CN102174579A - Reducible and biodegradable comb type high polymer gene vector and preparation method of same - Google Patents

Abstract

The invention discloses a reductively degradable comb-shaped polymer gene carrier and a preparation method thereof. The structure is as follows: In the formula, CP is a low-molecular-weight cationic polymer with a molecular weight between 600-30000, F1 and F2 are groups that can react with each other, and F1 or F2 contains a disulfide bond, M is the main chain unit structure of the polymer, and n= 10-500; m<n, m=0-400, l=0-800; l, m, n are integers. The comb-shaped macromolecule gene carrier of the present invention has low cytotoxicity, can well bind and compound gene DNA under physiological conditions, and can release the contained gene due to the rapid breakage of the reducing environment in the cell when it enters the cell. The results showed that it had higher transfection efficiency and lower cytotoxicity than 25kDa PEI. Moreover, the preparation method is simple, and the structure control is easy.

Description

Reductively degradable comb-shaped polymer gene carrier and preparation method thereof

technical field

The invention relates to a reductively degradable comb-shaped polymer gene carrier and a preparation method thereof, belonging to the field of gene therapy.

Background technique

The completion of the human genome project draft has enabled people to better understand the relationship between genes and diseases. As of 2008, there have been more than 1,400 clinical cases, using genes to correct genetic defects or treat acquired diseases (http://www.wiley.co.uk/genmed/clinical/). The field of genetics has identified many genes with the potential to treat and prevent diseases, but the progress of gene delivery systems is limited, which is the bottleneck of gene therapy at present. Gene delivery vectors mainly include viral vectors and non-viral vectors. Because of the high transfection efficiency, viral vectors were first applied in the clinical research of gene therapy; however, their application and promotion were limited due to safety hazards and complicated and expensive preparation process. Therefore, it is of great significance to research and develop efficient and safe non-viral gene vectors.

Cationic macromolecular polymers are non-viral gene carriers that have been studied more at present, including polylysine (polylysine, PLL), polyethyleneimine (polyethyleneimine, PEI), chitosan and its derivatives, polyamide-amine ( polyamidoamine, PAMAM) dendrimers and polymethacrylate cationic derivatives, etc. However, traditional polymer gene carriers cannot be used clinically due to relatively low transfection efficiency and high toxicity. For example, the accumulation of non-degradable cationic polymers in cells will lead to high toxicity. The transfection efficiency and toxicity of polymer gene vectors generally increase with the increase of molecular weight. For example, the DNA complex with PEI as the carrier increases its transfection efficiency with the increase of molecular weight within 0.6-70kDa [Godbey W.T., et al., J.Biomed.Mater.Res.1999,45,268], The cytotoxicity of PEI also increases with the increase of molecular weight. It has been reported that PEI with a molecular weight lower than 1.8kDa has almost no transfection effect, and the molecular weight range of PEI suitable as a gene carrier is roughly 11.9-70kDa [Godbey W.T.; Wu K.K.; Mikos A.G. Size matters: molecular weight affects the efficiency of poly( ethyleneimine) as a genedelivery vehicle.J.Biomed.Mater.Res.1999,45,268], the most commonly used molecular weight is 25kDa, therefore, in most studies, the branched PEI of 25KDa is selected as the reference standard.

In order to solve the contradictory effect of polymer molecular weight on vector transfection efficiency and toxicity, Lee's research group first reported the introduction of reductively degradable disulfide bonds, which reacted with active amino groups to cross-link PEI with a molecular weight of 0.8 kDa to obtain high molecular weight PEI derivatives. [MA.Gosselin, W-J.Guo, R.J.Lee, Efficient genetransfer using reversibly cross-linked low molecular weight polyethyleneimine, Bioconjugate Chem.12 (2001) 989-994], the PEI derivative has higher transfection efficiency and very low toxicity. Similar studies have been discussed in our previous invention patent [Chinese patent application publication number: CN101812178A]. The main disadvantage of this type of PEI derivatives is that the degree of crosslinking is not easy to control, and the resulting polymer has poor solubility and contains The microgel must be removed with a column, the process is complicated, and the yield is low Cytotoxic and highly efficient PEI gene transfection non-viral vectors with a controllable chain length and structure? J. Control. Release 140 (2009) 40-46.].

The invention prepares a comb-shaped high molecular weight cationic polymer containing a disulfide bond by reacting a low-molecular-weight polycation containing a reactive terminal group through a side group reaction. The preparation method of this type is simple and does not produce crosslinking. The prepared polymers can be rapidly degraded into lower molecular weight polymers in a reducing environment, and at the same time have higher gene transfection efficiency and lower cytotoxicity.

Contents of the invention

The technical problem to be solved by the present invention is to provide a comb-shaped polymer gene carrier with high gene transfection efficiency and low cytotoxicity and no cross-linking and a preparation method thereof.

The polymer gene carrier provided by the present invention is a comb-shaped cationic polymer modified with a disulfide bond, and its structure is shown in the following formula:

In the formula, CP is a low-molecular-weight cationic polymer with a molecular weight between 600-30000, F1 and F2 are groups that can react with each other, and F1 or F2 contains a disulfide bond, M is the main chain unit structure of the polymer, and n= 10-500; m<n, m=0-400, l=0-800; l, m, n are integers.

The value range of n is 10-500, which is mainly controlled by the ratio of monomer and initiator added when preparing the main chain polymerization. The value range of l is 0-800, which is mainly controlled by the composition of the monomer raw materials added during the main chain polymerization or the composition of the side chain modified raw materials. m<n, the value range of m is 0-400, which is mainly controlled by adding side chain modified raw material composition.

The molecular weight range of the raw material low molecular weight cationic polymer is between 600-30000. If PDE is used, the molecular weight is preferably between 2000-30000. If PEI is used, the molecular weight is preferably between 600-5000.

A more specific solution could be:

CP is linear polymethacrylate-2-N,N-dimethylaminoethyl ester (L-PDE-N ₃ ) or polyethyleneimine (PEI).

The main chain unit structure of the polymer is polyaspartic acid derivative, polymethacrylamide or polyacrylamide derivative.

The present invention also provides a preparation method of the above-mentioned polymer gene carrier, comprising the following steps:

(1) synthesis of low molecular weight (low molecular-weight) cationic polymer CP-F1 with a reactive end group F1;

(2) Synthesize the main chain polymer, its side chain contains the group F2 that can react with the F1 end group, and F1 or F2 contains a disulfide bond, as shown in the right formula:

(3) The above two polymers are mixed, reacted and purified to prepare a comb-shaped cationic polymer containing disulfide bonds.

A further scheme is: F1 is an azide-containing group, F2 is a disulfide bond-containing and alkynyl group; or F1 is a disulfide bond-containing and alkynyl group, and F2 is an azide-containing group.

The functionalized low molecular weight (low molecular-weight) cationic polymer CP-F1 is synthesized by ordinary free radical polymerization or controlled living polymerization, or by modification of commercial low molecular weight cationic polymer.

The main chain polymer, at least a part of its side chain contains a group F2 that can react with the end group of F1, and F1 or F2 contains a disulfide bond. The synthesis method includes but is not limited to ordinary free radical polymerization or controlled living polymerization method, either by side chain modification of commercial polymers or side chain modification of synthetic polymers.

In the polymer gene carrier provided by the present invention, the low-molecular-weight cationic polymer CP-F1 with the reactive end group F1 is a linear polymethacrylic acid-2-N, N-dimethylaminoethylene functionalized with an azide end group. Ester (poly(2-(dimethylamino)ethyl methacrylate), PDE), when the main chain polymer is a polyaspartic acid derivative with a disulfide bond and an alkynyl group in the side chain,

Concrete preparation method is as follows:

(1) The preparation method of linear polymethacrylic acid-2-N, N-dimethylaminoethyl ester (L-PDE-N ₃ ) containing an azide end group functionalization: adopt atom transfer radical polymerization method, control The ratio of initiator and monomer is used to prepare L-PDE-N ₃ with molecular weight between 2000-30000;

(2) The preparation method of the polyaspartic acid derivative that side chain contains disulfide bond and alkynyl group: add propargyl carbonylimidazole compound and cystamine in molar ratio 0.5-1.5: 1 in the organic solvent, in 20 The reaction was stirred at -60°C for 2-48 hours, and purified to obtain (allyl carbamate ethyl) dithioethylamine compound (PPA-cyst); then (allyl carbamate ethyl) disulfide Ethylamine compound (PPA-cyst) and polysuccinimide (PSI) are added to the solvent at a molar ratio of 0.2-2:1, stirred and reacted at 20-70°C for 2-48 hours, and the side chain disulfide-containing Polyaspartic acid derivative P(Asp-ss-Al) of bond and alkynyl group;

