004962 physiological condition Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 239000007821 HATU Substances 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
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- 229910052739 hydrogen Inorganic materials 0.000 description 2
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- 230000036039 immunity Effects 0.000 description 2
- 239000012948 isocyanate Substances 0.000 description 2
- 150000002513 isocyanates Chemical class 0.000 description 2
- 229940116298 l- malic acid Drugs 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
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- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 2
- OAOSXODRWGDDCV-UHFFFAOYSA-N n,n-dimethylpyridin-4-amine;4-methylbenzenesulfonic acid Chemical compound CN(C)C1=CC=NC=C1.CC1=CC=C(S(O)(=O)=O)C=C1 OAOSXODRWGDDCV-UHFFFAOYSA-N 0.000 description 2
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 238000006845 Michael addition reaction Methods 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- OOTFVKOQINZBBF-UHFFFAOYSA-N cystamine Chemical compound CCSSCCN OOTFVKOQINZBBF-UHFFFAOYSA-N 0.000 description 1
- 229940099500 cystamine Drugs 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
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- 239000007789 gas Chemical group 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
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- 238000001819 mass spectrum Methods 0.000 description 1
- 238000001254 matrix assisted laser desorption--ionisation time-of-flight mass spectrum Methods 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 229920001601 polyetherimide Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- ZDYVRSLAEXCVBX-UHFFFAOYSA-N pyridinium p-toluenesulfonate Chemical compound C1=CC=[NH+]C=C1.CC1=CC=C(S([O-])(=O)=O)C=C1 ZDYVRSLAEXCVBX-UHFFFAOYSA-N 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- FESDUDPSRMWIDL-UHFFFAOYSA-N tert-butyl n,n'-di(propan-2-yl)carbamimidate Chemical compound CC(C)NC(OC(C)(C)C)=NC(C)C FESDUDPSRMWIDL-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C235/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The application discloses a gene delivery vector, a preparation method and application thereof. The structural formula of the gene delivery vector is as follows:wherein n=1 to 50. The gene delivery carrier has the characteristics of single molecular weight, low cost, large-scale repeatability, low cytotoxicity, excellent and stable transfection efficiency and the like, and has wide application prospect in the field of gene therapy.
Description
Technical Field
The application particularly relates to a gene delivery vector, a preparation method and application thereof, and belongs to the technical field of biochemistry.
Background
Gene therapy (Genetherapy) refers to the process of introducing a foreign gene into a specific cell or tissue of a patient and properly expressing the gene, thereby correcting or compensating a disease caused by a gene defect or abnormality, and finally achieving the therapeutic purpose. The research result of gene therapy has been widely used in the treatment of vascular diseases, genetic diseases, tumors and other diseases, and the new medical means is an important component of current biological therapy and becomes a new research focus in the medical field.
Currently, the most commonly used delivery vectors in gene therapy include viral vectors and non-viral vectors. Although the viral vector has better transgenic performance under most conditions, the application of the viral vector in clinical treatment, basic research and other aspects is restricted due to the defects of high immunogenicity, high toxicity, low gene load, poor specificity and targeting, complex preparation process, difficult realization of large-scale production and the like, and particularly the safety problem of the viral vector is more and more emphasized by researchers. Compared with the viral vector, the non-viral vector has the advantages of good biocompatibility, low immunogenicity, large gene load capacity, strong targeting specificity, easiness in preparation and the like, so that the non-viral vector becomes an important component of the current gene therapy transfer vector.
Polyethyleneimine (Poly (ethylene imine), PEI) has been successfully reported by Boussif et al in the nineties of the last century to have the potential to be a gene transfer vector, a material that has been of great interest. Through continuous research, polyethyleneimine has been developed as one of the most superior materials for transfection performance in synthetic polycation type gene therapy vectors.
However, studies have shown that the cytotoxicity and transgenic properties of polyethyleneimine are strongly related to their molecular weight. PEI with high molecular weight has relatively better transfection properties, but has a larger molecular weight, too high a positive charge density and cannot be decomposed by the body, thus exhibiting greater cytotoxicity. PEI with lower molecular weight has a remarkably reduced transfection performance, although it has low cytotoxicity and good biocompatibility. Meanwhile, the PEI carrier with high molecular weight and low molecular weight is polymerized, so that the PEI carrier has molecular weight polydispersity, meanwhile, the preparation process and batch have the problems of instability, incomplete repeatability and the like, and the application of PEI is affected to a certain extent.
