CN102174579A - Reducible and biodegradable comb type high polymer gene vector and preparation method of same - Google Patents
Reducible and biodegradable comb type high polymer gene vector and preparation method of same Download PDFInfo
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Abstract
The invention discloses a reducible and biodegradable comb type high polymer gene vector and a preparation method of the same. The structure of the vector is as shown in the following formula, wherein CP is a cationic polymer with low molecular weight between 600 and 30000; F1 and F2 are groups capable of interaction; F1 or F2 contains a disulfide bond; M is a main chain unit structure of the polymer; n equals to 10-500; m is smaller than n; m equals to 0-400; l equals to 0-800; and l, m and n are integers. In the invention, the comb type high polymer gene vector has small cytotoxicity, gene DNA can be well bound and compounded under physical conditions so as to enter cells; and the carried genes can be released as the reducing environment in the cells can rupture quickly. The result show that the reducible and biodegradable comb type high polymer gene vector has higher transfection efficiency and lower cytotoxicity compared with 25kDa PEI (polyether imide); moreover, the preparation method is simple, and the structure is easy to control.
Description
Technical field
The present invention relates to comb type polymer genophore of a kind of reducible degraded and preparation method thereof, belong to field of gene.
Background technology
Finishing of human genome engineering draft makes people better understand the relation of gene and disease.By the end of 2008, clinical case more than 1400 has been arranged, utilize gene to correct genetic deficiency or treatment acquired disease (http://www.wiley.co.uk/genmed/clinical/).The gene of treatment and preventing disease potentiality has been determined much to have in the genetics field, but that gene transports the progress of system is limited, is the bottleneck of present gene therapy.The carrier that transports gene mainly comprises virus vector and non-virus carrier.Because transfection efficiency height, virus vector at first are applied to the clinical study of gene therapy; Yet,, limited its application and popularization owing to there is the preparation process of potential safety hazard and complex and expensive.Therefore, the non-viral gene vector of research and development highly effective and safe has very important significance.
The cation high molecular polymkeric substance is to study more non-viral gene vector at present, comprise polylysine (polylysine, PLL), polymine (polyethylenimine, PEI), chitosan and derivative thereof, polyamide-amide (polyamidoamine, PAMAM) tree-like polymer and polymethacrylate cationic derivative etc.But traditional polymer genophore is because low relatively transfection efficiency and high toxicity and can not be used for clinically, and for example nondegradable cation high molecular can cause very high toxicity etc. at cell inner accumulation.Polymer genophore transfection efficiency and toxicity generally improve with the increase of molecular weight.For example, be that the DNA mixture of carrier its transfection efficiency of increase along with molecular weight in 0.6-70kDa increases [Godbey W.T., et al. with PEI, J.Biomed.Mater.Res.1999,45,268], the cytotoxicity of PEI also improves along with the increase of molecular weight.The PEI that has research reporter molecule amount to be lower than 1.8kDa does not almost have the transfection effect, and the relatively more suitable PEI molecular weight ranges of making genophore is roughly 11.9-70kDa[Godbey W.T.; Wu K.K.; Mikos A.G.Size matters:molecular weight affects the efficiency of poly (ethylenimine) as a genedelivery vehicle.J.Biomed.Mater.Res.1999,45,268], the most frequently used molecular weight is 25kDa, therefore, in great majority research, all be to select for use the branching PEI of 25KDa as reference standard.
In order to solve polymericular weight to carrier transfection efficiency and the influence of toxic contradiction, Lee seminar has at first reported the cystine linkage of introducing reducible degraded, with active amino cross-linking reaction molecular weight be that the PEI of 0.8kDa makes high-molecular weight PEI derivative [MA.Gosselin, W-J.Guo, R.J.Lee, Efficient genetransfer using reversibly cross-linked low molecular weight polyethylenimine, Bioconjugate Chem.12 (2001) 989-994], this PEI derivative has than high transfection efficiency and very low toxicity.Similarly research is discussed in our last piece of writing patent of invention [Chinese patent application publication No.: CN101812178A], the main drawback of this class PEI derivative is that degree of crosslinking is wayward, the polymer dissolution that obtains is bad, the microgel that contains must be removed with pillar, complex process, low [the R.Deng of productive rate, Y.N Yue, F.Jin, Y.C.Chen, HF Kung, M.C.M.Lin, C.Wu, Revisit the complexation of PEI andDNA-How to make low cytotoxic and highly efficient PEI gene transfectionnon-viral vectors with a controllable chain length and structure? J.Control.Release140 (2009) 40-46.].
The present invention will contain the lower molecular weight polycation that can react end group and prepare the comb type high-molecular cationic polymer that contains cystine linkage by reaction of pendant group, and such preparation method is simple, can not produce crosslinked.The polymkeric substance that makes can be degraded into the polymkeric substance of lower molecular weight fast in reducing environment, have higher gene transfection efficient simultaneously and than low cytotoxicity.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of have higher gene transfection efficient and than low cytotoxicity, simultaneously can be not crosslinked comb type polymer genophore and preparation method thereof.
Polymer genophore provided by the present invention is a kind of comb shape cationic polymers that contains the cystine linkage modification, and structure is shown below:
CP is the low molecular weight cationic polymkeric substance in the formula, molecular weight between 600-30000, F1 and F2 be can interreaction group, and F1 or F2 contain cystine linkage, M is the backbone units structure of polymkeric substance, n=10-500; M＜n, m=0-400, l=0-800; L, m, n are integer.
The n span is 10-500, and the ratio of added monomer and initiator is controlled during mainly by the polymerization of preparation main chain.The l span is 0-800, and the raw material of the composition of added raw material monomer or modified side chain is formed and controlled during mainly by the polymerization of preparation main chain.M＜n, the m span is 0-400, mainly forms by the raw material that adds modified side chain and controls.
The molecular weight ranges of raw material low molecular weight cationic polymkeric substance if it is excellent using PDE, its molecular weight, as is used PEI between 2000-30000 between 600-30000, its molecular weight is excellent between 600-5000.
Scheme can be more specifically:
CP is linear polymethyl acrylic acid-2-N, N-dimethylaminoethyl (L-PDE-N
3) or polymine (PEI).
The backbone units structure of polymkeric substance is poly-asparagus fern amino acid derivative, PMAm or polyacrylamide analog derivative.
The present invention also provides the preparation method of above-mentioned polymer genophore, comprises following steps:
(1) lower molecular weight (low molecular-weight) the cationic polymers CP-F1 of a reaction of anamorphic zone end group F1;
(2) synthetic trunk polymer, its side chain contain can with the group F2 of F1 end group reaction, and F1 or F2 contain cystine linkage, shown in right formula:
(3) with above-mentioned two kinds of mixed with polymers reaction, purifying, make the comb type cationic polymers that contains cystine linkage.
Further scheme is: F1 is for containing azido group, and F2 is for containing cystine linkage and alkynyl group; Or F1 is for containing cystine linkage and alkynyl group, and F2 is for containing azido group.
The synthetic method of the lower molecular weight of functionalization (low molecular-weight) cationic polymers CP-F1 is common radical polymerization or controlled living polymerization method, perhaps the modification by business-like low molecular weight cationic polymkeric substance.
Described trunk polymer, its at least a portion side chain contains the group F2 that can react with the F1 end group, and F1 or F2 contain cystine linkage, synthetic method includes but not limited to common radical polymerization or controlled living polymerization method, perhaps modified side chain by business-like polymkeric substance or synthetic polymer modified side chain again.