(3) The above-mentioned L-PDE-N ₃ and P(Asp-ss-Al) are dissolved in an organic solvent or water or a mixture of water and an organic solvent according to the molar ratio of azide end group to alkynyl group of 0.5-3:1 , react at 20-80°C for 1-48 hours, if the product still has an unopened ring structure, then add excess N,N-dimethylpropylenediamine or N,N-dimethylethylene Diamine, or ethanolamine, propanolamine, butanolamine or pentanolamine, reacted for 24 hours to obtain a comb-shaped cationic polymer P(Asp-ss-Al-Az-PDE) containing a disulfide bond, and its structural representation can be is the following formula:

In the polymer carrier provided by the present invention, the low-molecular-weight cationic polymer CP-F1 is a polyethyleneimine (PEI) derivative with a molecular weight between 600-5000 functionalized by an azide end group, wherein the weight-average molecular weight is between The schematic structure of polyethyleneimine between 600-5000 is:

When the main chain polymer is a degradable polyaspartic acid derivative, and its side chain contains disulfide bonds and alkynyl groups, the specific preparation method is as follows:

(1) The preparation method of polyethyleneimine functionalized with azide end group: the molar ratio of propyl azidocarbonylimidazole compound and polyethyleneimine PEI with a weight average molecular weight between 600-5000 is 0.3-1.1:1 Soluble in an organic solvent, stir and react at 25-60°C for 2-24 hours, remove the solvent under reduced pressure to obtain azide-end functionalized polyethyleneimine containing imidazole small molecules, each of which is polyethyleneimine The number of azide-containing end groups is 0.3-1.1;

(2) The preparation method of the polyaspartic acid derivative that side chain contains disulfide bond and alkynyl group: add propargyl carbonylimidazole compound and cystamine in molar ratio 0.5-1.5: 1 in the organic solvent, in 20 The reaction was stirred at -60°C for 2-48 hours, and the (allyl carbamate ethyl) dithioethylamine compound ([(propargylcarbamate) ethyl]-dithioethylamine, PPA-cyst) was obtained after purification; Carbamate ethyl) dithioethylamine compound (PPA-cyst) and polysuccinimide (PSI) are added to the solvent at a molar ratio of 0.2-2:1, and the reaction is stirred at 20-70°C for 2-48 hours , synthesize and prepare a polyaspartic acid derivative P(Asp-ss-Al) with a molar percentage of alkynyl group in the side chain of 15%-100%;

(3) Dissolve the above-mentioned P(Asp-ss-Al) in an organic solvent. If there is still an unopened ring structure, add excess N,N-dimethylpropylenediamine or N,N- Dimethylethylenediamine, or ethanolamine, propanolamine, butanolamine or pentanolamine, reacted for 24 hours, and then the above-mentioned polyethyleneimine functionalized with azide end group according to the ratio of azide end group and alkynyl group The molar ratio is 0.5-3:1 dissolved in an organic solvent or water or a mixture of water and an organic solvent, and reacted at 20-80°C for 1-48 hours to obtain a comb-shaped cationic polymer P(Asp- ss-Al-Az-PEI), its structure diagram can be:

In the polymer carrier provided by the present invention, the low molecular weight cationic polymer CP-F1 is polymethacrylic acid-2-N,N-dimethylamine with a molecular weight between 2000-30000 containing an azide end group Ethyl ester (L-PDE-N ₃ ); when the main chain is methacrylamide or acrylamide derivatives containing disulfide bonds and alkynyl groups in the polymer side chain, the specific preparation method is as follows:

(1) The preparation method of linear polymethacrylic acid-2-N, N-dimethylaminoethyl ester (L-PDE-N ₃ ) containing an azide end group functionalization: adopt atom transfer radical polymerization method, control The ratio of initiator and monomer is used to prepare L-PDE-N ₃ with molecular weight between 2000-30000;

(2) The preparation method of polymethacrylamide or polyacrylamide derivatives containing disulfide bonds and alkynyl groups in the side chain: adding propargyl carbonylimidazole compound and cystamine in a molar ratio of 0.5-1.5:1 In an organic solvent, stir and react at 20-60°C for 2-48 hours, and purify to obtain (allyl carbamate ethyl) dithioethylamine compound (PPA-cyst); then (allyl carbamate Ethyl ester) dithioethylamine compound (PPA-cyst) reacts with methacryloyl chloride or acryloyl chloride for purification, then adds an initiator such as azobisisobutyronitrile AIBN and a solvent, and stirs the reaction at 50-80°C 2- 24 hours, or synthesized by ATRP polymerization to prepare polymethacrylamide derivatives P(PPA-cyst-Mam) or polyacrylamide derivatives P(PPA-cyst-Mam) containing disulfide bonds and alkynyl groups in the side chain -Am);

(3) Dissolve the above-mentioned L-PDE-N ₃ and P(PPA-cyst-MAm) or P(PPA-cyst-Am) in an organic solvent according to the molar ratio of azide end group to alkynyl group of 0.5-3:1 Or in water or a mixture of water and organic solvents, react at 20-80°C for 1-48 hours, and purify to obtain a comb-shaped cationic polymer P(PPA-cyst-MAm-PDE) containing a disulfide bond, its structure schematic diagram for:

In the polymer carrier provided by the present invention, the low-molecular-weight functionalized cationic polymer CP-F1 is a polyethyleneimine derivative with a molecular weight between 600-5000 containing a disulfide bond and an alkynyl end group; the main chain is polymerized The compound is a degradable polyaspartic acid derivative with an azide group in its side chain. The specific steps are as follows:

(1) The preparation method of the polyethyleneimine PEI-(PPA-cyst) of alkyne end group functionalization: add propargyl ester carbonylimidazole compound and cystamine in molar ratio 0.5-1.5: 1 in the organic solvent, in 20- Stir the reaction at 60°C for 2-48 hours, and obtain (allyl carbamate ethyl) dithioethylamine compound (PPA-cyst) after purification; then (allyl carbamate ethyl) dithioethylamine Amine compound (PPA-cyst) and 1,1'-carbonyldiimidazole molar ratio 0.4-0.75:1 are added to the organic solvent, stirred and reacted at 20-70°C for 2-48 hours, and purified to obtain (allylaminomethyl Ethyl ester) dithioethyl 1-carboxamide imidazole compound (PPA-cyst-CI); Compound PPA-cyst-CI and polyethyleneimine with a weight average molecular weight between 600-5000 in molar ratio 0.2- 1.1:1 dissolved in an organic solvent, stirred and reacted at 20-60°C for 2-48 hours, and the solvent was removed under reduced pressure to obtain a polyethylenimine functionalized with an alkyne end group containing a small molecule of imidazole, in which each polyethyleneimine The number of alkyne-containing end groups of imines ranges from 0.2 to 1.1 (the vast majority of each polyethyleneimine contains 1 or 0 alkyne-containing end groups, and the average number of alkyne-containing end groups of polyethyleneimine PEI is 0.2-1.1);

(2) The preparation method of the polyaspartic acid derivative that side chain contains azide group: 2-azidoethylamine and polysuccinimide (PSI) are added in the solvent in molar ratio 0.4-2: 1, in Stir and react at 20-70°C for 2-200 hours, if the product still has an unopened ring structure, then add excess N,N-dimethylpropylenediamine or N,N-dimethylethylenediamine, Or ethanolamine, propanolamine, butanolamine or pentanolamine, and react for 24 hours to synthesize a polyaspartic acid derivative P(Asp-Az) containing an azide group in the side chain;

(3) The polyethyleneimine PEI-(PPA-cyst) and P(Asp-Az) functionalized by the above-mentioned alkyne end group are dissolved in an organic solvent according to the molar ratio of the alkyne end group to azide of 0.5-3:1 Or in water or a mixture of water and an organic solvent, the reaction is carried out at 20-80 ° C for 1-96 hours to obtain a modified reduction-sensitive comb-shaped high-molecular-weight cationic polymer P(Asp-Az-Al- ss-PEI), its structure diagram can be one of the following:

The reduction-sensitive comb-type high molecular cationic polymer of the present invention can be combined with exogenous genes and used as a gene carrier.

The gene carrier is a carrier used for gene transduction of human body or animal and plant cells, and can be compounded with the target gene (DNA or RNA) to form a stable complex small particle by using a suitable compounding method. The complex is co-cultivated with human or animal cells, and the target gene (DNA or RNA) can be brought into human or animal cells for release, transfected and expressed, and used for human gene therapy and gene transduction of animals and plants.