Researchers have found that if high molecular weight PEI (HMW-PEI) is modified with PEG, surface charge can be effectively shielded, toxicity can be significantly reduced, and in vivo circulation can be prolonged, but the modification can lose the 'sponge effect' of HMW-PEI and prevent the HMW-PEI from escaping from endosomes, so that transfection efficiency is reduced. There have also been attempts by researchers to graft low molecular weight PEI (LMW-PEI) onto a dextran backbone or a biodegradable polycarbonate backbone using isocyanate or the like as a linking agent to overcome the drawbacks of LMW-PEI. However, isocyanates are relatively toxic and difficult to remove entirely, otherwise additional cytotoxicity can occur. Polycarbonates have acid instability, and in particular degrade faster in the environment surrounding cancer cell tissue, and thus have shorter in vivo circulation times.
And researchers use LMW-PEI to initiate NCA ring opening to obtain PEI-polypeptide graft copolymer, and the polymer has low toxicity and good transfection efficiency, but has low preparation efficiency, has strict requirements on raw material purity and the like, has high cost, and is not beneficial to large-scale production. In addition, researchers also prepare a reduction-sensitive cross-linked PEI vector by Michael addition of Cystamine Bisacrylamide (CBA) and 800 Da PEI, the optimal transfection effect of the vector is similar to that of PEI 25kDa, the toxicity is obviously reduced, but the molecular weight distribution of the product is difficult to control, and the process stability is not high, so that the vector has no practical value.
Disclosure of Invention
The main purpose of the application is to provide a gene delivery vector, a preparation method and application thereof, so as to overcome the defects in the prior art.
In order to achieve the purpose of the application, the technical scheme adopted by the application comprises the following steps:
one aspect of the present application provides a gene delivery vector having the structural formula:
wherein n=1 to 50.
The preparation method of the gene delivery vector provided by the other aspect of the application comprises the following steps: reacting alpha-polymalic acid with polyethyleneimine to graft polyethyleneimine residues onto an alpha-polymalic acid skeleton, thereby preparing the gene delivery vector; wherein the polymerization degree of the alpha-polymalic acid is 1-50, and the number average molecular weight of the polyethyleneimine is 200-5000 Da.
In another aspect, the application provides the use of the gene delivery vector in the manufacture of a medicament for gene therapy.
Another aspect of the present application provides a gene therapy drug comprising: the gene delivery vector; and, an exogenous gene associated with the gene delivery vehicle.
Compared with the prior art, the application has at least the following advantages:
(1) According to the application, the single molecular weight alpha-polymalic acid is used as a skeleton, and the LMW-PEI is modified to obtain the single molecular weight gene delivery vector (defined as MAL-PEI), so that the molecular weight of each batch can be ensured to be consistent, the same quality standard can be achieved, and the problem of unstable transfection efficiency of the polydisperse high molecular vector can be effectively solved.
(2) The application is characterized in that the alpha-polymalic acid is polymerized by ester bonds by taking tricarboxylic acid cycle intermediate product L-malic acid as a monomer, the alpha-polymalic acid is a high molecular polyester, and the alpha-polymalic acid serving as a skeleton of a gene delivery carrier can be finally degraded into water and carbon dioxide which are metabolized out of the body in vivo, and the alpha-polymalic acid has the characteristics of no immunogenicity, high spontaneous degradation rate, high solubility and the like under physiological conditions, is low in toxicity, does not generate immunity, and meanwhile, the malic acid is cheap and easy to obtain, thereby being beneficial to realizing mass production of the alpha-polymalic acid skeleton.