In polymer genophore provided by the present invention, the low molecular weight cationic polymkeric substance CP-F1 of band reaction end group F1 is the linear polymethyl acrylic acid-2-N of nitrine end group functional, N-dimethylaminoethyl (poly (2-(dimethylamino) ethyl methacrylate), PDE), trunk polymer is that side chain is when containing the poly-asparagus fern amino acid derivative of cystine linkage and alkynyl group
Concrete preparation method is as follows:
(1) contains the linear polymethyl acrylic acid-2-N of a nitrine end group functional, N-dimethylaminoethyl (L-PDE-N
3) the preparation method: adopt Transfer Radical Polymerization, control initiator and monomeric ratio make the L-PDE-N of molecular weight between 2000-30000
3
(2) side chain contains the preparation method of the poly-asparagus fern amino acid derivative of cystine linkage and alkynyl group: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst); Again with (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst) and polysuccinimide (PSI) 0.2-2 in molar ratio: 1 adds in the solvent, at 20-70 ℃ of following stirring reaction 2-48 hour, synthetic make the poly-asparagus fern amino acid derivative P (Asp-ss-Al) that side chain contains cystine linkage and alkynyl group;
(3) with above-mentioned L-PDE-N
3And P (Asp-ss-Al) is by the mol ratio 0.5-3 of nitrine end group and alkynyl: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, under 20-80 ℃, reacted 1-48 hour, if product also has the structure of not open loop to exist, then add excessive N again, N-dimethylated propyl diethylenetriamine or N, the N-dimethyl-ethylenediamine, perhaps thanomin, Propanolamine, butanolamine or amylalcohol amine, reacted 24 hours, obtain containing the comb type cationic polymers P (Asp-ss-Al-Az-PDE) of cystine linkage, its structural representation can be following formula:
In polymer carrier provided by the present invention, low molecular weight cationic polymkeric substance CP-F1 is polymine (PEI) derivative of molecular weight between 600-5000 of nitrine end group functional, and wherein the structural representation of the polymine of weight-average molecular weight between 600-5000 is:
Trunk polymer is degradable poly-asparagus fern amino acid derivative, and when its side chain contained cystine linkage and alkynyl group, concrete preparation method was as follows:
(1) preparation method of the polymine of nitrine end group functional: the 0.3-1.1 in molar ratio of the polymine PEI between 600-5000 with nitrine propyl ester carbonylic imidazole compound and weight-average molecular weight: 1 is dissolved in the organic solvent, at 25-60 ℃ of following stirring reaction 2-24 hour, removal of solvent under reduced pressure, obtain containing the polymine of the micromolecular nitrine end group functional of imidazoles, the quantity that each polymine wherein contains the nitrine end group is 0.3-1.1;
(2) side chain contains the preparation method of the poly-asparagus fern amino acid derivative of cystine linkage and alkynyl group: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtain (allyl amino manthanoate ethyl) two thioethylamine compounds ([(propargylcarbamate) ethyl]-dithioethylamine, PPA-cyst); Again with (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst) and polysuccinimide (PSI) 0.2-2 in molar ratio: 1 adds in the solvent, at 20-70 ℃ of following stirring reaction 2-48 hour, synthetic make side chain and contain the poly-asparagus fern amino acid derivative P (Asp-ss-Al) of alkynyl group molar percentage at 15%-100%;
(3) above-mentioned P (Asp-ss-Al) is dissolved in organic solvent, if it also has the structure of not open loop to exist, then add excessive N earlier, N-dimethylated propyl diethylenetriamine or N, the N-dimethyl-ethylenediamine, perhaps thanomin, Propanolamine, butanolamine or amylalcohol amine, reacted 24 hours, again with the polymine of the above-mentioned nitrine end group functional mol ratio 0.5-3 by nitrine end group and alkynyl: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, under 20-80 ℃, reacted 1-48 hour, obtain containing the comb type cationic polymers P (Asp-ss-Al-Az-PEI) of cystine linkage, its structural representation can be:
In polymer carrier provided by the present invention, low molecular weight cationic polymkeric substance CP-F1 is the polymethyl acrylic acid-2-N of molecular weight between 2000-30000 that contains a nitrine end group, N-dimethyl amine ethyl ester (L-PDE-N
3); Main chain is a polymer lateral chain when containing the methacryloyl amine of cystine linkage and alkynyl group or acrylamide analog derivative, and concrete preparation method is as follows:
(1) contains the linear polymethyl acrylic acid-2-N of a nitrine end group functional, N-dimethylaminoethyl (L-PDE-N
3) the preparation method: adopt Transfer Radical Polymerization, control initiator and monomeric ratio make the L-PDE-N of molecular weight between 2000-30000
3
(2) side chain contains the preparation method of the PMAm or the polyacrylamide analog derivative of cystine linkage and alkynyl group: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst); Again with (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst) and methacrylic chloride or acrylate chloride reaction purification, add initiator such as Diisopropyl azodicarboxylate AIBN and solvent again, at 50-80 ℃ of following stirring reaction 2-24 hour, or make PMAm analog derivative P (PPA-cyst-Mam) or the polyacrylamide analog derivative P (PPA-cyst-Am) that side chain contains cystine linkage and alkynyl group by the ATRP polymerization is synthetic;
(3) with above-mentioned L-PDE-N
3And P (PPA-cyst-MAm) or P (PPA-cyst-Am) are by the mol ratio 0.5-3 of nitrine end group and alkynyl: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, under 20-80 ℃, reacted 1-48 hour, purifying obtains containing the comb type cationic polymers P (PPA-cyst-MAm-PDE) of cystine linkage, and its structural representation is:
In polymer carrier provided by the present invention, lower molecular weight functionalization cationic polymers CP-F1 is the polyethylenimine derivates of molecular weight between 600-5000 that contains cystine linkage and alkynyl end group; Trunk polymer is degradable poly-asparagus fern amino acid derivative, and its side chain contains azido group.Concrete step is as follows:
(1) preparation method of the polymine PEI-(PPA-cyst) of alkynes end group functional: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst); Again with (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst) and 1,1 '-carbonyl dimidazoles is 0.4-0.75 in molar ratio: 1 adds in the organic solvent, at 20-70 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two sulphur ethyl 1-carboxamide imidazolium compoundss (PPA-cyst-CI); The 0.2-1.1 in molar ratio of the polymine between 600-5000 with Compound P PA-cyst-CI and weight-average molecular weight: 1 is dissolved in the organic solvent, at 20-60 ℃ of following stirring reaction 2-48 hour, removal of solvent under reduced pressure, obtain containing the polymine of the micromolecular alkynes end group functional of imidazoles, the quantity span that each polymine wherein contains the alkynes end group is 0.2-1.1 (it is 1 or 0 that each most polymines contains the alkynes end group, and the mean number value that polymine PEI contains the alkynes end group is 0.2-1.1);
(2) side chain contains the preparation method of the poly-asparagus fern amino acid derivative of azido group: with 2-nitrine ethamine and polysuccinimide (PSI) 0.4-2 in molar ratio: 1 adds in the solvent, at 20-70 ℃ of following stirring reaction 2-200 hour, if product also has the structure of not open loop to exist, then add excessive N again, N-dimethylated propyl diethylenetriamine or N, the N-dimethyl-ethylenediamine, perhaps thanomin, Propanolamine, butanolamine or amylalcohol amine, reacted 24 hours, and synthetic made the poly-asparagus fern amino acid derivative P (Asp-Az) that side chain contains azido group;
(3) with the polymine PEI-(PPA-cyst) of above-mentioned alkynes end group functional and P (Asp-Az) the mol ratio 0.5-3 by alkynyl end group and nitrine: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, under 20-80 ℃, reacted 1-96 hour, obtain containing the responsive sex comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) of reductive modification of cystine linkage, it is one of following that its structural representation can be:
Reduction-sensitive comb type macromolecule cation polymer of the present invention can combine with foreign gene, as genophore.
Described genophore is the carrier that is used for human body or the conduction of animal and plant cells gene, with suitable complex method, can be compounded to form the stabilized complex small-particle with target gene (DNA or RNA).With this mixture and human body or zooblast co-cultivation, target gene (DNA or RNA) can be brought into release in human body or the zooblast, and transfection expression, be used for human body gene treatment and the conduction of vegeto-animal gene.
The reduction-sensitive comb type polymer that contains the cystine linkage modification of the present invention is during as genophore, introduces polyoxyethylene glycol, target group and/or component that can be crosslinked.Introduce polyoxyethylene glycol and can reduce the rejection of living things system (such as human body), reduce the absorption of protein, cell and germ greatly, can also reduce the speed of being removed by kidney (because its volume is bigger) on the gene vector material surface to gene vector material.Can introduce target group such as folic acid and antibody, improve the targeted delivery characteristic of gene vector system, target enters behind the tumour cell because the interior reducing environment of cell can very fast degraded discharge contained genomic medicine.Can introduce on a small quantity can be crosslinked component, after the comb type macromolecule cation polymer of this modification loads genomic medicine, carry out crosslinkedly, can improve the stability of loading the genomic medicine system.