When the modified reduction-sensitive comb polymer containing disulfide bonds of the present invention is used as a gene carrier, polyethylene glycol, targeting groups and/or components that can be cross-linked are introduced. The introduction of polyethylene glycol can reduce the rejection of biological systems (such as the human body) to gene carrier materials, greatly reduce the adsorption of proteins, cells and bacteria on the surface of gene carrier materials, and also reduce the rate of clearance by the kidneys (because its volume is relatively small) big). Targeting groups such as folic acid and antibodies can be introduced to improve the targeted delivery characteristics of the gene carrier system. After targeting into tumor cells, the intracellular reducing environment will quickly degrade and release the loaded gene drug. A small amount of components that can be cross-linked can be introduced, and cross-linking can be carried out after the modified comb-type high molecular cationic polymer is loaded with gene drugs, which can improve the stability of the system loaded with gene drugs.

The high molecular polymer of the present invention has the characteristic of bioreduction and degradability. It can bind and compound gene DNA very well outside the cell, and enter the cell because the reducing environment in the cell can quickly break into a polymer with a low molecular weight similar to the raw material and release the contained gene, so it has a high gene transfection at the same time High efficiency and low cytotoxicity, it is a promising polymer gene carrier.

The preparation method of the invention uses the low molecular weight cationic polymer as the main raw material, and utilizes a highly selective reaction such as a click chemical reaction to prepare the final product. The advantage is that the method is simple and does not produce cross-linking. Due to the use of highly selective reactions such as click chemistry, the structure control is easy, the positively charged groups that disappear in the reaction are few or no, and the utilization rate of positively charged groups such as amino groups is high.

Description of drawings

Fig. 1 is (A) monoalkyne end group functionalized polyethyleneimine PEI-(PPA-cyst) (embodiment 4, each polyethyleneimine molecule contains 0.33 alkyne groups on average) in CDCl ₃ , (B) P(Asp-Az) _0.65 (Asp-EA) _0.35 (Example 6) in D ₂ O and (C) comb-shaped high molecular weight cationic polymer P(Asp-Az) modified with disulfide bonds -Al-ss-PEI)(Asp-EA) _0.35 (Example 11) in D ₂ O NMR spectrum

Fig. 2 is the comb type high molecular weight cationic polymer P(PPA-cyst-MAm-PDE)-1 that contains disulfide bond modification of the synthesis of the present invention before (A) after adding 50mmol/L DTT (B) and L- The GPC chromatogram of PDE-N ₃ -2 (C) (embodiment 12)

Fig. 3 is that dynamic light scattering detects comb type high molecular weight cationic polymer P (PPA-cyst-MAm-PDE) and DNA composite particle particle size change with time before and after adding DTT (embodiment 12)

Fig. 4 is the comb type high molecular weight cationic polymer P(Asp-Az-Al-ss-PEI)(Asp-EA) _0.67 containing disulfide bond modification synthesized by the present invention, P(Asp-Az-Al-ss- PEI) (Asp-EA) _0.35 and P (Asp-Az-Al-ss-PEI) and reference polymer PEI 25kDa to the cytotoxicity comparison of 293T (embodiment 13, the graph data is the average value of 4 hole parallel experiment and standard deviation).

Fig. 5 is that the present invention synthesizes and contains disulfide bond modified comb type high molecular weight 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 ₃ -1, the HMPDE of linear high molecular weight and contrast polymer PEI 25kDa are to the cytotoxicity comparison of 293T (embodiment 13, the figure data is the average value of 4 hole parallel experiment and standard deviation).

Fig. 6 is that the present invention synthesizes the cationic polymer P(Asp-Az-Al-ss-PEI) (Asp-EA) _0.67 of the comb type high molecular weight modified containing disulfide bond, P(Asp-Az-Al-ss-PEI )(Asp-EA) _0.35 and P(Asp-Az-Al-ss-PEI) were expressed to 293T cells by luciferase transfection in the absence of serum at different polymer/DNA mass ratios (Example 14, The data shown are the mean and standard deviation of 3 well parallel experiments).

Fig. 7 is the comb type high molecular weight cationic polymer P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az-PDE)-2 containing disulfide bond modification synthesized by the present invention , low-molecular-weight polymer L-PDE-N ₃ -1 and linear high-molecular-weight HMPDE were expressed in luciferase transfection in 293T cells in the absence of serum at different polymer/DNA mass ratios (PEI 25kDa and DNA The ratio of nitrogen to phosphorus is 10, in Example 14, the data shown are the mean and standard deviation of 3 well parallel experiments).

Fig. 8 is the comb-shaped high molecular weight cationic polymer P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az-PDE)-2 containing disulfide bond modification synthesized by the present invention , the low molecular weight polymer L-PDE-N ₃ -1 was expressed by luciferase transfection in HeLa cells in the absence of serum at different polymer/DNA mass ratios (the nitrogen-phosphorus ratio of PEI 25kDa to DNA was 10, Example 14, the illustrated data are the mean and standard deviation of 3 well parallel experiments).

Fig. 9 is the comb-type high molecular weight cationic polymer P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az-PDE)-2 containing disulfide bond modification synthesized by the present invention , the low molecular weight polymer L-PDE-N ₃ -1 was expressed by luciferase transfection in 293T cells in the presence of serum at different polymer/DNA mass ratios (the nitrogen-phosphorus ratio of PEI 25kDa to DNA was 10, Example 15, the illustrated data are the mean and standard deviation of 3 well parallel experiments).

Detailed ways

The present invention is further explained by the following examples, but they are not limiting to the claims.

The structure of the modified comb-type polymer cationic polymer was tested by nuclear magnetic resonance spectroscopy, and the molecular weight of the polymer was tested by gel permeation chromatography (GPC).

Embodiment 1 (allyl carbamate ethyl) dithioethylamine ((propargyl carbamate) ethyldisulfide ethylamine, PPA-cyst) and (allyl carbamate ethyl) dithioethyl 1-carbon amide Synthesis of imidazole ((propargyl carbamate) ethyl disulfide ethyl 1-carbamide-imidazole, PPA-cyst-CI)

(Allyl carbamate ethyl) dithioethylamine ((propargyl carbamate) ethyl disulfideethylamine, PPA-cyst) synthesis: According to the literature [XLJiang, MCLok, WEHennink, Degradable-Brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery, Bioconjugate Chem.18 (2007) 2077-2084] synthesize propargyl ester of carbonyl-imidazole (PPA-CI) by propargyl alcohol and 1,1'-carbonyldiimidazole. First desalt cystamine hydrochloride, weigh 4.40 grams of cystamine and 3.47 grams of propargyl carbonylimidazole in 50 milliliters of chloroform, stir and react at room temperature for 24 hours, remove the solvent, add 80 milliliters of sodium dihydrogen phosphate solution (pH 4.0 ) was dissolved, extracted 3 times with diethyl ether, adjusted the pH of the obtained lower aqueous phase to 9.0, then extracted 3 times with 40 ml of ethyl acetate, combined the organic phases, washed once with 40 ml of pH=9 sodium phosphate solution, and obtained The organic phase was dried with anhydrous magnesium sulfate, filtered, and distilled under reduced pressure to obtain 1.9 g of a yellow oily liquid with a yield of 35%. ¹ H-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 is replaced with other organic solvents without active protons such as dichloromethane, acetone, tetrahydrofuran, and DMF is also carried out and similar results can be obtained.

(Allyl carbamate ethyl) dithioethyl 1-carbamide imidazole ((propargylcarbamate) ethyl disulfide ethyl 1-carbamide-imidazole, PPA-cyst-CI) synthesis: Weigh 1,1'-carbonyl di Dissolve 3.94 g of imidazole (CDI) in 40 ml of chloroform, drop 1.9 g of the above-mentioned PPA-cyst into the chloroform solution of CDI, react at room temperature for 4 hours, and the reaction solution changes from turbid to transparent. Water was added and washed three times, the organic phase was dried with anhydrous magnesium sulfate, filtered, and distilled under reduced pressure to obtain 1.9 g of white solid PPA-cyst-CI with a yield of 71%. ¹ H-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 is replaced with other organic solvents without active protons such as dichloromethane, acetone, tetrahydrofuran, and DMF is also carried out and similar results can be obtained.