(3) The MAL-PEI provided by the application can achieve stable transfection efficiency, and meanwhile, the cell transfection efficiency can reach or even be better than PEI 25 kDa.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the operation of a gene delivery vehicle according to an embodiment of the present application;
FIG. 2 is a synthetic route diagram of a gene delivery vehicle according to one embodiment of the application;
FIG. 3 shows a compound MA-1 according to an embodiment of the application 1 H NMR chart;
FIG. 4 shows an embodiment of the present application of a compound MA-2 1 H NMR chart;
FIG. 5 shows a compound MA-3 according to an embodiment of the application 1 H NMR chart;
FIG. 6 shows a compound MA-COOH according to one embodiment of the application 1 H NMR chart;
FIG. 7 shows an embodiment of the present application of a compound MA-OH 1 H NMR chart;
FIG. 8 shows a compound oMA according to an embodiment of the application 2 A kind of electronic device 1 H NMR chart;
FIG. 9 shows a compound oMA according to an embodiment of the application 2 -1 1 H NMR chart;
FIG. 10 shows a compound oMA according to an embodiment of the application 2 -2 1 H NMR chart;
FIG. 11 shows a compound oMA according to an embodiment of the application 4 A kind of electronic device 1 H NMR chart;
FIG. 12 shows a compound oMA according to an embodiment of the application 4 -1 1 H NMR chart;
FIG. 13 shows a compound oMA according to an embodiment of the application 4 -2 1 H NMR chart;
FIG. 14 shows a compound oMA according to an embodiment of the application 8 A kind of electronic device 1 H NMR chart;
FIG. 15 is a MALDI-TOF-MS spectrum of MAL-PEI according to one embodiment of the present application;
FIG. 16A shows the toxicity of MAL-PEI to 293T cells according to an embodiment of the present application;
FIG. 16B shows the toxicity of MAL-PEI to SH-SY5Y cells in accordance with one embodiment of the present application;
FIG. 16C shows the toxicity of MAL-PEI to HeLa cells in one embodiment of the present application;
FIG. 17 is a fluorescence micrograph of an embodiment of the application after transfection of SH-SY5Y cells with MAL-PEI;
FIG. 18 is a graph showing the detection of acid degradation of MAL-PEI in an embodiment of the present application.
Detailed Description
In view of the shortcomings of the prior art, the inventor of the present application has long studied and practiced to propose the technical solution of the present application, as will be described in more detail below.
The structural formula of the gene delivery vector (defined as MAL-PEI) provided by the application is as follows:
wherein n=1 to 50.
In one embodiment, the gene delivery vector is of the formula:
the application provides application of the gene delivery vector in preparing a gene therapy drug.
The application provides a gene therapy drug, which comprises: the gene delivery vector; and, an exogenous gene associated with the gene delivery vehicle.
The MAL-PEI provided by the application is taken as a single molecular weight gene carrier based on an alpha-polymalic acid skeleton, is grafted with modified PEI, has a series of hydrogen bond effects, can be combined with plasmid DNA through electrostatic interaction under normal physiological conditions, effectively compresses the DNA to form composite nano particles, and simultaneously has good ion sponge effect, so that the composite nano particles have good endosome escape, and thus, good transfection efficiency is achieved. Because of the main chain polymalic acid structure, the MAL-PEI carrier has good acid degradability, so the cytotoxicity is obviously reduced. The principle of operation of this MAL-PEI as a gene delivery vehicle can be seen in FIG. 1.
The preparation method of the gene delivery vector provided by the application comprises the following steps: reacting alpha-polymalic acid with polyethyleneimine to graft low molecular weight polyethyleneimine residues onto the alpha-polymalic acid backbone to produce the gene delivery vehicle; wherein the polymerization degree of the alpha-polymalic acid is 1-50, preferably 8-32, and the number average molecular weight of the polyethyleneimine is 200-5000 Da.
In one embodiment, the preparation method specifically includes: the gene delivery vehicle is prepared by reacting alpha-polymalic acid with acrylonitrile-modified polyethyleneimine in the presence of a condensation catalyst to obtain an intermediate product, and then reducing cyano groups in the intermediate product to amino groups.
In one embodiment, the condensation catalyst includes 2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate, and is not limited thereto.
In one embodiment, the preparation method specifically includes: the cyano groups in the intermediate product are reduced to amino groups using a hydrogenation catalyst. Wherein the hydrogenation catalyst includes Raney nickel, and is not limited thereto.