High molecular polymer of the present invention has the degradable characteristic of biological reducing.Bind well and complex gene DNA in the cell external enwergy, enter in the cell and discharge contained gene owing to intracellular reducing environment can comparatively fast fragment into the approximate low-molecular-weight polymkeric substance of raw material, thereby have higher gene transfection efficient simultaneously and, be a kind of very promising polymer genophore than low cytotoxicity.
Preparation method of the present invention is to be main raw material with the low molecular weight cationic polymkeric substance, utilizes the reaction of highly selective such as click chemistry to react, and makes final product.Advantage is that method is simple, can not produce crosslinked.Because used the reaction such as the click chemistry of highly selective, structure control is easy, the positively charged group of loss for reaction seldom or do not have, positively charged group such as amino utilization ratio are very high.
Description of drawings
Fig. 1 for the polymine PEI-(PPA-cyst) (embodiment 4, and each polyethyleneimine: amine molecule on average contains 0.33 ethynylene group) of the single alkynes end group functional of synthetic of the present invention (A) at CDCl
3, (B) P (Asp-Az)
0.65(Asp-EA)
0.35(embodiment 6) are at D
2O and the comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that (C) contains the cystine linkage modification are (Asp-EA)
0.35(embodiment 11) are at D
2The nucleus magnetic hydrogen spectrum figure of O
Fig. 2 contains comb type high-molecular cationic polymer P (PPA-cyst-MAm-PDE)-1 (A) back (B) and L-PDE-N before adding 50mmol/L DTT of cystine linkage modification for synthetic of the present invention
3The GPC color atlas of-2 (C) (embodiment 12)
Fig. 3 is adding DTT front and back (embodiment 12) over time for dynamic light scattering detects comb type high-molecular cationic polymer P (PPA-cyst-MAm-PDE) with DNA composite particles particle diameter
The comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that Fig. 4 contains the cystine linkage modification for synthetic of the present invention (Asp-EA)
0.67, P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.35And P (Asp-Az-Al-ss-PEI) and with reference to polymer P EI 25kDa to the cytotoxicity of 293T relatively (embodiment 13, and data in graph form is the mean value and the standard variance of 4 hole parallel laboratory tests).
Fig. 5 contains cystine linkage modification comb type high molecular polycation P (Asp-ss-Al-Az-PDE)-1 and P (Asp-ss-Al-Az-PDE)-2 (embodiment 8), low-molecular weight polymer L-PDE-N for the present invention is synthetic
3-1, linear high-molecular weight HMPDE and comparison polymer PEI 25kDa compare (embodiment 13, and data in graph form is the mean value and the standard variance of 4 hole parallel laboratory tests) to the cytotoxicity of 293T.
The comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that Fig. 6 contains the cystine linkage modification for the present invention synthesizes (Asp-EA)
0.67, P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.35With P (Asp-Az-Al-ss-PEI) different polymkeric substance/DNA quality than the time to the luciferase transfection expression (embodiment 14, data in graph form be mean value and the standard variance of 3 hole parallel laboratory tests) of 293T cell when serum-free exists.
Fig. 7 contains the comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE)-1 and the P (Asp-ss-Al-Az-PDE)-2 of cystine linkage modification, low-molecular weight polymer L-PDE-N for synthetic of the present invention
3-1 with linear high-molecular weight HMPDE different polymkeric substance/DNA quality than the time to the luciferase transfection expression of 293T cell when serum-free exists (N/P ratio of PEI 25kDa and DNA is 10, embodiment 14, and data in graph form is the mean value and the standard variance of 3 hole parallel laboratory tests).
Fig. 8 contains the comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE)-1 and the P (Asp-ss-Al-Az-PDE)-2 of cystine linkage modification, low-molecular weight polymer L-PDE-N for synthetic of the present invention
3-1 different polymkeric substance/DNA quality than the time to the luciferase transfection expression (N/P ratio of PEI 25kDa and DNA be 10, embodiment 14, data in graph form be mean value and the standard variance of 3 hole parallel laboratory tests) of HeLa cell when serum-free exists.
Fig. 9 contains the comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE)-1 and the P (Asp-ss-Al-Az-PDE)-2 of cystine linkage modification, low-molecular weight polymer L-PDE-N for synthetic of the present invention
3-1 different polymkeric substance/DNA quality than the time to the luciferase transfection expression (N/P ratio of PEI 25kDa and DNA be 10, embodiment 15, data in graph form be mean value and the standard variance of 3 hole parallel laboratory tests) of 293T cell when having serum to exist.
Embodiment
Further explain the present invention by the following examples, but they not the restrictions to claim.
By the structure of nuclear magnetic resonance spectrum test modification comb type macromolecule cation polymer, by the molecular weight of gel permeation chromatography (GPC) test polymer.
Embodiment 1 (allyl amino manthanoate ethyl) two thioethylamines ((propargyl carbamate) ethyldisulfide ethylamine, PPA-cyst) and (allyl amino manthanoate ethyl) two sulphur ethyl 1-carboxamide imidazoles ((propargyl carbamate) ethyl disulfide ethyl 1-carbamide-imidazole, PPA-cyst-CI) synthetic
(allyl amino manthanoate ethyl) two thioethylamines ((propargyl carbamate) ethyl disulfideethylamine, synthesizing PPA-cyst): according to document [X.L.Jiang, M.C.Lok, W.E.Hennink, Degradable-Brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistryfor gene delivery, Bioconjugate Chem.18 (2007) 2077-2084] by propargyl alcohol and 1,1 '-carbonyl dimidazoles synthesize alkynes propyl ester carbonylic imidazole (propargyl ester ofcarbonyl-imidazole, PPA-CI).Earlier with the desalination of cystamine hydrochloride, claim the cystamine and the 3.47 gram alkynes propyl ester carbonylic imidazoles of 4.40 grams to be dissolved in 50 milliliters of chloroforms, stirring reaction is 24 hours under the room temperature, remove and desolvate, add 80 milliliters of sodium dihydrogen phosphates (pH 4.0) dissolving, with extracted with diethyl ether 3 times, gained lower floor aqueous portion is regulated pH to 9.0, use 40 milliliters ethyl acetate extraction 3 times again, merge organic phase, wash once with 40 milliliters of pH=9 sodium radio-phosphate,P-32 solutions, the organic phase anhydrous magnesium sulfate drying that obtains, filter, underpressure distillation obtains yellow oily liquid 1.9 grams, and productive rate is 35%.
1H-NMR?in?CDCl
3：δ(ppm)2.45(s，1H，CH≡C)，2.75(m，4H，S-CH
2-C)，2.98(m，2H，NH
2-CH
2-C)，3.50(m，2H，NH-CH
2-C)，4.37(b，2H，NH
2-C)，4.64(s，2H，O-CH
2-C≡C)，5.58(s，1H，NH-C＝O)。The chloroform of this reaction changes other organic solvent such as the methylene dichloride of no active proton into, and acetone, tetrahydrofuran (THF), DMF carry out too and can obtain similar result.
(allyl amino manthanoate ethyl) two sulphur ethyl 1-carboxamide imidazoles ((propargylcarbamate) ethyl disulfide ethyl 1-carbamide-imidazole, PPA-cyst-CI) synthetic: as to take by weighing 1,1 '-carbonyl dimidazoles (CDI), 3.94 grams are dissolved in the 40ml chloroform, above-mentioned PPA-cyst 1.9 grams are splashed in the chloroformic solution of CDI, normal-temperature reaction 4h, reaction solution is become transparent by muddiness.Add water washing three times, the organic phase anhydrous magnesium sulfate drying filters, and underpressure distillation gets white solid PPA-cyst-CI 1.9 grams, and productive rate is 71%.
1H-NMR?in?CDCl
3：δ(ppm)2.49(s，1H，CH≡C)，2.78(t，2H，S-CH
2-CH
2-NH-COO)，3.00(t，2H，S-CH
2-CH
2-NH-CON)，3.50(m，2H，-OCO-NH-CH
2-C)，3.55(m，2H，-NCO-NH-CH
2-C)，4.66(s，2H，O-CH
2-C≡C)，5.39(b，1H，NH-COO)，7.05(s，1H，CH＝N-CH＝CH)，7.49(b，1H，NH-CON)，7.56(s，1H，CH＝CH-N)，8.25(s，1H，N-CH＝N)。The chloroform of this reaction changes other organic solvent such as the methylene dichloride of no active proton into, and acetone, tetrahydrofuran (THF), DMF carry out too and can obtain similar result.