Example 2 Mono-azide terminated poly(2-(dimethylamino)ethyl methacrylate), L-PDE-N ₃ )Synthesis:

According to the literature [XLJiang, MCLok, WEHennink, Degradable-BrushedpHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery, Bioconjugate Chem.18(2007) 2077-2084.] first synthesize the initiator 2-bromo-isobutyric acid-3- Propylazide (BiBAP). 6.29 grams of monomer methacrylate-2-N, N-dimethylaminoethyl ester, 260 mg of ligand 1,1,4,7,7,-pentamethyldiethylenetriamine (PMDETA) and 250 mg of initiator BiBAP was dissolved in 15 mL of 1,2-dichlorobenzene, 210 mg of cuprous bromide was added under nitrogen, and then the oxygen in the system was removed through three vacuum-nitrogen cycles, and the mixture was heated in an oil bath at 50°C for reaction 2 Hour. Add 200 ml of n-hexane to stop the reaction and precipitate the product, then repeat the precipitation twice for purification, and dry in a vacuum oven to obtain 5.22 g of the product L-PDE-N ₃ with a yield of 83%. Its number-average molecular weight recorded by proton nuclear magnetic spectrum is 8.6kDa, and its weight-average molecular weight recorded by gel permeation chromatography (GPC) coupled with a multi-angle light scattering detector is 12.7kDa, and the number-average molecular weight is 10.7kDa, and the polydispersity index The PDI is 1.19, represented by L-PDE-N ₃ -1.

In another synthesis formula and steps of L-PDE-N ₃ , the reaction mixture was heated and polymerized in an oil bath at 55°C for 2.5 hours, precipitated, purified and dried in vacuum to obtain 5.79 grams of L-PDE-N ₃ with a yield of 92%. , the number average molecular weight measured by proton nuclear magnetic spectrum is 12.8kDa, the weight average molecular weight measured by GPC coupled with multi-angle light scattering detector is 19.8kDa, the number average molecular weight is 18.5kDa, and the PDI is 1.07. -N ₃ -2 means. By controlling the ratio of initiator and monomer and the reaction conditions, a series of linear polydimethylammonium methacrylate functionalized with different L-PDE-N ₃ azide end groups with a molecular weight of 2000-30000 can be prepared ethyl ester.

The synthesis of the polyethylenimine of embodiment 3 azide end group functionalization

Synthesis of polyethyleneimine PEI ₈₀₀ -(N ₃ ) ₁ functionalized with azide end groups (the subscript number 800 indicates that the weight average molecular weight of PEI is 800, and the subscript number 1 indicates the azide end group of each polyethyleneimine The average is about 1): Reference [2008-033s Chem Comm, 20071219, N2, 190-192, Biodegradable microcapsules designed via'click'chemistry, Bruno G.De Geest; Wim Van Camp, Filip E.Du Prez; Stefaan C . De Smedt; Jo Demeester; Wim E. Hennink] Synthesis of liquid azidopropyl ester of carbonylimidazole (3-azidopropyl ester of carbonylimidazole, AP-CI) via azidopropanol and 1,1'-carbonyldiimidazole. 0.30 gram of propyl azidocarbonylimidazole (AP-CI) was dissolved in 60 milliliters of chloroform, and 1.23 grams of polyethyleneimine with a weight average molecular weight of 800 was dissolved in 20 milliliters of chloroform, and they were mixed and reacted at room temperature for 2 hours, and then heated up Reflux for 10 hours, and remove the solvent chloroform under reduced pressure to obtain 1.2 g of a yellow liquid containing imidazole-containing azide-end functionalized polyethyleneimine. Infrared spectrum analysis showed that the product had an obvious absorption peak at 2099cm ^-1 , indicating that it contained azide. Proton nuclear magnetic spectrum showed that the raw material propyl azido carbonylimidazole had reacted completely, and it was measured that each polyethyleneimine molecule contained 1 azide group. The chloroform of this reaction is replaced with other organic solvents without active protons such as dichloromethane, acetone, tetrahydrofuran, and DMF is also carried out, and the reaction temperature is stirred at 30-80 ° C for 2-48 hours, and similar results can be obtained.

Synthesis of polyethyleneimine PEI _1800- (N ₃ ) ₁ functionalized with azide end groups (the subscript number 1800 indicates that the weight average molecular weight of PEI is 1800, and the subscript number 1 indicates the azide end group of each polyethyleneimine The average is about 1): 0.42 gram of propyl azido carbonyl imidazole is dissolved in 10 milliliters of chloroform, and 4.0 grams of polyethyleneimine with a weight average molecular weight of 1800 is dissolved in 40 milliliters of chloroform, and they are mixed and reacted at room temperature for 2 hours, The temperature was raised to reflux for 10 hours, and the solvent chloroform was removed under reduced pressure to obtain 4.4 g of a yellow oily viscous liquid, which was polyethyleneimine functionalized with azide end groups containing imidazole small molecules. Infrared spectrum analysis showed that the product had an obvious absorption peak at 2099cm ^-1 , indicating that it contained azide. The hydrogen nuclear magnetic spectrum shows that the raw material azidopropyl ester carbonylimidazole has been completely reacted, and it is measured that each polyethyleneimine molecule contains an average of 1 azide group on average. The chloroform of this reaction is replaced with other organic solvents without active protons such as dichloromethane, acetone, tetrahydrofuran, and DMF is also carried out, and the reaction temperature is stirred at 30-80 ° C for 2-48 hours, and similar results can be obtained.

Polyethyleneimine PEI with a weight average molecular weight between 600-5000 is dissolved in an organic solvent at a molar ratio of 0.3-1.1:1, stirred and reacted at 25-60°C for 2-24 hours, and the solvent is removed under reduced pressure to obtain imidazole-containing The azide end group functionalized polyethyleneimine of small molecules, wherein each polyethyleneimine has an average number of azide end groups of 0.3-1.1.

The synthesis of the polyethylenimine of embodiment 4 alkyne end group functionalization

Synthesis of polyethyleneimine PEI _800- (PPA-cyst) ₁ functionalized with alkyne end groups (the subscript number 800 indicates that the weight average molecular weight of PEI is 800, and the subscript number 1 indicates the average number of alkyne end groups of each polyethyleneimine The number is about 1): 1.26 grams of PPA-cyst-CI prepared in Example 1 are dissolved in 100 milliliters of chloroform, and 3.07 grams of polyethyleneimine with a weight average molecular weight of 800 are dissolved in 50 milliliters of chloroform, and they are mixed at room temperature Reacted for 2 hours, then raised the temperature and refluxed for 24 hours, and removed the solvent chloroform under reduced pressure to obtain 4.26 g of polyethylenimine PEI _800- (PPA-cyst) ₁ yellow viscous liquid containing the alkyne end group functionalization of imidazole small molecules, producing The rate is 98%. Proton nuclear magnetic spectrum showed that the raw material PPA-cyst-CI had reacted completely, and it was measured that each polyethyleneimine molecule contained 1.0 alkyne groups. The chloroform of this reaction is replaced by other organic solvents without active protons such as dichloromethane, acetone, tetrahydrofuran, DMF, and the same is carried out, and the reaction temperature is 30-80 ° C and the reaction is stirred for 2-48 hours.

Synthesis of polyethyleneimine PEI1 ₈₀₀ -(PPA-cyst) ₁ functionalized with alkyne end group (subscript number 1800 indicates PEI weight average molecular weight is 1800, subscript number 1 indicates the average number of alkyne end groups of each polyethyleneimine The number is about 1): same as above-mentioned synthetic PEI _800- (PPA-cyst) ₁ , 0.50 gram of PPA-cyst-CI prepared in Example 1 is dissolved in 50 milliliters of chloroform, and 2.74 grams of polyethylene with a weight average molecular weight of 1800 The imine was dissolved in 50 ml of chloroform, and they were mixed and reacted at room temperature for 2 hours, then heated and refluxed for 24 hours, and the solvent chloroform was removed under reduced pressure to obtain alkyne-end-functionalized polyethyleneimine PEI _1800- (PPA-cyst) ₁ Yellow viscous liquid 2.96g. Proton nuclear magnetic spectrum showed that the raw material PPA-cyst-CI had reacted completely, and it was measured that each polyethyleneimine molecule contained 1.0 alkyne groups. The chloroform of this reaction is replaced by other organic solvents without active protons such as dichloromethane, acetone, tetrahydrofuran, DMF, and the same is carried out, and the reaction temperature is 30-80 ° C and the reaction is stirred for 2-48 hours.