In one embodiment, the preparation method specifically comprises the following steps:
(1) Protecting one hydroxyl group in malic acid with benzyl to obtain a compound MA-3;
(2) Protecting one hydroxyl group of one part of the compound MA-3 to obtain a compound MA-COOH, and protecting the carboxyl group of the other part of the compound MA-3 to obtain a compound MA-OH;
(3) Condensation reaction of compound MA-COOH with compound MA-OH to obtain compound oMA 2 ；
(4) For a part of the compound oMA 2 Deprotection of the hydroxyl groups in (a) to give compound oMA 2 -1 and for another part of the compounds oMA 2 Deprotection of one of the carboxyl groups to give compound oMA 2 -2，
(5) Allowing compound oMA 2 -1 and Compound oMA 2 -2 to obtain compound oMA 4 ；
(6) In the form of compound oMA 4 Repeating the operations of the steps (1) - (5) for more than one time by using an iterative exponential synthesis method as a raw material to finally prepare the alpha-polymalic acid;
wherein the structural formula of the compound MA-3 isThe structural formula of the compound MA-COOH is +.>The structural formula of the compound MA-OH is +.>Compound oMA 2 Is of the formula->Compound oMA 2 -1 has the formula->Compound oMA 2 -2 has the formula->Compound oMA 4 Is of the formula->
In one embodiment, step (6) comprises: in the form of compound oMA 4 Repeating the steps (1) - (5) once by iterative exponential synthesis method to obtain compound oMA 8 The compound oMA 8 Is of the structure ofThereafter subjecting the compound oMA 8 The benzyl is removed to obtain the alpha-polymalic acid, and the structural formula of the alpha-polymalic acid is as follows:
in one example, the synthetic route of the gene delivery vehicle can be seen in FIG. 2, i.e., starting from natural Malic Acid (MA), using 2, 2-dimethoxypropane to protect the hydroxyl group to give compound MA-1, which can ensure efficient production of another benzyl (Bn) -protected compound MA-2, followed by deprotection with acetic acid, and quantitative production of compound MA-3. The compound MA-3 was then divided equally into two parts, one part protected with tert-butyl-diphenylchlorosilane (TBDPSCl) to give MA-COOH with exposed carboxyl end and one part with 2-tert-ButyleneButyl-1, 3-diisopropylisourea is used for blocking carboxyl end to obtain MA-OH with exposed hydroxyl, and then the two parts are condensed by an N, N-cyclohexyl carbodiimide (DCC) system and an N, N-dimethylaminopyridine p-toluenesulfonate (DTPS) system to generate a compound (oMA) 2 Alpha-polymapple dimer) followed by compound oMA 2 Dividing two parts, and removing tert-butyl-diphenyl chlorosilane-based protecting group (TBDPS) by tetrabutylammonium fluoride to obtain compound oMA 2 -1. By using a twice iterative exponential synthesis method, one part of silica gel is used for removing tert-butyl protecting group (t-Bu) to obtain a compound oMA 2 -2, condensing the two moieties by N, N-cyclohexylcarbodiimide (DCC) and N, N-dimethylaminopyridine p-toluenesulfonate (DTPS) system to give compound oMA 4 (alpha-apple tetramer). The compound oMA is obtained by repeating the iterative exponential synthesis process once 8 (alpha-apple octamer). Compound oMA 8 Removal of Bn protection by palladium Hydrocarbon followed by in situ and acrylonitrile modified PEI (Compound PEI-CN) by condensation of 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) gives Compound oMA with side chain modified PEI-CN 8 -CONHPEI-CN followed by reduction of cyano to amino group by Raney Ni hydrogenation to finally obtain MAL-PEI.
In the above examples of the present application, α -polymalic acid is a polymer polyester polymerized by ester bonds using a tricarboxylic acid cycle intermediate L-malic acid as a monomer, and is finally degraded in vivo into water and carbon dioxide which are metabolized out of the body, and has the characteristics of no immunogenicity, high spontaneous degradation rate, high solubility, etc. under physiological conditions, and is low in toxicity and does not generate immunity.
In the above embodiment of the application, MAL-PEI can be stably produced in each batch, the molecular weight can be ensured to be consistent, the quality control is stable, and the problems of unstable transfection efficiency and the like caused by molecular weight distribution can be solved.
The following more detailed description of the technical solution of the present application is given with reference to the accompanying drawings and examples, but it should be understood that the following examples are only for the purpose of explaining and illustrating the synthetic method, but not limiting the scope of the present application. Also, unless otherwise specified, various materials, reaction equipment, detection equipment, methods, and the like used in the following examples are known in the art.
The preparation method of the gene delivery vector provided by the embodiment comprises the following steps:
1) The synthesis route of MAL-PEI is as follows:
MA-1: into a 500mL eggplant-shaped bottle was charged 25g of malic acid, 4.215g of pyridinium p-toluenesulfonate (0.09 eq), followed by dropwise addition of 100mL of 2, 2-dimethoxypropane (4V) and stirring overnight at room temperature. Removing solvent in the reaction system by rotary evaporation, uniformly mixing the remainder and silica gel powder (200-300 meshes) according to the proportion of 2:3, loading the mixture into a dry method, and performing column chromatography to obtain the product MA-1 by PE:EA=20:1-3:2. 1 H NMR (300 mhz, dmso) δ12.59 (s, 1H), 4.79 (t, j=4.8 hz, 1H), 2.87-2.62 (m, 2H), 1.53 (d, j=4.3 hz, 6H) (see fig. 3).