Linear polymethyl acrylic acid-the 2-N of embodiment 2 single nitrine end group functionals, N-dimethylaminoethyl (mono-azide terminated poly (2-(dimethylamino) ethyl methacrylate), L-PDE-N
3) synthetic:
According to document [X.L.Jiang, M.C.Lok, W.E.Hennink, Degradable-BrushedpHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery, Bioconjugate Chem.18 (2007) 2077-2084.] earlier synthetic initiator 2-bromo-isopropylformic acid-3-nitrine third fat (BiBAP).With 6.29 gram monomer methacrylic acid-2-N, the N-dimethylamino ethyl ester, 260 milligrams of ligand 1s, 1,4,7,7 ,-five methyl diethylentriamine (PMDETA) and 250 milligrams of initiator B iBAP are dissolved in 15mL1, the 2-dichlorobenzene, under nitrogen, add the 210mg cuprous bromide, remove oxygen in the system by three vacuum-nitrogen circulation then, mixed solution reacting by heating 2 hours in 50 ℃ of oil baths.Add 200 ml n-hexane stopped reaction and be settled out product, repeated precipitation is carried out purifying 2 times then, and drying obtains product L-PDE-N in vacuum drying oven
35.22 gram, productive rate 83%.Recording its number-average molecular weight by nucleus magnetic hydrogen spectrum is 8.6kDa, and recording its weight-average molecular weight by gel permeation chromatography (GPC) coupling multi-angle light scattering detector is 12.7kDa, and number-average molecular weight is 10.7kDa, and polydispersity index PDI is 1.19, uses L-PDE-N
3-1 expression.
Another L-PDE-N
3Synthesizing formula and step on, just with reaction mixture heated polymerizable reaction 2.5 hours in 55 ℃ of oil baths, deposition and purification vacuum-drying obtains L-PDE-N
35.79 gram, productive rate 92%, recording its number-average molecular weight by nucleus magnetic hydrogen spectrum is 12.8kDa, and recording its weight-average molecular weight by GPC coupling multi-angle light scattering detector is 19.8kDa, and number-average molecular weight is 18.5kDa, and PDI is 1.07, uses L-PDE-N
3-2 expressions.By control initiator and monomeric ratio and reaction conditions, can make the different L-PDE-N of a series of molecular weight between 2000-30000
3The linear polymethyl acrylic acid dimethylamino ethyl ester of nitrine end group functional.
Synthesizing of the polymine of embodiment 3 nitrine end group functionals
The polymine PEI of nitrine end group functional
800-(N
3)
1Synthetic (index number 800 expression PEI weight-average molecular weight are 800, the nitrine end group mean number of index number 1 each polymine of expression is about 1): reference literature [2008-033s Chem Comm, 20071219, N2,190-192, Biodegradablemicrocapsules designed via ' click ' chemistry, Bruno G.De Geest; Wim Van Camp, Filip E.Du Prez; Stefaan C.De Smedt; Jo Demeester; Wim E.Hennink] by nitrine propyl alcohol and 1,1 '-carbonyl dimidazoles synthetic fluid nitrine propyl ester carbonylic imidazole (3-azidopropyl ester ofcarbonylimidazole, AP-CI).0.30 gram nitrine propyl ester carbonylic imidazole (AP-CI) is dissolved in 60 milliliters of chloroforms, 1.23 being 800 polymine, the grammes per square metre average molecular weight is dissolved in 20 milliliters of chloroforms, with they hybrid reactions 2 hours at room temperature, the temperature rising reflux reaction is 10 hours again, the removal of solvent under reduced pressure chloroform obtains containing polymine yellow liquid 1.2 grams of the micromolecular nitrine end group functional of imidazoles.Infrared spectroscopy shows that this product is at 2099cm
-1There is tangible absorption peak at the place, illustrates that it contains nitrine.Nucleus magnetic hydrogen spectrum shows that raw material nitrine propyl ester carbonylic imidazole reacts completely, and records each polyethyleneimine: amine molecule and contain 1 azido group.The chloroform of this reaction changes other organic solvent such as the methylene dichloride of no active proton into, acetone, and tetrahydrofuran (THF), DMF carry out too, and temperature of reaction is at 30-80 ℃ of following stirring reaction 2-48 hour, and can obtain similar result.
The polymine PEI of nitrine end group functional
1800-(N
3)
1Synthetic (index number 1800 expression PEI weight-average molecular weight are 1800, the nitrine end group mean number of index number 1 each polymine of expression is about 1): 0.42 gram nitrine propyl ester carbonylic imidazole is dissolved in 10 milliliters of chloroforms, 4.0 being 1800 polymine, the grammes per square metre average molecular weight is dissolved in 40 milliliters of chloroforms, with they hybrid reactions 2 hours at room temperature, temperature rising reflux 10 hours, the removal of solvent under reduced pressure chloroform, obtain yellow oily thick liquid 4.4 grams, for containing the polymine of the micromolecular nitrine end group functional of imidazoles.Infrared spectroscopy shows that this product is at 2099cm
-1There is tangible absorption peak at the place, illustrates that it contains nitrine.Nucleus magnetic hydrogen spectrum shows that raw material nitrine propyl ester carbonylic imidazole reacts completely, and records average each polyethyleneimine: amine molecule and on average contain 1 azido group.The chloroform of this reaction changes other organic solvent such as the methylene dichloride of no active proton into, acetone, and tetrahydrofuran (THF), DMF carry out too, and temperature of reaction is at 30-80 ℃ of following stirring reaction 2-48 hour, and can obtain similar result.
The polymine PEI of weight-average molecular weight between 600-5000 be 0.3-1.1 in molar ratio: 1 is dissolved in the organic solvent, at 25-60 ℃ of following stirring reaction 2-24 hour, removal of solvent under reduced pressure, obtain containing the polymine of the micromolecular nitrine end group functional of imidazoles, the average quantity that each polymine wherein contains the nitrine end group is 0.3-1.1.
Synthesizing of the polymine of embodiment 4 alkynes end group functionals
The polymine PEI of alkynes end group functional
800-(PPA-cyst)
1Synthetic (index number 800 expression PEI weight-average molecular weight are 800, the alkynes end group mean number of index number 1 each polymine of expression is about 1): PPA-cyst-CI 1.26 grams that embodiment 1 is made are dissolved in 100 milliliters of chloroforms, 3.07 being 800 polymine, the grammes per square metre average molecular weight is dissolved in 50 milliliters of chloroforms, with they hybrid reactions 2 hours at room temperature, the temperature rising reflux reaction is 24 hours again, the removal of solvent under reduced pressure chloroform obtains containing the polymine PEI of the micromolecular alkynes end group functional of imidazoles
800-(PPA-cyst)
1Yellow thick liquid 4.26g, productive rate is 98%.Nucleus magnetic hydrogen spectrum shows that raw material PPA-cyst-CI reacts completely, and records each polyethyleneimine: amine molecule and contain 1.0 ethynylene groups.The chloroform of this reaction changes other organic solvent such as the methylene dichloride of no active proton into, acetone, and tetrahydrofuran (THF), DMF carry out too, and temperature of reaction was at 30-80 ℃ of following stirring reaction 2-48 hour.
The polymine PEI1 of alkynes end group functional
800-(PPA-cyst)
1Synthetic (index number 1800 expression PEI weight-average molecular weight are 1800, and the alkynes end group mean number of index number 1 each polymine of expression is about 1): with above-mentioned synthetic PEI
800-(PPA-cyst)
1Identical, PPA-cyst-CI 0.50 gram that embodiment 1 is made is dissolved in 50 milliliters of chloroforms, 2.74 being 1800 polymine, the grammes per square metre average molecular weight is dissolved in 50 milliliters of chloroforms, with they hybrid reactions 2 hours at room temperature, the temperature rising reflux reaction is 24 hours again, the removal of solvent under reduced pressure chloroform obtains containing the polymine PEI of the micromolecular alkynes end group functional of imidazoles
1800-(PPA-cyst)
1Yellow thick liquid 2.96g.Nucleus magnetic hydrogen spectrum shows that raw material PPA-cyst-CI reacts completely, and records each polyethyleneimine: amine molecule and contain 1.0 ethynylene groups.The chloroform of this reaction changes other organic solvent such as the methylene dichloride of no active proton into, acetone, and tetrahydrofuran (THF), DMF carry out too, and temperature of reaction was at 30-80 ℃ of following stirring reaction 2-48 hour.