Synthesis of polyethyleneimine PEI _1800- (PPA-cyst) functionalized by mono-alkyne end group (subscript number 1800 represents PEI weight-average molecular weight is 1800): same as above-mentioned synthetic PEI _1800- (PPA-cyst) ₁ , will 0.33 grams of PPA-cyst-CI prepared in Example 1 was dissolved in 30 milliliters of chloroform, and 5.40 grams of polyethyleneimine with a weight average molecular weight of 1800 was dissolved in 80 milliliters of chloroform, and they were mixed and reacted at room temperature for 1 hour, and then heated to reflux After reacting for 24 hours, the solvent chloroform was removed under reduced pressure to obtain 5.61 g of a yellow viscous liquid of polyethyleneimine PEI ₁₈₀₀ -(PPA-cyst) functionalized with a monoalkyne end group containing a small imidazole molecule. The proton nuclear magnetic spectrum (Figure 1) shows that the raw material PPA-cyst-CI has reacted completely, and it is measured that each polyethyleneimine molecule contains an average of 0.33 alkyne groups, ensuring that most of each polyethyleneimine contains an alkyne end The base is 1 or 0. The chloroform of this reaction is replaced by other organic solvents without active protons such as dichloromethane, acetone, tetrahydrofuran, DMF, and the same is carried out, and the reaction temperature is 30-80 ° C and the reaction is stirred for 2-48 hours. Polyethyleneimine PEI with a weight-average molecular weight between 600-5000 can be modified by the above method to obtain alkyne-end functionalized polyethyleneimine PEI, wherein each polyethyleneimine contains an average of alkyne-end groups The quantity is 0.3-1.1.

Example 5 The preparation method of the polyaspartic acid derivative P(Asp-ss-Al) containing a disulfide bond and an alkynyl group in the side chain:

Dissolve 0.375 grams of self-made polysuccinimide (PSI) in 12 milliliters of DMF, then (allyl carbamate ethyl) dithioethylamine compound (PPA-cyst) 1.36 g into the DMF solution and reacted at 55°C for 24 hours. Precipitate and purify three times with 200 ml of diethyl ether, and dry in vacuo to obtain 0.92 g of a yellow powder solid. Infrared spectrum analysis showed that the product had an obvious absorption peak at 2121cm-1, indicating that it contained an alkynyl group. A new characteristic peak appears at 4.0-4.8ppm in the proton nuclear magnetic spectrum of the product, and the calculated ring-opening rate of polysuccinimide is 65%. The product is represented by P(Asp-ss-Al)-1.

The preparation of the polyaspartic acid derivative P (Asp-ss-Al) that side chain contains 100% alkynyl group: Experimental procedure is the same as above, weighs 1.0 grams of PSI and dissolves in 20 milliliters of DMF, then the preparation of Example 1 4.56 g of (allyl carbamate ethyl) dithioethylamine compound (PPA-cyst) was added into the DMF solution, and reacted at 65° C. for 48 hours. It was purified by precipitation three times with 300 ml of diethyl ether, and dried in vacuo to obtain 1.74 g of a yellow powder solid. Infrared spectrum analysis showed that the product had an obvious absorption peak at 2121cm-1, indicating that it contained an alkynyl group. The proton nuclear magnetic spectrum shows that the characteristic peak of polysuccinimide at 5.0-5.2 disappears, and the ring-opening rate of polysuccinimide is 100%. The product is represented by P(Asp-ss-Al)-2.

According to the molar ratio of PPA-cyst and PSI at 0.2-2:1, stirring and reacting at 20-70°C for 2-48 hours, the polyhexine with a molar percentage of alkynyl groups in the side chain of 15%-100% can be prepared. Aspartic Amino Acid Derivative P(Asp-ss-Al)

Example 6 The preparation method of polyaspartic acid derivatives containing azide groups in the side chain:

0.28 grams of self-made polysuccinimide (PSI) and 0.1, 0.2 or 0.5 grams of self-made 2-azidoethylamine (according to the molar ratio of azide and PSI repeating unit respectively 0.4:1, 0.8:1 and 2: 1) Add in the solvent, mix and react at room temperature for 96 hours, reprecipitate and purify to obtain polyaspartic acid derivatives P(Asp-Az) with different proportions of azide groups in the side chain, and the products are 0.29 grams, 0.30 grams, or 0.33 grams. The PSI rings of the latter have been completely opened as detected by proton nuclear magnetic spectroscopy, indicating that all side chains have been connected with azide groups. The products of the former two were detected by proton nuclear magnetic spectrum, and there were unopened ring structures, and then an excessive amount of ethanolamine was added for ring-opening reaction for 24 hours to obtain polyaspartic acid derivatives containing azide groups and ethanol groups in the side chains. P(Asp-Az), the percentages of azide-containing groups in _its side chain detected by proton nuclear magnetic spectrum are 33% and 65% respectively (Fig. 1). EA) _0.67 and P(Asp-Az) _0.65 (Asp-EA) _0.35 . Infrared spectrum analysis shows that this kind of product has obvious absorption peak at 2100cm ^-1 , indicating that it contains azide. Adding excessive ethanolamine to open the ring can also be replaced by N,N-dimethylpropylenediamine or N,N-dimethylethylenediamine, or propanolamine, butanolamine or pentanolamine, and the side chain containing For the polyaspartic acid derivative P(Asp-Az) with different ratios of azide groups, the reaction can be stirred at 20-70°C for 2-200 hours.

Example 7 The preparation method of polymethacrylamide or polyacrylamide derivatives containing disulfide bonds and alkynyl groups in the side chain:

The synthesis of the methacrylamide monomer PPA-cyst-MAm that side chain contains disulfide bond and alkynyl group: the (allyl carbamate ethyl) dithioethylamine compound that embodiment 1 makes (PPA-cyst) 2.5 g, triethylamine 1.62 g were dissolved in 40 ml of dry dichloromethane, cooled with an ice-water bath, a solution of 1.67 g of methacryloyl chloride in dichloromethane (20 ml) was added dropwise, and dissolved in an ice-water bath Reacted for 2 hours at room temperature, then reacted at room temperature for 15 hours, purified and recrystallized to obtain white crystal PPA-cyst-MAm, the yield was 46%. ¹ H-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 & 5.74 (2H, CH ₂ =C).

Weigh 0.907 grams of monomer PPA-cyst-MAm, 9.85 milligrams of initiator azobisisobutyronitrile (AIBN) are dissolved in 4 milliliters of dry DMF, remove the oxygen in the system by two vacuum-nitrogen cycles, and React in an oil bath at 70°C for 24 hours under protection. Precipitate and purify three times with 200 ml of diethyl ether respectively to obtain 0.68 g of white powder. The weight-average molecular weight measured by gel permeation chromatography (GPC) coupled with a multi-angle light scattering detector is 15.1 kDa, and the polydispersity index PDI is 1.98. Infrared spectrum analysis showed that the product had an obvious absorption peak at 2127cm-1, indicating that it contained an alkynyl group.

When preparing the above-mentioned monomers, acryloyl chloride is used instead of methacryloyl chloride for reaction, and then polyacrylamide derivative P(PPA-cyst-Am) is also obtained in the repolymerization reaction. Add an initiator such as azobisisobutyronitrile AIBN and a solvent during the polymerization reaction, and stir the reaction at 50-80°C for 2-24 hours, or it can also be prepared by ATRP polymerization containing disulfide bonds and alkynyl groups in the side chain. Polymethacrylamide derivative P(PPA-cyst-Mam) or polyacrylamide derivative P(PPA-cyst-Am)

Examples 8-11 are click chemical synthesis of reduction-sensitive comb-type high molecular weight cationic polymer series containing disulfide bond modification

Embodiment 8 Preparation of comb-type high molecular weight polycation P (Asp-ss-Al-Az-PDE) containing disulfide bond modification:

Click chemistry synthesis of comb-shaped polydimethylaminoethyl methacrylate P(Asp-ss-Al-Az-PDE)-1 containing disulfide bonds: functionalized monoazide end group prepared in Example 2 Linear polymethacrylic acid-2-N, N-dimethylaminoethyl ester L-PDE-N ₃ -1 200 milligrams, the side chain containing alkynyl group mole percentage that makes in the embodiment 5 is the polyhexene of 65% Aspartic amino acid derivative P(Asp-ss-Al)-130 mg was dissolved in 10 ml of DMF, 15 mg of cuprous bromide was added under nitrogen, and reacted in an oil bath at 50°C for 2 days under nitrogen protection, and packed Put into the dialysis bag (MWCO 8k-12kDa) and dialyze with water, freeze-drying obtains comb-shaped polydimethylaminoethyl methacrylate cationic polymer P(Asp-ss-Al-Az-PDE)-1, and its nuclear magnetic spectrum is in A new characteristic peak appeared at 8.15ppm, which was the characteristic peak of the proton on the triazole ring generated by click chemistry. There is no obvious absorption peak around 2100cm ^-1 in the infrared spectrum, indicating that the alkynyl and azide groups basically disappear after the click chemical reaction, indicating that the click chemistry has been basically completed. The weight-average molecular weight measured by gel permeation chromatography (GPC) coupled with a multi-angle light scattering detector is 243kDa, and the PDI is 1.63.