MA-2: into a 500mL eggplant-shaped bottle was charged 30.77g MA-1, dissolved with 300mL acetone, followed by sequentially adding 31.38mL triethylamine (1.2 eq), 27.38mL benzyl bromide (1.23 eq), and refluxed at 50℃for 24 hours. Removing solvent in the reaction system by rotary evaporation, uniformly mixing the remainder and silica gel powder (200-300 meshes) according to the proportion of 2:3, loading the mixture into a dry method, and performing column chromatography to obtain the product MA-2 by PE:EA=100:0-10:1. 1 H NMR(300MHz，CDCl 3 ) Delta 7.35 (d, j=2.7 hz,5 h), 5.16 (t, j=6.8 hz,2 h), 4.74 (dd, j=6.4, 3.9hz,1 h), 2.92 (ddd, j=23.5, 17.0,5.2hz,2 h), 1.57 (d, j=5.7 hz,6 h) (see fig. 4).
MA-3: into a 500mL eggplant-shaped bottle were charged 27.24g MA-2, 157.76mL acetic acid, 78.88mL water, and stirred at 60℃for 2h. The solvent was removed from the reaction by rotary evaporation, the residue was dried by rotary drying with 200mL of dichloromethane and then dissolved in 300mL of ethyl acetate, and the organic phase was washed with 200mL of saturated brine, 200mL of 2 and 200mL of water, 1. The organic phase was collected, dried over anhydrous sodium sulfate, evaporated to dryness and the residue was dried in an oven to give the product MA-3. 1 H NMR(300MHz，CDCl 3 ) Delta 7.36 (t, j=2.8 hz, 4H), 5.28-5.09 (m, 2H), 4.58 (dd, j=6.3, 4.6hz, 1H), 2.95 (qd, j=17.1, 5.4hz, 2H) (see fig. 5).
MA-COOH: into a 250mL eggplant-shaped bottle were added 12.04g MA-3 and 5.48g imidazole (1.5 eq) and dissolved with 70mL DMF followed by dropwise addition of 14.75g TBDPSCl (dissolved with 70mL DMF, 1 eq) and stirring overnight at 45 ℃. To the reaction solution, 200mL of ethyl acetate a was added, and the mixture was washed with 200mL of saturated aqueous ammonium chloride solution and 200mL of saturated brine. The organic phase was collected and dried over anhydrous sodium sulfate, and the residue after evaporation was subjected to column chromatography, PE: ea=50:1-4:1 to give the product MA-COOH. 1 H NMR(300MHz，CDCl 3 ) Delta 7.71-7.54 (m, 4H), 7.51-7.27 (m, 11H), 5.15-4.97 (m, 3H), 4.49 (dd, j=5.4, 4.0hz, 1H), 2.83 (dd, j=16.4, 4.0hz, 1H), 2.56 (dd, j=16.4, 5.4hz, 1H), 1.08 (d, j=13.0 hz, 9H) (fig. 6).
MA-OH: into a 500mL eggplant-shaped bottle were charged 11g MA-3, 39.27g 2-tert-butyl-1, 3-diisopropylisourea (4 eq) and 200mL dichloromethane, and stirred overnight at room temperature. The solids were removed by suction filtration and the filter cake was washed with 200mL of dichloromethane, the organic phase was collected and the residue was subjected to column chromatography, PE: ea=100:1-10:1 to give the product MA-OH. 1 HNMR(300MHz，CDCl 3 ) Delta 7.35 (s, 5H), 5.15 (s, 2H), 4.39 (dd, j=6.0, 4.7hz, 1H), 2.82 (qd, j=16.2, 5.4hz, 2H), 1.46 (s, 9H) (see fig. 7).
oMA 2 : into a 250mL eggplant-shaped bottle was added 7.02g MA-OH (1 eq), 11.58g MA-COOH (1 eq), 3.68g DPTS (0.5 eq), 120mL DCM was added for dissolution, and 7.24g DCC (30 mL DCM for dissolution, 1.5 eq) was added dropwise and stirred overnight at room temperature. The solids were removed by suction filtration and the filter cake was washed with 100mL of dichloromethane, the organic phase was collected and the residue was subjected to column chromatography, PE: ea=100:1-8:1 to give product oMA. 1H NMR (300 MHz, CDCl 3) delta 7.81-7.54 (m, 4H), 7.44-7.28 (m, 16H), 5.21 (t, J=6.0 Hz, 1H)) 5.13-4.93 (m, 4H), 4.68 (dd, j=6.4, 5.2hz, 1H), 2.82 (dd, j=5.9, 1.8hz, 2H), 2.65 (dd, j=6.0, 3.8hz, 2H), 1.37 (s, 9H), 1.04 (s, 9H) (see fig. 8).