The polymine PEI of single alkynes end group functional
1800-(PPA-cyst) synthetic (index number 1800 expression PEI weight-average molecular weight are 1800): with above-mentioned synthetic PEI
1800-(PPA-cyst)
1Identical, PPA-cyst-CI 0.33 gram that embodiment 1 is made is dissolved in 30 milliliters of chloroforms, 5.40 being 1800 polymine, the grammes per square metre average molecular weight is dissolved in 80 milliliters of chloroforms, with they hybrid reactions 1 hour at room temperature, the temperature rising reflux reaction is 24 hours again, the removal of solvent under reduced pressure chloroform obtains containing the polymine PEI of the micromolecular single alkynes end group functional of imidazoles
1800-(PPA-cyst) yellow thick liquid 5.61g.Nucleus magnetic hydrogen spectrum (Fig. 1) shows that raw material PPA-cyst-CI reacts completely, and records each polyethyleneimine: amine molecule and on average contain 0.33 ethynylene group, guarantees that it is 1 or 0 that each most polymines contains the alkynes end group.The chloroform of this reaction changes other organic solvent such as the methylene dichloride of no active proton into, acetone, and tetrahydrofuran (THF), DMF carry out too, and temperature of reaction was at 30-80 ℃ of following stirring reaction 2-48 hour.The polymine PEI of weight-average molecular weight between 600-5000 all can make the polymine PEI of alkynes end group functional with the aforesaid method modification, and the average quantity that each polymine wherein contains the alkynes end group is 0.3-1.1.
Homemade polysuccinimide (PSI) 0.375 gram is dissolved among 12 milliliters of DMF, and (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst) 1.36 grams that again embodiment 1 made added in the DMF solution, in 55 ℃ of reactions 24 hours.Use 200 milliliters of ether sedimentation purifying three times respectively, vacuum-drying obtains yellow powder solid 0.92 gram.Infrared spectroscopy shows that this product has tangible absorption peak at the 2121cm-1 place, illustrates that it contains alkynyl.The product nucleus magnetic hydrogen spectrum new characteristic peak occurs at 4.0-4.8ppm, and the open loop rate that calculates polysuccinimide is 65%, and this product is with P (Asp-ss-Al)-1 expression.
Side chain contains the preparation of the poly-asparagus fern amino acid derivative P (Asp-ss-Al) of 100% alkynyl group: experimental procedure is the same, taking by weighing PSI 1.0 grams is dissolved among 20 milliliters of DMF, (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst) 4.56 grams that again embodiment 1 made add in the DMF solution, and 65 ℃ were reacted 48 hours.With 300 milliliters of ether sedimentation purifying three times, vacuum-drying obtains yellow powder solid 1.74 grams.Infrared spectroscopy shows that this product has tangible absorption peak at the 2121cm-1 place, illustrates that it contains alkynyl.Nucleus magnetic hydrogen spectrum shows that polysuccinimide disappears at the characteristic peak of 5.0-5.2, and the open loop rate of polysuccinimide is 100%, and this product is represented with P (Asp-ss-Al)-2.
Can be according to the mol ratio of PPA-cyst and PSI at 0.2-2: 1, at 20-70 ℃ of following stirring reaction 2-48 hour, make side chain and contain the poly-asparagus fern amino acid derivative P (Asp-ss-Al) of alkynyl group molar percentage at 15%-100%
Embodiment 6 side chains contain the preparation method of the poly-asparagus fern amino acid derivative of azido group:
With homemade polysuccinimide (PSI) 0.28 gram and homemade 2-nitrine ethamine 0.1,0.2 or 0.5 gram (is pressed the mol ratio 0.4: 1 of nitrine and PSI repeating unit respectively, 0.8: 1 and 2: 1) add in the solvent, at room temperature hybrid reaction is 96 hours, the redeposition purifying makes the poly-asparagus fern amino acid derivative P (Asp-Az) that side chain contains the azido group of different ratios, product is respectively 0.29 gram, 0.30 gram, perhaps 0.33 gram.The latter detects its all open loop of PSI ring through nucleus magnetic hydrogen spectrum, illustrates that all side chains have connected azido group.The above two product detects its structure that also has not open loop through nucleus magnetic hydrogen spectrum and exists, added excess ethanol amine ring-opening reaction again 24 hours, make the poly-asparagus fern amino acid derivative P (Asp-Az) that side chain contains azido group and ethanol group, detect the per-cent that contains azido group in its side chain through nucleus magnetic hydrogen spectrum and be respectively 33% and 65% (Fig. 1), use skeleton symbol P (Asp-Az) respectively
0.33(Asp-EA)
0.67And P (Asp-Az)
0.65(Asp-EA)
0.35Expression.Infrared spectroscopy shows that such product is at 2100cm
-1There is tangible absorption peak at the place, illustrates that it contains nitrine.Add the also available N of excess ethanol amine open loop, N-dimethylated propyl diethylenetriamine or N, the N-dimethyl-ethylenediamine, perhaps Propanolamine, butanolamine or amylalcohol amine replace, synthetic make the poly-asparagus fern amino acid derivative P (Asp-Az) that side chain contains the azido group of different ratios, this reaction can be at 20-70 ℃ of following stirring reaction 2-200 hour.
Embodiment 7 side chains contain the preparation method of the PMAm or the polyacrylamide analog derivative of cystine linkage and alkynyl group:
Side chain contains methacryloyl amine monomer PPA-cyst-MAm synthetic of cystine linkage and alkynyl group: (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst) 2.5 grams that embodiment 1 is made, triethylamine 1.62 grams are dissolved in 40 milliliters of dry methylene chloride, cool off with ice-water bath, methylene dichloride (20 milliliters) solution that dropwise adds 1.67 gram methacrylic chlorides, reaction is 2 hours in ice-water bath, again room temperature reaction 15 hours, the purifying recrystallization obtains white crystal PPA-cyst-MAm, and productive rate is 46%.
1H-NMR inCDCl
3: δ (ppm) 1.98 (s, 3H, ≡ C-CH
2-O), 2.47 (s, 1H, CH ≡ C), 2.83 (m, 4H, S-CH
2-C), 3.52 (q, 2H, NH-CH
2-C), 3.66 (q, 2H, O-CO-NH-CH
2-C), 4.68 (s, 2H, O-CH
2-C ≡ C), 5.36 ﹠amp; 5.74 (2H, CH
2=C).
Take by weighing monomer PPA-cyst-MAm 0.907 gram, initiator Diisopropyl azodicarboxylate (AIBN) is dissolved among 4 milliliters of exsiccant DMF for 9.85 milligrams, removes oxygen in the system by twice vacuum-nitrogen circulation, and under nitrogen protection 70 ℃ of oil baths reactions 24 hours.Obtain white powder 0.68 gram for three times with 200 milliliters of ether sedimentation purifying respectively.Recording its weight-average molecular weight by gel permeation chromatography (GPC) coupling multi-angle light scattering detector is 15.1kDa, and polydispersity index PDI is 1.98.Infrared spectroscopy shows that this product has tangible absorption peak at the 2127cm-1 place, illustrates that it contains alkynyl.
Replace the methacrylic chloride reaction with acrylate chloride when preparing above-mentioned monomer, the repolymerization reaction then makes polyacrylamide analog derivative P (PPA-cyst-Am) equally.Add initiator such as Diisopropyl azodicarboxylate AIBN and solvent during polyreaction, at 50-80 ℃ of following stirring reaction 2-24 hour, perhaps also can make PMAm analog derivative P (PPA-cyst-Mam) or the polyacrylamide analog derivative P (PPA-cyst-Am) that side chain contains cystine linkage and alkynyl group by the ATRP polymerization
Embodiment 8-11 is the synthetic reduction-sensitive comb type macromolecule cation polymer series that contains the cystine linkage modification of click chemistry
The synthetic comb shape polymethyl acrylic acid dimethylamino ethyl ester P (Asp-ss-Al-Az-PDE)-1 that contains cystine linkage of click chemistry: the linear polymethyl acrylic acid-2-N of single nitrine end group functional that embodiment 2 is made, N-dimethylaminoethyl L-PDE-N
3-1 200 milligram; the side chain that makes among the embodiment 5 contains the alkynyl group molar percentage and is 65% poly-asparagus fern amino acid derivative P (Asp-ss-Al)-130 milligram and is dissolved among 10 milliliters of DMF; under nitrogen, add 15 milligrams of cuprous bromides; and under nitrogen protection; reaction is 2 days in 50 ℃ of oil baths; dialysis tubing (MWCO 8k-12kDa) water of packing into dialysis; lyophilize obtains comb shape polymethyl acrylic acid dimethylamino ethyl ester cationic polymers P (Asp-ss-Al-Az-PDE)-1; its nucleus magnetic hydrogen spectrum new characteristic peak occurs at 8.15ppm, is the proton characteristic peak on the click chemistry generation triazole ring.Infrared spectra is at 2100cm
-1Near do not have tangible absorption peak, show the basic disappearance after click chemistry reaction of alkynyl and azido group, illustrate that click chemistry finishes substantially.Recording its weight-average molecular weight by gel permeation chromatography (GPC) coupling multi-angle light scattering detector is 243kDa, and PDI is 1.63.