Synthesis of another comb-shaped polydimethylaminoethyl methacrylate P(Asp-ss-Al-Az-PDE)-2 containing a disulfide bond: the steps are the same as above, the difference is that L-PDE-N ₃ -1 Consumption is reduced to 102 milligrams, and the prepared comb-shaped polydimethylaminoethyl methacrylate cationic polymer is represented by P(Asp-ss-Al-Az-PDE)-2, and its weight-average molecular weight is 157kDa, and PDI is 1.80. This click chemistry reaction can also be performed using the solvent DMSO instead of DMF. CuBr in this reaction can be replaced by CuCl; this reaction can also not use CuBr, but adopts raising reaction temperature (80 ℃) and prolonging reaction time (48 hours).

Example 9 Preparation of comb-shaped high molecular weight cationic polymer P(Asp-ss-Al-Az-PEI) containing disulfide bond modification: the side chain prepared in Example 5 contains disulfide bond and alkynyl group The polyaspartic acid derivative P(Asp-ss-Al)-181 mg, 40 mg of N,N-dimethylpropylenediamine were dissolved in 10 milliliters of DMF, reacted at room temperature for 12 hours, and then added the preparation in Example 3 The polyethyleneimine PEI _1800- (N ₃ ) ₁ 1.07 grams of the obtained azide end group functionalization, removes the oxygen in the system by three vacuum-nitrogen circulations, adds cuprous bromide 18 milligrams under nitrogen, and under nitrogen Under protection, it was reacted in an oil bath at 50°C for 2 days, put into a dialysis bag (MSCO3.5kDa) and dialyzed with water, filtered the precipitate that appeared during the dialysis, and freeze-dried to obtain 70 mg of P(Asp-ss-Al-Az-PEI). The N,N-dimethylpropylenediamine here can also be replaced by excess N,N-dimethylethylenediamine, ethanolamine, propanolamine, butanolamine or pentanolamine.

Embodiment 10 Preparation of comb-type high molecular weight cationic polymer P (PPA-cyst-MAm-PDE) containing disulfide bond modification:

Weigh 20 mg of P(PPA-cyst-MAm) prepared in Example 7, L-PDE-N ₃ -2216 or 325 mg obtained in Example 2, dissolve in 10 ml of DMF, and pass through three vacuum-nitrogen cycles The oxygen in the system was removed, and 15 mg of cuprous bromide was added under nitrogen, and reacted in an oil bath at 50°C for 2 days under the protection of nitrogen. The prepared polymer is dissolved and loaded into a dialysis bag (MWCO 8k-12kDa) for water dialysis, and freeze-dried to obtain 191 mg of comb-shaped polydimethylaminoethyl methacrylate cationic polymer (using P(PPA-cyst-MAm- PDE)-1) and 305 mg (expressed as P(PPA-cyst-MAm-PDE)-2), P(PPA-cyst-MAm-PDE)-1 was measured by GPC with a multi-angle light scattering detector The weight average molecular weight is 266kDa, the PDI is 1.24, and the number of connected branched chains is about 13; the weight average molecular weight of P(PPA-cyst-MAm-PDE)-2 is 88.5kDa, the PDI is 1.60, and the connected branched chains The number is about 4.

Embodiment 11 Preparation of comb-shaped high molecular weight polycation P (Asp-Az-Al-ss-PEI) containing disulfide bond modification:

The P(Asp-Az) _0.33 (Asp-EA) _0.67 50 mg prepared in Example 6, and the polyethyleneimine PEI-(PPA-cyst) of the mono-alkyne terminal functionalization prepared in Example 4 1.18 g (each polyethyleneimine molecule contains an average of 0.33 alkyne groups, according to the molar ratio of alkynyl end group to azide 2:1), dissolved in a mixed solvent of 5 ml water and 5 ml DMF, added CuBr 20 mg was reacted in an oil bath at 50°C for 2 days under the protection of nitrogen. The obtained polymer solution was loaded into a dialysis bag (MWCO 3.5kDa) and dialyzed with water for 5 days, and freeze-dried to obtain the comb-shaped high molecular weight cationic polymer P(Asp-Az-Al-ss-PEI) containing disulfide bond modification ( Asp-EA) _0.67 . Its H NMR spectrum has an obvious peak at 7.9ppm, which is the H of the triazole ring, indicating that the triazole ring has been generated by click chemistry. Infrared spectrum analysis shows that the product has no obvious absorption peak at 2100cm ^-1 , indicating that the azide side basic reaction. This click chemistry reaction can also be performed using the solvent DMSO instead of DMF. CuBr in this reaction can be replaced by CuCl; this reaction can also not use CuBr, but adopts raising reaction temperature (80 ℃) and prolonging reaction time (48 hours).

The P(Asp-Az) _0.65 (Asp-EA) _0.35 50 mg prepared in Example 6, and the polyethyleneimine PEI-(PPA-cyst) of the monoalkyne terminal functionalization prepared in Example 4 2.41 g (each polyethylenimine molecule contains an average of 0.33 alkyne groups, according to the molar ratio of alkynyl end group to azide 2:1), dissolved in a mixed solvent of 5 ml water and 5 ml DMF, added CuBr 20 mg was reacted in an oil bath at 50°C for 2 days under the protection of nitrogen. The prepared polymer solution was loaded into a dialysis bag (MWCO 3.5kDa) and dialyzed with water, and freeze-dried to obtain a comb-shaped high-molecular-weight cationic polymer P(Asp-Az-Al-ss-PEI) (Asp-Az-Al-ss-PEI) (Asp- EA) _0.35 . Its H NMR spectrum (Figure 1) has an obvious peak at 7.9ppm which is the H of the triazole ring, indicating that the triazole ring was generated by click chemistry. This click chemistry reaction can also be performed using the solvent DMSO instead of DMF. CuBr in this reaction can be replaced by CuCl; this reaction can also not use CuBr, but adopts raising reaction temperature (80 ℃) and prolonging reaction time (48 hours).

50 milligrams of P(Asp-Az) made in embodiment 6, and 2.62 grams of polyethyleneimine PEI-(PPA-cyst) of mono-alkyne terminal functionalization made in embodiment 4 (each polyethylene The imine molecule contains an average of 0.33 alkyne groups, according to the molar ratio of alkyne end group to azide (2:1), dissolve in 10 ml of DMF, add 20 mg of CuBr, and react in an oil bath at 50°C under nitrogen protection 2 days. The prepared polymer solution is loaded into a dialysis bag (MWCO 3.5kDa) and dialyzed with water, and freeze-dried to obtain a comb-shaped high-molecular-weight cationic polymer P(Asp-Az-Al-ss-PEI) containing a disulfide bond modification whose nuclear magnetic hydrogen There is an obvious peak at 7.9ppm in the spectrum, which is the H of the triazole ring, indicating that the triazole ring is generated by click chemistry, and its elution time is much earlier than that of the raw material PEI-1.8kDa as measured by gel permeation chromatography (GPC). The time shows that the molecular weight of the comb-shaped high molecular weight cationic polymer P(Asp-Az-Al-ss-PEI) modified with disulfide bonds is much larger than that of the raw material PEI-1.8kDa. This click chemistry reaction can also be performed using the solvent DMSO instead of DMF. CuBr in this reaction can be replaced by CuCl; this reaction can also not use CuBr, but adopts raising reaction temperature (80 ℃) and prolonging reaction time (48 hours).

Example 12 Reduction Sensitive Properties of Comb-type High Molecular Weight Cationic Polymers Containing Disulfide Bond Modification

The P(PPA-cyst-MAm-PDE)-1 prepared in Example 10 was dissolved in 0.3 mol/L NaAc, pH 4.4 buffer solution to prepare a 5.0 mg/mL solution. Take 1mL of this polymer solution, add 53 microliters of 1.0mol/L 1,4-dithiothreitol (DTT) aqueous solution to mix, place it at room temperature for 1 hour, and measure its concentration with GPC coupled with a multi-angle light scattering detector. The weight-average molecular weight is 24.4kDa, and the PDI is 1.11, as shown in Figure 2, which shows that the molecular weight of the degradation product is closer to the molecular weight ( _{Mw 19.8kDa} , PDI 1.07) of the raw material L-PDE-N used, indicating that the Sulfur-bonded modified comb-type high molecular weight cationic polymers with reduction-sensitive properties.