oMA 2 -1: into a 25mL eggplant-shaped bottle was added 0.68g oMA 2 62uL of acetic acid (1.1 eq), 1.02mL of TBAF and 15mL of THF were added in this order, and the mixture was stirred at room temperature for 1h. The solvent was removed from the reaction by rotary evaporation, 20mL of ethyl acetate was added to the residue, and the organic phase was washed with 20mL of water 1 and 20mL of saturated brine. The organic phase is collected and dried over anhydrous sodium sulfate, and then column chromatography is carried out after evaporation, PE: EA=50:1-7:5, thus obtaining the product oMA 2 -1。 1 H NMR(300MHz，CDCl 3 ) Delta 7.34 (t, j=3.8 hz, 10H), 5.45 (dd, j=7.0, 5.2hz, 1H), 5.23-5.06 (m, 4H), 4.58 (dd, j=7.7, 3.9hz, 1H), 3.02-2.70 (m, 4H), 1.43 (s, 9H) (see fig. 9).
oMA 2 -2: into a 25mL eggplant-shaped bottle was added 0.5g oMA 2 0.5g of silica gel powder (1 eq), 0.025g of p-toluenesulfonic acid monohydrate (0.2 eq) and 10mL of toluene were added in this order and stirred at 110℃for 20min. Removing silica gel powder by suction filtration, flushing filter cake with 10mL of ethyl acetate, collecting organic phase, evaporating the residue, and performing column chromatography to obtain a product oMA by PE:EA=50:1-5:1 2 -2。 1 H NMR(300MHz，CDCl 3 ) Delta 7.80-7.53 (m, 4H), 7.53-7.29 (m, 16H), 5.46 (s, 1H), 5.20-4.94 (m, 4H), 4.58 (s, 1H), 3.02-2.62 (m, 4H), 1.04 (s, 9H) (see FIG. 10).
oMA 4 : into a 25mL eggplant-shaped bottle was added 3.135g oMA 2 -1(1eq)，4.18g oMA 2 -2 (1 eq), 0.978g dpts (0.5 eq), 60mL DCM was dissolved, followed by 1.92g DCC (20 mL DCM dissolved, 1.5 eq) and stirred overnight at room temperature. The solids were removed by suction filtration and the filter cake was washed with 30mL of dichloromethane, the residue was collected after evaporation of the organic phase and subjected to column chromatography with PE: ea=60:1-1:1 to give the product oMA 4 。 1 H NMR(300MHz，CDCl 3 )δ7.64(ddd，J＝16.0，7.8，1.6Hz，4H)，7.49-7.27(m，26H)，5.55(dd，J＝8.6，4.0Hz，1H)，5.46-5.29(m，2H)，5.23-4.88(m，8H)，4.66(t，J＝5.7Hz，1H)，3.13-2.55(m，8H)，1.39(s，9H) 1.03 (s, 9H) (see fig. 11).
oMA 4 -1: 100mg oMA was added to a 25mL eggplant-shaped bottle 4 11uL of acetic acid (2.2 eq), 0.192mL of TBAF and 3mL of THF were added in this order at 0deg.C, and the mixture was stirred overnight at 10deg.C. The solvent was removed from the reaction by rotary evaporation, 5mL of ethyl acetate was added to the residue, and the organic phase was washed with 5mL of water 5mL of x 2 and 5mL of saturated brine 1. The organic phase is collected and dried over anhydrous sodium sulfate, and after evaporation, the column chromatography is carried out, PE: EA=30:1-1:1 to obtain the product oMA 4 -1。 1 H NMR(300MHz，CDCl 3 ) Delta 7.43-7.29 (m, 20H), 5.59 (td, j=8.6, 3.8hz, 2H), 5.38 (t, j=5.9 hz, 1H), 5.26-5.04 (m, 8H), 4.54 (dd, j=7.6, 3.9hz, 1H), 3.12-2.67 (m, 8H), 1.40 (s, 9H). (see FIG. 12).
oMA 4 -2: 170mg oMA was added to a 25mL eggplant-shaped bottle 4 170mg of silica gel powder (1 eq), 0.008g of p-toluenesulfonic acid monohydrate (0.3 eq) and 6mL of toluene were added in this order and stirred at 110℃for 20min. Removing silica gel powder by suction filtration, flushing filter cake with 5mL of ethyl acetate, collecting organic phase, evaporating the residue, and performing column chromatography to obtain a product oMA by PE:EA=50:1-1:1 4 -2。 1 H NMR(300MHz，CDCl 3 ) Delta 7.74-7.55 (m, 4H), 7.47-7.27 (m, 26H), 5.52 (dt, j=11.8, 5.4hz, 2H), 5.38 (t, j=6.1 hz, 1H), 5.19-4.89 (m, 8H), 4.66 (t, j=5.7 hz, 1H), 3.07-2.63 (m, 8H), 1.03 (s, 9H) (see fig. 13).