Another contains comb shape polymethyl acrylic acid dimethylamino ethyl ester P (Asp-ss-Al-Az-PDE)-2 synthetic of cystine linkage: step is the same, and difference is L-PDE-N
3-1 consumption reduces to 102 milligrams, and the comb shape polymethyl acrylic acid dimethylamino ethyl ester cationic polymers that makes represents that with P (Asp-ss-Al-Az-PDE)-2 its weight-average molecular weight is 157kDa, and PDI is 1.80.Use solvent DMSO to replace DMF, this click chemistry reaction also can be carried out.CuBr in this reaction can use CuCl instead; This reaction also can not used CuBr, improves temperature of reaction (80 ℃) and prolongs the reaction times (48 hours) and adopt.
Embodiment 9 contains the preparation of the comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PEI) of cystine linkage modification: the side chain that embodiment 5 is made contains cystine linkage and alkynyl group poly-asparagus fern amino acid derivative P (Asp-ss-Al)-181 milligram, N, the N-dimethylated propyl diethylenetriamine is dissolved among 10 milliliters of DMF for 40 milligrams, normal-temperature reaction 12 hours adds the polymine PEI of the nitrine end group functional that makes among the embodiment 3 then
1800-(N
3)
11.07 gram; remove oxygen in the system by three vacuum-nitrogen circulation; under nitrogen, add 18 milligrams of cuprous bromides; and under nitrogen protection, in 50 ℃ of oil baths, reacted 2 days; dialysis tubing (MSCO3.5kDa) water of packing into dialysis; filter the precipitation that occurs in the dialysis, lyophilize obtains 70 milligrams of P (Asp-ss-Al-Az-PEI).The N here, the also available excessive N of N-dimethylated propyl diethylenetriamine, the N-dimethyl-ethylenediamine, thanomin, Propanolamine, butanolamine or amylalcohol amine replace.
Take by weighing 20 milligrams of the P (PPA-cyst-MAm) that embodiment 7 makes, the L-PDE-N that embodiment 2 makes
3-2216 or 325 milligrams, be dissolved among 10 milliliters of DMF, remove oxygen in the system by three vacuum-nitrogen circulation, under nitrogen, add 15 milligrams of cuprous bromides, and reaction 2 days in 50 ℃ of oil baths under nitrogen protection.Pack into dialysis tubing (MWCO 8k-12kDa) water dialysis of the polymer dissolution that makes, lyophilize obtains 191 milligrams of comb shape polymethyl acrylic acid dimethylamino ethyl ester cationic polymerss (with P (PPA-cyst-MAm-PDE)-1 expression) and 305 milligrams (with P (PPA-cyst-MAm-PDE)-2 expression) respectively, recording P (PPA-cyst-MAm-PDE)-1 weight-average molecular weight by GPC coupling multi-angle light scattering detector is 266kDa, PDI is 1.24, and the moon is 13 to the side chain number that connects approximately; The weight-average molecular weight 88.5kDa of P (PPA-cyst-MAm-PDE)-2, PDI is 1.60, the side chain number that connects is about 4.
With the P (Asp-Az) that makes among the embodiment 6
0.33(Asp-EA)
0.6750 milligrams; (each polyethyleneimine: amine molecule on average contains 0.33 ethynylene group with polymine PEI-(PPA-cyst) 1.18 gram of the single alkynes end group functional that makes among the embodiment 4; press the mol ratio 2: 1 of alkynyl end group and nitrine); be dissolved in the mixed solvent of 5 ml waters and 5 milliliters of DMF; add 20 milligrams of CuBr; under nitrogen protection, reaction is 2 days in 50 ℃ of oil baths.The comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that the polymers soln that makes dialysis tubing (MWCO 3.5kDa) the water dialysis 5 days of packing into, lyophilize must contain the cystine linkage modification (Asp-EA)
0.67It is the H of triazole ring that its nucleus magnetic hydrogen spectrum has tangible peak at the 7.9ppm place, illustrates by click chemistry to have generated triazole ring, and Infrared spectroscopy shows that this product is at 2100cm
-1The place does not have tangible absorption peak, and nitrine side group primitive reaction is described.Use solvent DMSO to replace DMF, this click chemistry reaction also can be carried out.CuBr in this reaction can use CuCl instead; This reaction also can not used CuBr, improves temperature of reaction (80 ℃) and prolongs the reaction times (48 hours) and adopt.
With the P (Asp-Az) that makes among the embodiment 6
0.65(Asp-EA)
0.3550 milligrams; (each polyethyleneimine: amine molecule on average contains 0.33 ethynylene group with polymine PEI-(PPA-cyst) 2.41 gram of the single alkynes end group functional that makes among the embodiment 4; press the mol ratio 2: 1 of alkynyl end group and nitrine); be dissolved in the mixed solvent of 5 ml waters and 5 milliliters of DMF; add 20 milligrams of CuBr; under nitrogen protection, reaction is 2 days in 50 ℃ of oil baths.The comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that pack into dialysis tubing (MWCO 3.5kDa) water dialysis of the polymers soln that makes, lyophilize must contain the cystine linkage modification (Asp-EA)
0.35It is the H of triazole ring that its nucleus magnetic hydrogen spectrum (Fig. 1) has tangible peak at the 7.9ppm place, illustrates by click chemistry to have generated triazole ring.Use solvent DMSO to replace DMF, this click chemistry reaction also can be carried out.CuBr in this reaction can use CuCl instead; This reaction also can not used CuBr, improves temperature of reaction (80 ℃) and prolongs the reaction times (48 hours) and adopt.
With 50 milligrams of the P (Asp-Az) that make among the embodiment 6; (each polyethyleneimine: amine molecule on average contains 0.33 ethynylene group with polymine PEI-(PPA-cyst) 2.62 gram of the single alkynes end group functional that makes among the embodiment 4; press the mol ratio 2: 1 of alkynyl end group and nitrine); be dissolved among 10 milliliters of DMF; add the CuBr20 milligram; under nitrogen protection, reaction is 2 days in 50 ℃ of oil baths.Pack into dialysis tubing (MWCO 3.5kDa) water dialysis of the polymers soln that makes, its nucleus magnetic hydrogen spectrum of comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that lyophilize must contain the cystine linkage modification has tangible peak at the 7.9ppm place be the H of triazole ring, explanation has generated triazole ring by click chemistry, record its elution time greatly prior to the appearance time of raw material PEI-1.8kDa by gel permeation chromatography (GPC), illustrate that the molecular weight of the comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that contains the cystine linkage modification is the molecular weight much larger than raw material PEI-1.8kDa.Use solvent DMSO to replace DMF, this click chemistry reaction also can be carried out.CuBr in this reaction can use CuCl instead; This reaction also can not used CuBr, improves temperature of reaction (80 ℃) and prolongs the reaction times (48 hours) and adopt.
The P (PPA-cyst-MAm-PDE)-1 that embodiment 10 is made is dissolved in 0.3mol/L NaAc, and the buffered soln of pH 4.4 is configured to the solution of 5.0mg/mL.Get this polymers soln of 1mL, add 1 of 53 microlitre 1.0mol/L, 4-dithiothreitol (DTT) (DTT) aqueous solution, normal temperature was placed 1 hour down, recording its weight-average molecular weight with GPC coupling multi-angle light scattering detector is 24.4kDa, PDI is 1.11, sees Fig. 2, the molecular weight and the raw materials used L-PDE-N of this explanation degraded product
3-2 molecular weight (M
w19.8kDa PDI 1.07) more approaching, illustrate that this modification comb type high-molecular cationic polymer that contains cystine linkage has the reduction sensitivity characteristic.