Dissolve P(PPA-cyst-MAm-PDE)-1 and P(PPA-cyst-MAm-PDE)-2 in HBS buffer (20mM Hepes, 130mM NaCl, pH 7.4) respectively to configure 62.5mg/L solution, Add 600 microliters of polymer solution to 150 microliters of pcDNA3-Luc plasmid DNA solution (50 mg/L in HBS), mix well and compound at 37°C for 30 minutes, then add 8.3 microliters of 1.0mol/L 1,4-dithiothreitol (DTT) aqueous solution, and then use dynamic light scattering to detect the change of particle size in the solution with time, the results are shown in Figure 3. It can be seen from the figure that the composite particles are stable without adding DTT, but the composite particles rapidly increase and become unstable after adding DTT, which also shows that the modified comb-shaped high molecular weight cationic polymers containing disulfide bonds have reduction-sensitive properties.

Example 13 Cationic polymer cytotoxicity in vitro

The toxicity of cationic polymers to cells is an important evaluation index of polymer gene delivery carriers, which is generally measured by the inhibition rate of cells under the action of different concentrations of polymers. Here, the in vitro cytotoxicity of the disulfide bond-modified comb-shaped high-molecular-weight cationic polymers synthesized in Examples 8-11 on 293T cells was measured by a tetramethylazolium salt (MTT) colorimetric method.

293T cells were seeded at 3000 cells/well in a 96-well culture plate and added with 100 μL of serum-containing DMEM medium, and cultured at 37°C for 48 hours in a 5% _CO2 incubator. Add filter-sterilized polymer solutions of different concentrations (0.01 to 0.20 mg/mL) (100 μL and 100 μL fresh DMEM medium) to each well of cells, respectively. Continue culturing for 48 h until the end of the material action period, remove the medium in all wells containing cells, and add 200 μL of fresh DMEM medium. 20 μL of MTT (5 mg/ml) was added to all wells. Wrap the culture plate with aluminum foil and incubate for 4 h at 37°C in a humid environment. The culture medium and MTT in the wells were discarded, 200 μL of DMSO was added to each well and shaken at room temperature, and the absorbance value was recorded at 570 nm with a microplate reader (550 BIO-RAD, USA). The relative cell survival rate was calculated according to the absorbance value.

The comb-shaped high molecular weight cationic polymer P(Asp-Az-Al-ss-PEI)(Asp-EA) prepared in Example 11 containing disulfide bond modification _0.67 , P(Asp-Az-Al-ss-PEI )(Asp-EA) _0.35 and P(Asp-Az-Al-ss-PEI) and the toxicity of the control polymer PEI 25kDa are shown in Figure 4, which shows that when the concentration of the polymer is lower than 0.03 mg/ml, adding disulfide Bond-modified comb-type high molecular weight cationic polymer P(Asp-Az-Al-ss-PEI)(Asp-EA) _0.67 , P(Asp-Az-Al-ss-PEI)(Asp-EA) _0.35 and P (Asp-Az-Al-ss-PEI) relative cell viability was about 80%, while relative cell viability was less than 20% when 0.03 mg/ml 25kDa PEI was added.

The disulfide bond-containing modified comb-type high molecular weight polycation P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az-PDE)-2 prepared in Example 8 and the control polymerization The toxicity of substance PEI 25kDa is shown in Fig. 5, 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 The toxicity of _3-1 and linear high molecular weight PDE (HMPDE, weight average molecular weight 154kDa, PDI 1.58.4) synthesized by ordinary free radical polymerization is also shown in Fig. 5 as a control. The figure shows that when the concentration of the polymer is lower than 0.03 mg/ml, the addition of the disulfide bond-modified comb-shaped high molecular weight cationic polymers P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss- The relative cell survival rate was greater than 90% when Al-Az-PDE)-2 was added, while the relative cell survival rate was less than 10% when 0.03 mg/ml 25kDa PEI was added. , the toxicity of the raw material low molecular weight polymer L-PDE-N ₃ -1 is very low, but the toxicity of P(Asp-ss-Al-Az-PDE)-2 is much lower than that of linear high molecular weight HMPDE with comparable molecular weight many. This indicates that the comb-type high molecular weight cationic polymers modified with disulfide bonds are less toxic.

The in vitro transfection efficiency of embodiment 14 polymer/DNA complex when there is no serum

The comb-shaped high-molecular-weight cationic polymers containing disulfide bonds modified in Examples 8-11 were respectively dissolved in NaCl solution (150mmol/L, pH7.4), filtered and sterilized, and prepared into solutions of corresponding concentrations. Add 50 μL solution containing 1.0 μg plasmid DNA (pcDNA3-Luc is the reporter gene of luciferase) to a 1.5 mL sterile centrifuge tube, add corresponding amount of 50 μL cationic polymer solution according to N/P ratio of 10-50, and DNA Composite, shaking for 5s, the complex was used for cell transfection experiments after standing at 37°C for half an hour. Using 25kDa PEI and 1.8kDa PEI as controls, the in vitro transfection efficiency of the complex on 293T and HeLa cells was compared (after adding the complex and serum-free DMEM medium, transfect at 37°C for 4 hours, and the medium was replaced with 1 mL of complete After DMEM medium, culture was continued at 37°C for 48h). Figure 6 shows the comb-shaped high molecular weight cationic polymer P(Asp-Az-Al-ss-PEI)(Asp-EA) _0.67 modified with disulfide bonds, P(Asp-Az-Al-ss-PEI)( Asp-EA) _0.35 and P(Asp-Az-Al-ss-PEI) at different polymer/DNA mass ratios on the in vitro luciferase transfection expression efficiency of 293T cells. The gene transfection efficiency of the complex of P(Asp-Az-Al-ss-PEI)(Asp-EA) _0.67 is higher than that of the common polymer gene carrier reference polymer 25kDa PEI. Figure 7 shows P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az-PDE)-2, low molecular weight polymer L-PDE-N ₃ -1 and linear high molecular weight The transfection efficiency of HMPDE to 293T cells in vitro luciferase at different polymer/DNA mass ratios, and the gene transfection efficiency of the control polymer PEI 25kDa and DNA at a nitrogen-to-phosphorus ratio of 10 were also used. Figure 8 shows the comb-shaped high molecular weight cationic polymers P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az-PDE)-2 modified with disulfide bonds, low molecular weight Polymer L-PDE-N ₃ -1 was expressed by luciferase transfection in HeLa cells in the absence of serum at different polymer/DNA mass ratios (Nitrogen-Phosphorus ratio of PEI 25kDa to DNA was 10). These figures illustrate that the gene transfection efficiency of complexes containing disulfide bond-modified comb-shaped high-molecular-weight cationic polymer P(Asp-ss-Al-Az-PDE) is much higher than that of the starting low-molecular-weight polymer L-PDE-N The complex of ₃ -1 (2-3 orders of magnitude) is also higher than the gene transfection efficiency of the complex of the reference polymer 25kDa PEI of the commonly used high molecular gene carrier. The results showed that this kind of reduction-sensitive disulfide bond-modified comb-shaped high-molecular-weight cationic polymer had high transfection efficiency to 293T and HeLa cells.

The in vitro transfection efficiency of embodiment 15 polymer/DNA complex when serum exists

The steps were the same as in Example 14, except that the transfection was at 37°C for 4 hours after adding the complex and DMEM medium with 10% serum, and the culture medium was replaced with 1 mL of complete DMEM medium and continued to culture at 37°C for 48 hours). Figure 9 shows the comb-shaped high molecular weight cationic polymers P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az-PDE)-1 modified with disulfide bonds in the presence of serum 2. Low-molecular-weight polymer L-PDE-N ₃ -1 at different polymer/DNA mass ratios on the transfection and expression efficiency of the complex to 293T cell luciferase. The results showed that the transfection efficiency of the reduction-sensitive disulfide bond-modified comb-shaped high-molecular-weight cationic polymer complex to 293T cells was also high in the presence of serum.

Example 16 polymer/DNA complex in vitro green fluorescent protein gene transfection efficiency

Using the comb-type high molecular weight cationic polymer P(Asp-ss-Al-Az-PDE)-1 and P(Asp-ss-Al-Az modified by disulfide bonds obtained in Example 8 and Example 11 -PDE)-2, P(Asp-Az-Al-ss-PEI)(Asp-EA) _0.67 , P(Asp-Az-Al-ss-PEI)(Asp-EA) _0.35 and P(Asp-Az- Al-ss-PEI), P(PPA-cyst-MAm-PDE)-1 and P(PPA-cyst-MAm-PDE)-2 were respectively combined with plasmid DNA pEGFP (green fluorescent protein gene) to transfect 293T cells in vitro. After 48 hours of transfection, the expression of green fluorescent protein was observed with a fluorescence microscope. The results showed that all of them could effectively transduce the cells.

