阅读说明：本技术 一种pH敏感纳米载体及其在基因药物递送中的应用 (pH sensitive nano-carrier and application thereof in gene drug delivery ) 是由 赵秀丽 史梦浩 张九龙 于 2020-06-01 设计创作，主要内容包括：本发明涉及医药技术领域,具体涉及一种pH敏感纳米载体及其在基因药物递送系统中的应用。本发明提供一种pH敏感纳米载体,所述的pH敏感纳米载体为PEG-PHis-PSD三嵌段共聚物修饰在阳离子脂质体的表面,即所述的pH敏感纳米载体由阳离子脂质体与聚乙二醇-聚组氨酸-聚磺胺二甲氧嘧啶(PEG-PHis-PSD)三嵌段共聚物正负电荷吸附连接,在生理环境中(pH7.3～7.4),阳离子脂质体与聚乙二醇-聚组氨酸-聚磺胺二甲氧嘧啶(PEG-PHis-PSD)三嵌段共聚物正负电荷吸附,即得一种新型的pH敏感纳米载体。所述的PEG-PHis-PSD三嵌段共聚物结构如下：本发明的pH敏感纳米载体中,PEG增加其在血液中循环时间,PHis达到溶酶体逃逸功能,另外,由于pH敏感的PSD链的存在,其可以快速的在肿瘤部位释放基因药物。(The invention relates to the technical field of medicines, in particular to a pH sensitive nano-carrier and application thereof in a gene drug delivery system. The invention provides a pH sensitive nano-carrier, wherein the pH sensitive nano-carrier is a novel pH sensitive nano-carrier which is obtained by modifying a PEG-PHIS-PSD triblock copolymer on the surface of a cationic liposome, namely the pH sensitive nano-carrier is in adsorption connection with positive and negative charges of the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer by the cationic liposome, and the positive and negative charges of the cationic liposome and the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer are adsorbed in a physiological environment (pH 7.3-7.4). The PEG-PHIS-PSD triblock copolymer has the following structure: in the pH sensitive nano-carrier, PEG prolongs the circulation time of the pH sensitive nano-carrier in blood, PHIs achieves the escape function of lysosomes, and in addition, due to the existence of a pH sensitive PSD chain, the pH sensitive nano-carrier can quickly release gene drugs at tumor parts.)

A PSD-phi-PEG triblock copolymer having the structural formula:

n＝40～50。

2. the method of claim 1, wherein the PSD-PHIS-PEG triblock copolymer is prepared by the following steps,

(1) synthesis of Polysulfamediethoxypyrimidine: synthesizing a pH sensitive polymer polysulfonamide dimethoxy pyrimidine by adopting a free radical initiation solvent polymerization method;

(2) the polysulfanediaxine is catalyzed by NHS and DCC and then is subjected to amidation reaction with polyhistidine to obtain the product polysulfanediaxine-polyhistidine;

(3) one end of carboxylated polyethylene glycol is connected with the poly-sulfadimethoxine-poly-histidine through amidation reaction, and the final product of polyethylene glycol-poly-histidine-poly-sulfadimethoxine is obtained.

3. A pH-sensitive nano-carrier is characterized in that the pH-sensitive nano-carrier is connected with the polyethylene glycol-polyhistidine-polysulfonamidodimethoxine triblock copolymer of claim 1 through positive and negative charge adsorption.

4. The pH-sensitive nanocarrier of claim 3, wherein the cationic liposome comprises at least DOTAP, and the cationic liposome can be a combination of DOTAP and one or more of the following substances: soybean lecithin, cholesterol, egg yolk lecithin, etc.

5. The pH-sensitive nanocarrier of claim 4, wherein the cationic liposome is a combination of DOTAP, soy lecithin, cholesterol, DOTAP: cholesterol is 4-8: 2-4: 1.

6. The pH-sensitive nanocarrier of claim 5, wherein the cationic liposome is prepared by the following method: dissolving DOTAP, soybean phospholipid and cholesterol in a mixed solvent of chloroform and methanol, wherein the ratio of chloroform: the volume ratio of methanol is 3-4:1, and the organic solvent is removed through reduced pressure rotary evaporation to form a dry lipid film; adding water for hydration, carrying out ultrasonic treatment, and finishing the particles of a 0.22 mu m polycarbonate film to obtain the cationic liposome.