Respectively P (PPA-cyst-MAm-PDE)-1 and P (PPA-cyst-MAm-PDE)-2 are dissolved in HBS damping fluid (20mM Hepes, 130mM NaCl, pH 7.4) be configured to 62.5 milligrams/L solution, get 600 microlitre polymers solns and join (50 milligrams/L in HBS) in the 150 microlitre pcDNA3-Luc plasmid plasmid DNA solutions, mix and descended compound 30 minutes at 37 ℃, add 1 of 8.3 microlitre 1.0mol/L then, 4-dithiothreitol (DTT) (DTT) aqueous solution, detect particle diameter change in time in the solution with dynamic light scattering then, the results are shown in Figure 3.It is stable not add the DTT composite particles as known in the figure, and composite particles increases instability rapidly after adding DTT, and this illustrates that also such modification comb type high-molecular cationic polymer that contains cystine linkage has the reduction sensitivity characteristic.
The toxicity of cation high molecular pair cell is an important evaluation index of polymer gene delivery vector, and the inhibiting rate of general test pair cell under the effect of different concns polymkeric substance is weighed.Here the comb type high-molecular cationic polymer that synthetic among the embodiment 8-11 is contained the cystine linkage modification is measured by tetramethyl-azo azoles salt (MTT) colorimetry 293T cells in vitro cytotoxicity.
The 293T cell is inoculated with 3000 cells/well in 96 well culture plates and is added the DMEM substratum that 100 μ L contain serum, at 5%CO
2Cultivated 48 hours in 37 ℃ in the incubator.Polymers soln (0.01 to 0.20mg/mL) the 100 μ L and the fresh DMEM substratum of 100 μ L that in every porocyte, add the different concns of filtration sterilization respectively).Continue to cultivate 48h to the end of material effects phase, remove the substratum in all holes of containing cell, add the fresh DMEM substratum of 200 μ L.Respectively add 20 μ L MTT (5mg/ml) during institute is porose.Wrap up culture plate with aluminium foil, incubation 4h in 37 ℃ of moist environments.Discard substratum and MTT in the hole, each adds 200 μ L DMSO wherein and at room temperature vibrates, and writes down light absorption value at the 570nm place with microplate reader (550 BIO-RAD, the U.S.).Calculate the cell relative survival according to light absorption value.
The comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) that contains the cystine linkage modification of preparation (Asp-EA) among the embodiment 11
0.67, P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.35And the toxicity of P (Asp-Az-Al-ss-PEI) and comparison polymer PEI 25kDa is seen Fig. 4, this figure explanation is lower than 0.03 mg/ml when the concentration of polymkeric substance, and the comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) of adding cystine linkage modification (Asp-EA)
0.67, P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.35Relative cell survival rate is about 80% during with P (Asp-Az-Al-ss-PEI), and when adding 0.03 mg/ml 25kDaPEI relative cell survival rate less than 20%.
The toxicity that contains cystine linkage modification comb type high molecular polycation P (Asp-ss-Al-Az-PDE)-1 and P (Asp-ss-Al-Az-PDE)-2 and comparison polymer PEI 25kDa of preparation is seen Fig. 5 among the embodiment 8, the raw material low-molecular weight polymer L-PDE-N of preparation P (Asp-ss-Al-Az-PDE)-1 and P (Asp-ss-Al-Az-PDE)-2
3-1 and also see Fig. 5 in contrast by the toxicity of the linear high-molecular weight PDE of common radical polymerization synthetic (HMPDE, weight-average molecular weight 154kDa, PDI 1.58.4).This figure explanation is lower than 0.03 mg/ml when the concentration of polymkeric substance, the comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE)-1 that adds the cystine linkage modification and P (Asp-ss-Al-Az-PDE)-2 o'clock relative cell survival rate be greater than 90%, and when adding 0.03 mg/ml 25kDa PEI relative cell survival rate less than 10%., raw material low-molecular weight polymer L-PDE-N
3-1 toxicity is very low, but the toxicity of P (Asp-ss-Al-Az-PDE)-2 is more much lower than the toxicity of the linear high molecular HMPDE suitable with its molecular weight.This toxicity that shows the comb type high-molecular cationic polymer that contains the cystine linkage modification is lower.
The embodiment 14 polymkeric substance/transfection efficiency in vitro of DNA mixture when serum-free exists
The comb type high-molecular cationic polymer that contains the cystine linkage modification that makes among the embodiment 8-11 is dissolved in NaCl solution (150mmol/L respectively, pH7.4), filtration sterilization, be prepared into the solution of respective concentration, in the aseptic centrifuge tube of 1.5mL, add 50 μ L and contain 1.0 μ g plasmid DNA (pcDNA3-Luc is the reporter gene of luciferase) solution, the 50 μ L cationic polymer solution that add respective amount according to the N/P ratio for 10-50, compound with DNA, concussion 5s, this mixture is used for the cell transfecting experiment after leaving standstill half an hour at 37 ℃.With the PEI of the PEI of 25kDa and 1.8kDa in contrast, compared mixture to 293T and HeLa cells in vitro transfection efficiency (at 37 ℃ of transfection 4h, substratum continues to cultivate 48h at 37 ℃ after changing the complete DMEM substratum of 1mL into behind adding mixture and the serum-free DMEM substratum).Fig. 6 showed contain the cystine linkage modification comb type high-molecular cationic polymer P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.67, P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.35With P (Asp-Az-Al-ss-PEI) different polymkeric substance/DNA quality than the time to the transfection expression efficient of 293T cells in vitro luciferase.Wherein P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.67The gene transfection efficient of mixture be higher than the mixture gene transfection efficient of polymer genophore commonly used with reference to polymkeric substance 25kDa PEI.Fig. 7 has showed P (Asp-ss-Al-Az-PDE)-1 and P (Asp-ss-Al-Az-PDE)-2, low-molecular weight polymer L-PDE-N
3-1 with linear high-molecular weight HMPDE different polymkeric substance/DNA quality than the time to the transfection expression efficient of 293T cells in vitro luciferase, also having used comparison polymer PEI 25kDa and DNA is the gene transfection efficient of 10 mixtures at N/P ratio.Fig. 8 has showed comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE)-1 and the P (Asp-ss-Al-Az-PDE)-2 that contains the cystine linkage modification, low-molecular weight polymer L-PDE-N
3-1 different polymkeric substance/DNA quality than the time to the luciferase transfection expression (N/P ratio of PEI 25kDa and DNA be 10) of HeLa cell when serum-free exists.These figure illustrate that the gene transfection efficient of the mixture of the comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE) that contains the cystine linkage modification is much higher than raw material low-molecular weight polymer L-PDE-N
3-1 mixture (2-3 the order of magnitude) also is higher than the gene transfection efficient of polymer genophore commonly used with reference to the mixture of polymkeric substance 25kDa PEI.The result shows: the comb type high-molecular cationic polymer of this class reduction-sensitive cystine linkage modification has very high transfection efficiency to 293T and HeLa cell.
The embodiment 15 polymkeric substance/transfection efficiency in vitro of DNA mixture when having serum to exist
Step is with embodiment 14, and at 37 ℃ of transfection 4h, substratum continued to cultivate 48h at 37 ℃ after changing the complete DMEM substratum of 1mL into after difference was to add mixture and 10% serum DMEM substratum is arranged).Fig. 9 has showed comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE)-1 and the P (Asp-ss-Al-Az-PDE)-2 that contains the cystine linkage modification when having serum to exist, low-molecular weight polymer L-PDE-N
3-1 different polymkeric substance/DNA quality than the time to the transfection expression efficient of mixture to the plain enzyme of 293T cell fluorescence.The result shows: when having serum to exist, it is also higher to the transfection efficiency of 293T cell that this class reduction-sensitive contains the comb type high-molecular cationic polymer mixture of cystine linkage modification.