Claims (10)

1. A comb-shaped macromolecule gene carrier, the structure of which is shown in the following formula:

In the formula, CP is a low-molecular-weight cationic polymer with a molecular weight between 600-30000, F1 and F2 are groups that can react with each other, and F1 or F2 contains a disulfide bond, M is the main chain unit structure of the polymer, and n= 10-500; m<n, m=0-400, l=0-800; l, m, n are integers. 2. The comb-shaped polymer gene carrier according to claim 1, characterized in that CP is linear polymethacrylate-2-N, N-dimethylaminoethyl ester or polyethyleneimine. 3. The comb-shaped polymer gene carrier according to claim 1 or 2, characterized in that the main chain unit structure of the polymer is polyaspartic acid derivatives, polymethacrylamide or polyacrylamide derivatives. 4. the preparation method of polymer gene carrier described in claim 1 is characterized in that, comprises the steps: (1) synthesis of a low molecular weight cationic polymer CP-F1 with a reactive end group F1; (2) Synthesize the main chain polymer, its side chain contains the group F2 that can react with the F1 end group, and F1 or F2 contains a disulfide bond, as shown in the right formula:

(3) The above two polymers are mixed, reacted and purified to prepare a comb-shaped cationic polymer containing disulfide bonds. 5. the preparation method of polymer gene carrier as claimed in claim 4 is characterized in that, F1 is containing azide group, F2 is containing disulfide bond and alkynyl group; Or F1 is containing disulfide bond and alkynyl group; base group, F2 is an azide-containing group. 6. preparation method as claimed in claim 4, is characterized in that: The step 1 is: using atom transfer radical polymerization method, controlling the ratio of initiator and monomer to prepare L-PDE-N ₃ with molecular weight between 2000-30000; The step 2 is: adding propargyl carbonylimidazole compound and cystamine into the organic solvent in a molar ratio of 0.5-1.5:1, stirring and reacting at 20-60°C for 2-48 hours, and purifying to obtain (allylaminomethyl Acid ethyl) dithioethylamine compound; then (allyl carbamate ethyl) dithioethylamine compound and polysuccinimide are added in the solvent at a molar ratio of 0.2-2:1, at 20- Stirring and reacting at 70°C for 2-48 hours to synthesize polyaspartic acid derivative P(Asp-ss-Al) containing disulfide bonds and alkynyl groups in the side chain; The step 3 is: dissolve the above-mentioned L-PDE-N ₃ and P(Asp-ss-Al) in an organic solvent or water or water and organic In the mixture of solvents, react at 20-80°C for 1-48 hours, if the product still has an unopened ring structure, then add excess N,N-dimethylpropylenediamine or N,N-di Methylethylenediamine, or ethanolamine, propanolamine, butanolamine or pentanolamine was reacted for 24 hours to obtain a comb-shaped cationic polymer P(Asp-ss-Al-Az-PDE) containing a disulfide bond. 7. preparation method as claimed in claim 4, is characterized in that: The step 1 is: dissolving the propyl azidocarbonylimidazole compound and polyethyleneimine with a weight average molecular weight between 600-5000 in a molar ratio of 0.3-1.1:1 in an organic solvent, stirring at 25-60°C After reacting for 2-24 hours, the solvent is removed under reduced pressure to obtain a polyethyleneimine functionalized with azide end groups containing imidazole small molecules, wherein the number of azide end groups in each polyethyleneimine is 0.3-1.1; The step 2 is: adding propargyl carbonylimidazole compound and cystamine into the organic solvent in a molar ratio of 0.5-1.5:1, stirring and reacting at 20-60°C for 2-48 hours, and purifying to obtain (allylaminomethyl Acid ethyl) dithioethylamine compound; then (allyl carbamate ethyl) dithioethylamine compound and polysuccinimide are added in the solvent at a molar ratio of 0.2-2:1, at 20- Stirring and reacting at 70°C for 2-48 hours to synthesize and prepare a polyaspartic acid derivative P(Asp-ss-Al) with a molar percentage of alkynyl-containing groups in the side chain of 15%-100%; The step 3 is: dissolve the above-mentioned P(Asp-ss-Al) in an organic solvent, if there is still an unopened ring structure, then first add an excess of N,N-dimethylpropylenediamine or N , N-dimethylethylenediamine, or ethanolamine, propanolamine, butanolamine or pentanolamine, reacted for 24 hours, and then the above-mentioned azide end group functionalized polyethyleneimine was combined with azide end group and The molar ratio of the alkynyl group is 0.5-3:1, which is dissolved in an organic solvent or water or a mixture of water and an organic solvent, and reacted at 20-80°C for 1-48 hours to obtain a comb-shaped cationic polymer P containing a disulfide bond (Asp-ss-Al-Az-PEI). 8. preparation method as claimed in claim 4, is characterized in that: The step 1 is: the preparation method of linear polymethacrylic acid-2-N, N-dimethylaminoethyl ester L-PDE- _N3 functionalized with an azide end group: using atom transfer radical polymerization method, Controlling the ratio of initiator and monomer to produce L-PDE-N ₃ with molecular weight between 2000-30000; The step 2 is: adding propargyl carbonylimidazole compound and cystamine into the organic solvent in a molar ratio of 0.5-1.5:1, stirring and reacting at 20-60°C for 2-48 hours, and purifying to obtain (allylaminomethyl ester ethyl) dithioethylamine compound (PPA-cyst); then (allyl carbamate ethyl) dithioethylamine compound and methacryloyl chloride or acryloyl chloride reaction purification; add initiator and Solvent, stirred at 50-80°C for 2-24 hours through ordinary free radical polymerization, or through atom transfer free radical polymerization, synthesized to obtain polymethacrylamide derivatives with side chains containing disulfide bonds and alkynyl groups Substance P (PPA-cyst-Mam) or polyacrylamide derivative P (PPA-cyst-Am); The step 3 is: dissolving the above-mentioned L-PDE-N ₃ and P(PPA-cyst-MAm) or P(PPA-cyst-Am) according to the molar ratio of azide end group to alkynyl group of 0.5-3:1 React in an organic solvent or water or a mixture of water and an organic solvent at 20-80° C. for 1-48 hours, and purify to obtain a comb-shaped cationic polymer P(PPA-cyst-MAm-PDE) containing a disulfide bond. 9. preparation method as claimed in claim 4, is characterized in that: The step 1 is: adding propargyl carbonylimidazole compound and cystamine into an organic solvent in a molar ratio of 0.5-1.5:1, stirring and reacting at 20-60°C for 2-48 hours, and purifying to obtain (allylaminomethyl ester ethyl) dithioethylamine compound; then (allyl carbamate ethyl) dithioethylamine compound and 1,1'-carbonyldiimidazole molar ratio 0.4-0.75:1 added to the organic solvent , stirred and reacted at 20-70°C for 2-48 hours, and purified to obtain (allyl carbamate ethyl) dithioethyl 1-carbonamide imidazole compound (PPA-cyst-CI); the compound PPA-cyst - CI and polyethyleneimine with a weight average molecular weight between 600-5000 are dissolved in an organic solvent in a molar ratio of 0.2-1.1:1, stirred and reacted at 20-60°C for 2-48 hours, and the solvent is removed under reduced pressure to obtain Polyethyleneimine functionalized with alkyne end groups containing imidazole small molecules, wherein the number of alkyne end groups in each polyethyleneimine ranges from 0.2 to 1.1; The step 2 is: add 2-azidoethylamine and polysuccinimide into the solvent at a molar ratio of 0.4-2:1, stir and react at 20-70°C for 2-200 hours, if the product is still unopened If the ring structure exists, then add excess N,N-dimethylpropylenediamine or N,N-dimethylethylenediamine, or ethanolamine, propanolamine, butanolamine or pentanolamine, and react for 24 hours , and synthesized the polyaspartic acid derivative P(Asp-Az) containing azide group in the side chain. The step 3 is: dissolving the above-mentioned polyethyleneimine PEI-(PPA-cyst) and P(Asp-Az) functionalized by the alkyne end group according to the molar ratio of the alkyne end group to azide of 0.5-3:1 In an organic solvent or water or a mixture of water and an organic solvent, react at 20-80°C for 1-96 hours to obtain a comb-shaped cationic polymer P(Asp-Az-Al-ss-PEI) containing a disulfide bond . 10. The comb-shaped macromolecule according to claim 1 is used as a gene carrier, characterized in that polyethylene glycol, targeting groups and/or cross-linkable components are introduced.

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