oMA 8 : 1g oMA was added to a 25mL eggplant-shaped bottle 4 -1(1eq)，1.2g oMA 4 2 (1 eq) and 0.163g DPTS, 30mL DCM was added followed by 0.32g DCC (10 mL DCM dissolved, 1.5 eq) and stirred overnight at room temperature. The solids were removed by suction filtration and the filter cake was washed with 5mL of dichloromethane, the residue was collected after evaporation of the organic phase and subjected to column chromatography with PE: ea=50:1-1:1 to give the product oMA 8 。 1 H NMR(300MHz，CDCl 3 )δ7.77-7.54(m，4H)，7.42-7.27(m，46H)，5.62-548 (m, 4H), 5.38 (dt, j=12.3, 5.0hz, 3H), 5.21-4.86 (m, 16H), 4.66 (t, j=5.7 hz, 1H), 3.11-2.58 (m, 16H), 1.39 (s, 9H), 1.03 (s, 9H) (see fig. 14).
oMA 8 -COOH: 1.5g oMA was added to a 100mL eggplant-shaped bottle 8 (1 eq) was dissolved in 30mL of ethanol, followed by addition of 0.3g of 10% palladium on carbon and gas substitution by bubbling through a hydrogen bag, and stirring was carried out at room temperature overnight. Removing solid by suction filtration, flushing filter cake with 50mL of ethanol, collecting organic phase and evaporating to dryness to obtain a product oMA 8 -COOH。
NHPEI-CN: 1.24g oMA was added to a 50mL eggplant-shaped bottle 8 -COOH (1 eq), dissolved in 30mL of DMF at 25℃followed by 3.80g of HATU (10 eq) and 1.93g of DIPEA (15 eq) were stirred at room temperature for 30min, followed by 4.54g of PEI-CN (10 eq) and stirred at room temperature for 3H. To the reaction solution was added 50mL of ethyl acetate, and the mixture was washed with 50mL of saturated aqueous ammonium chloride solution and 50mL of saturated brine. The organic phase is collected and dried over anhydrous sodium sulfate, and the residue after evaporation is subjected to column chromatography, PE: EA=50:1-1:5 to give the product oMA 8 -CONHPEI-CN。
MAL-PEI: 1.0g oMA was added to a 50mL eggplant-shaped bottle 8 CONHPEI-CN (1 eq) was dissolved in 30mL of methanol, then 2.0mL of Raney nickel (Raney Ni) aqueous ammonia (1.0 g) was added thereto, and the reaction system was stirred at room temperature overnight while being gas-replaced by introducing a hydrogen gas bag. The solids were removed by suction filtration and the filter cake was rinsed with 50mL of anhydrous methanol, the organic phase was collected, dried over anhydrous sodium sulfate, again suction filtered and evaporated to dryness to give the product MAL-PEI. Macromolecular mass spectra are characterized as single molecular weight macromolecules, andnot polydisperse polymers (see figure 15).
Finally, 1.0g MAL-PEI (1 eq) was added to a 50mL eggplant-shaped bottle, 10mL 10% trifluoroacetic acid/dichloromethane solution was added, and the mixture was stirred at room temperature for 3-5min. The solvent in the reaction system is removed by rotary evaporation, and the residue is dialyzed to obtain MAL-PEI.
2) MAL-PEI cytotoxicity test (MTT method)
Cytotoxicity experiments (MTT method) the biocompatibility of the vector MAL-PEI was studied and the toxicity of MAL-PEI on 293T (see FIG. 16A), SH-SY5Y (see FIG. 16B) and HeLa cells (see FIG. 16C) was evaluated with 25kDa PEI as a control. MAL-PEI showed lower cytotoxicity than 25kDa PEI with high molecular weight.