The outer green fluorescence protein gene transfection efficiency of embodiment 16 polymkeric substance/DNA composite body
Use the comb type high-molecular cationic polymer P (Asp-ss-Al-Az-PDE)-1 that contains the cystine linkage modification and the P (Asp-ss-Al-Az-PDE)-2 that make among embodiment 8 and the embodiment 11, P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.67, P (Asp-Az-Al-ss-PEI) (Asp-EA)
0.35And P (Asp-Az-Al-ss-PEI), P (PPA-cyst-MAm-PDE)-1 and P (PPA-cyst-MAm-PDE)-2 be complex plasmid DNA pEGFP (green fluorescence protein gene) in-vitro transfection 293T cell respectively, after the transfection 48 hours, with the expression of fluorescence microscope green fluorescent protein.The result shows, this cell of all effectively transduceing.
Claims (10)
1. comb type polymer genophore, structure is shown below:
CP is the low molecular weight cationic polymkeric substance in the formula, molecular weight between 600-30000, F1 and F2 be can interreaction group, and F1 or F2 contain cystine linkage, M is the backbone units structure of polymkeric substance, n=10-500; M＜n, m=0-400, l=0-800; L, m, n are integer.
2. the described comb type of claim 1 polymer genophore is characterized in that, CP is linear polymethyl acrylic acid-2-N, N-dimethylaminoethyl or polymine.
3. claim 1 or 2 described comb type polymer genophores is characterized in that, the backbone units structure of polymkeric substance is poly-asparagus fern amino acid derivative, PMAm or polyacrylamide analog derivative.
4. the preparation method of the described polymer genophore of claim 1 is characterized in that, comprises the steps:
(1) the low molecular weight cationic polymkeric substance CP-F1 of a reaction of anamorphic zone end group F1;
(2) synthetic trunk polymer, its side chain contain can with the group F2 of F1 end group reaction, and F1 or F2 contain cystine linkage, shown in right formula:
(3) with above-mentioned two kinds of mixed with polymers reaction, purifying, make the comb type cationic polymers that contains cystine linkage.
5. the preparation method of polymer genophore as claimed in claim 4 is characterized in that, F1 is for containing azido group, and F2 is for containing cystine linkage and alkynyl group; Or F1 is for containing cystine linkage and alkynyl group, and F2 is for containing azido group.
6. preparation method as claimed in claim 4 is characterized in that:
Described step 1 is: adopt Transfer Radical Polymerization, control initiator and monomeric ratio make the L-PDE-N of molecular weight between 2000-30000
3
Described step 2 is: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, and at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two thioethylamine compounds; Again with (allyl amino manthanoate ethyl) two thioethylamine compounds and polysuccinimide 0.2-2 in molar ratio: 1 adds in the solvent, at 20-70 ℃ of following stirring reaction 2-48 hour, synthetic make the poly-asparagus fern amino acid derivative P (Asp-ss-Al) that side chain contains cystine linkage and alkynyl group;
Described step 3 is: with above-mentioned L-PDE-N
3And P (Asp-ss-Al) is by the mol ratio 0.5-3 of nitrine end group and alkynyl: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, under 20-80 ℃, reacted 1-48 hour, if product also has the structure of not open loop to exist, then add excessive N again, N-dimethylated propyl diethylenetriamine or N, the N-dimethyl-ethylenediamine, perhaps thanomin, Propanolamine, butanolamine or amylalcohol amine, reacted 24 hours, and obtained containing the comb type cationic polymers P (Asp-ss-Al-Az-PDE) of cystine linkage.
7. preparation method as claimed in claim 4 is characterized in that:
Described step 1 is: the 0.3-1.1 in molar ratio of the polymine between 600-5000 with nitrine propyl ester carbonylic imidazole compound and weight-average molecular weight: 1 is dissolved in the organic solvent, at 25-60 ℃ of following stirring reaction 2-24 hour, removal of solvent under reduced pressure, obtain containing the polymine of the micromolecular nitrine end group functional of imidazoles, the quantity that each polymine wherein contains the nitrine end group is 0.3-1.1;
Described step 2 is: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, and at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two thioethylamine compounds; Again with (allyl amino manthanoate ethyl) two thioethylamine compounds and polysuccinimide 0.2-2 in molar ratio: 1 adds in the solvent, at 20-70 ℃ of following stirring reaction 2-48 hour, synthetic make side chain and contain the poly-asparagus fern amino acid derivative P (Asp-ss-Al) of alkynyl group molar percentage at 15%-100%;
Described step 3 is: above-mentioned P (Asp-ss-Al) is dissolved in organic solvent, if it also has the structure of not open loop to exist, then add excessive N earlier, N-dimethylated propyl diethylenetriamine or N, the N-dimethyl-ethylenediamine, perhaps thanomin, Propanolamine, butanolamine or amylalcohol amine, reacted 24 hours, again with the polymine of the above-mentioned nitrine end group functional mol ratio 0.5-3 by nitrine end group and alkynyl: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, under 20-80 ℃, reacted 1-48 hour, and obtained containing the comb type cationic polymers P (Asp-ss-Al-Az-PEI) of cystine linkage.
8. preparation method as claimed in claim 4 is characterized in that:
Described step 1 is: contain the linear polymethyl acrylic acid-2-N of a nitrine end group functional, N-dimethylaminoethyl L-PDE-N
3The preparation method: adopt Transfer Radical Polymerization, control initiator and monomeric ratio make the L-PDE-N of molecular weight between 2000-30000
3
Described step 2 is: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two thioethylamine compounds (PPA-cyst); Again with (allyl amino manthanoate ethyl) two thioethylamine compounds and methacrylic chloride or acrylate chloride reaction purification; Add initiator and solvent again, stir down by common Raolical polymerizable 2-24 hour at 50-80 ℃, or by atom transfer radical polymerization, syntheticly make PMAm analog derivative P (PPA-cyst-Mam) or the polyacrylamide analog derivative P (PPA-cyst-Am) that side chain contains cystine linkage and alkynyl group;
Described step 3 is: with above-mentioned L-PDE-N
3And P (PPA-cyst-MAm) or P (PPA-cyst-Am) are by the mol ratio 0.5-3 of nitrine end group and alkynyl: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, reacted under 20-80 ℃ 1-48 hour, purifying obtains containing the comb type cationic polymers P (PPA-cyst-MAm-PDE) of cystine linkage.
9. preparation method as claimed in claim 4 is characterized in that:
Described step 1 is: with alkynes propyl ester carbonylic imidazole compound and cystamine 0.5-1.5 in molar ratio: 1 adds in the organic solvent, and at 20-60 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two thioethylamine compounds; Again with (allyl amino manthanoate ethyl) two thioethylamine compounds and 1,1 '-carbonyl dimidazoles is 0.4-0.75 in molar ratio: 1 adds in the organic solvent, at 20-70 ℃ of following stirring reaction 2-48 hour, purifying obtained (allyl amino manthanoate ethyl) two sulphur ethyl 1-carboxamide imidazolium compoundss (PPA-cyst-CI); The 0.2-1.1 in molar ratio of the polymine between 600-5000 with Compound P PA-cyst-CI and weight-average molecular weight: 1 is dissolved in the organic solvent, at 20-60 ℃ of following stirring reaction 2-48 hour, removal of solvent under reduced pressure, obtain containing the polymine of the micromolecular alkynes end group functional of imidazoles, the quantity span that each polymine wherein contains the alkynes end group is 0.2-1.1;
Described step 2 is: with 2-nitrine ethamine and polysuccinimide 0.4-2 in molar ratio: 1 adds in the solvent, at 20-70 ℃ of following stirring reaction 2-200 hour, if product also has the structure of not open loop to exist, then add excessive N again, N-dimethylated propyl diethylenetriamine or N, N-dimethyl-ethylenediamine, perhaps thanomin, Propanolamine, butanolamine or amylalcohol amine, reacted 24 hours, and synthetic made the poly-asparagus fern amino acid derivative P (Asp-Az) that side chain contains azido group.
Described step 3 is: with the polymine PEI-(PPA-cyst) of above-mentioned alkynes end group functional and P (Asp-Az) the mol ratio 0.5-3 by alkynyl end group and nitrine: 1 is dissolved in organic solvent or the water or in the mixture of water and organic solvent, under 20-80 ℃, reacted 1-96 hour, and obtained containing the comb type cationic polymers P (Asp-Az-Al-ss-PEI) of cystine linkage.
10. the described comb type of claim 1 polymer is characterized in that as genophore: introduce polyoxyethylene glycol, target group and/or component that can be crosslinked.
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