3) MAL-PEI mediated in vitro gene transfection
In the POEI-mediated in vitro gene transfection study, pEGFP-N1 plasmid DNA is used as a reporter gene, and the in vitro gene transfection effect of the cationic polymer MAL-PEI on SH-SY5Y cells under the condition of different mass ratios (w/w=2/1, 4/1 and 8/1) is evaluated, and a complex of 25kDa PEI with N/P=10 (optimal ratio) and pEGFP-N1 plasmid is used as a positive control. And (5) detecting the transfection efficiency of the in-vitro gene by a fluorescence inversion microscope qualitative observation method. Fluorescence microscopy of MAL-PEI transfected with SH-SY5Y cells revealed that MAL-PEI gradually increased the number of cells expressing green yellow protein as the mass ratio of the MAL-PEI to pEGFP was increased, and the fluorescence intensity was also gradually increased, and at w/w=8, 25kDa PEI was higher than the optimal N/P ratio (see FIG. 17).
4) Acid degradation detection of MAL-PEI
MAL-PEI was added to acetic acid-sodium acetate buffer (pH=5.0) and the mixture was incubated at room temperature at a mass concentration of 5mg/mL, and degradation at various time points was detected by GPC and found to be substantially degraded to a small molecule state after 7.5 hours. (see FIG. 18).
The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the application are intended to be included within the scope of the application.
Claims (7)
1. A gene delivery vehicle characterized by the structural formula:
wherein n=1 to 50.
2. The gene delivery vector of claim 1, wherein the gene delivery vector has the structural formula:
3. a method of preparing a gene delivery vehicle comprising: reacting alpha-polymalic acid with acrylonitrile-modified polyethyleneimine in the presence of a condensation catalyst to obtain an intermediate product, and reducing cyano groups in the intermediate product into amino groups by adopting a hydrogenation catalyst to prepare the gene delivery vector; wherein the polymerization degree of the alpha-polymalic acid is 1-50, the number average molecular weight of the polyethyleneimine is 200-5000 Da, the condensation catalyst is 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, and the hydrogenation catalyst is Raney nickel.
4. A method of preparation according to claim 3, comprising the specific steps of:
(1) Protecting one hydroxyl group in malic acid with benzyl to obtain a compound MA-3;
(2) Protecting one hydroxyl group of one part of the compound MA-3 to obtain a compound MA-COOH, and protecting the carboxyl group of the other part of the compound MA-3 to obtain a compound MA-OH;
(3) Condensation reaction of compound MA-COOH with compound MA-OH to obtain compound oMA 2 ；
(4) For a part of the compound oMA 2 Deprotection of the hydroxyl groups in (a) to give compound oMA 2 -1 and for another part of the compounds oMA 2 Deprotection of one of the carboxyl groups to give compound oMA 2 -2，
(5) Allowing compound oMA 2 -1 and Compound oMA 2 -2 to obtain compound oMA 4 ；
(6) In the form of compound oMA 4 Repeating the operations of the steps (1) - (5) for more than one time by using an iterative exponential synthesis method as a raw material to finally prepare the alpha-polymalic acid;
wherein the structural formula of the compound MA-3 isThe structural formula of the compound MA-COOH is +.>The structural formula of the compound MA-OH is +.>Compound oMA 2 Is of the formula->Compound oMA 2 -1 has the formula->Compound oMA 2 -2 has the formula->Compound oMA 4 Is of the formula->
5. The method of claim 4, wherein step (6) comprises: in the form of compound oMA 4 Repeating the steps (1) - (5) once by iterative exponential synthesis method to obtain compound oMA 8 The compound oMA 8 Is of the structure ofThereafter subjecting the compound oMA 8 The benzyl is removed to obtain the alpha-polymalic acid, and the structural formula of the alpha-polymalic acid is as follows:
6. use of the gene delivery vector of any one of claims 1-2 in the manufacture of a gene therapy drug.
7. A gene therapy agent comprising: the gene delivery vector of any one of claims 1-2; and, an exogenous gene associated with the gene delivery vehicle.
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| CN112142972A (en) * | 2020-08-18 | 2020-12-29 | 北京大学 | Modified polyethyleneimine derivative and synthesis method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970591A (en) * | 2006-11-16 | 2007-05-30 | 南京慧基生物技术有限公司 | Biodegradable crosslinked polyethylenimine and its uses |
| CN101461947A (en) * | 2008-10-23 | 2009-06-24 | 刘湖 | Polypeptide tree-shaped macromolecule as gene vector and use thereof |
| CN106866960A (en) * | 2017-02-28 | 2017-06-20 | 广州医科大学 | Non-viral gene transfection carrier material that a kind of cytotoxicity is low, transfection efficiency is high and preparation method and application |
| CN112142972A (en) * | 2020-08-18 | 2020-12-29 | 北京大学 | Modified polyethyleneimine derivative and synthesis method and application thereof |
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