7. The drug-containing pH sensitive lipid nano-carrier is characterized in that a drug and cationic lipid with positive charges are combined through electrostatic interaction to form a drug-containing cationic liposome, and a PEG-PHIs-PSD triblock copolymer with negative charges is adsorbed with the positive and negative charges of the drug-containing cationic liposome.

8. The drug-containing pH-sensitive lipid nanocarrier of claim 7, comprising the pH-sensitive nanocarrier of claim 1 and a drug, wherein the drug is a gene drug, a nucleic acid drug, selected from the group consisting of: siRNA, miRNA, plasmid DNA.

9. The drug-containing pH-sensitive lipid nanocarrier of claim 8, wherein the weight ratio of the drug to the pH-sensitive nanocarrier is: 1-10: 1.

10. use of the polyethylene glycol-polyhistidine-polysulfonamidodimethoxine triblock copolymer of claim 1 for the preparation of pH sensitive and lysosomal escape vehicles.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a pH sensitive nano-carrier and application thereof in a gene drug delivery system.

Background

Malignant tumors have become one of the leading diseases causing human death worldwide, and become a worldwide problem seriously threatening the quality of human life. Gene therapy (gene therapy) has the advantages of high safety, high efficiency, specificity and the like, and is more and more widely applied. However, there are several major obstacles to siRNA delivery: first, siRNA is extremely unstable in physiological environments and is rapidly degraded by ribonuclease (RNase) in serum. In addition, siRNA is a negatively charged hydrophilic macromolecule, and the negative charge on the surface of the cell membrane causes the cell membrane to have very poor penetrating power. Thereafter, siRNA enters the target cell and escapes from lysosomes into the cytoplasm to avoid phagocytic degradation of the entering lysosome, before RNAi can be mediated. Therefore, it is imperative to develop a multifunctional delivery system to deliver siRNA efficiently.

The multifunctional gene delivery vector for delivering siRNA mainly comprises liposome, cationic micelle, protein polypeptide and the like, and is widely applied due to the characteristics of low toxicity, easy modification, good biocompatibility, high gene loading rate and the like of the cationic liposome. However, the application of cationic liposome has been limited by the disadvantages of poor stability, low transfection efficiency, poor tumor targeting property and the like. Therefore, the copolymer with certain functions is modified on the surface of the liposome to improve the stability, transfection efficiency and tumor targeting of a gene delivery system.

A number of documents report significant differences in the physiological environment of tumor sites from normal tissues. Including the weakly acidic, highly concentrated reducing substance Glutathione (GSH) and high levels of Reactive Oxygen Species (ROS). When the nanocarrier enters the endosome or lysosome in endocytosed form, the pH will decrease further (pH 5.0-6.0). Therefore, according to the characteristic of low pH of tumor cells, various pH-sensitive polymer-based drug delivery carriers can be designed.

Among many pH sensitive materials, polyhistidine (philidine), phils, is distinguished by its high biocompatibility, low toxicity, and in particular by its lysosomal escape function. The imidazole ring of histidine is hydrophobic in physiological environment (pH 7.4), and when the pH is lower than pKa, the imidazole ring becomes hydrophilic due to protonation, so that a 'proton sponge effect' is realized to achieve a lysosome escape effect, and the intracellular release of the drug is increased.

Polysulfonyldimethoxypyrimidine (PSD) has the general characteristics of sulfonamides, and exhibits weak acidity. In a high pH environment due to sulfonyl groups (-SO)₂NH-) has a high electronegativity, which attracts electrons of the sulfur atom and thus of the nitrogen atom, resulting in the electron cloud of the N-H bond moving towards the nitrogen atom and thus releasing a proton. The dimethoxypyrimidine group in SD is also an electron withdrawing group, which also facilitates the release of protons from the nitrogen atom. Therefore, under normal physiological pH conditions, the surface of the PSD has negative charges, and under the acidic environment of the lysosome, the surface charges of the PSD are converted from negative charges to neutral charges, so that charge reversal is realized.

Disclosure of Invention

The invention aims to provide a novel pH sensitive nano-carrier to improve the stability of gene drug delivery, improve specificity and quickly release the effect of gene drugs.

The invention realizes the aim through the following technical scheme:

the invention provides a PSD-PHIS-PEG triblock copolymer, which has the following structural formula:

wherein: n is 40 to 50.

The PEG-PHIS-PSD triblock copolymer is prepared by the following method:

(1) synthesis of Polysulfamediethoxypyrimidine (PSD): adopts a free radical initiation solvent polymerization method to synthesize a pH sensitive polymer, namely the polysulfonamide dimethoxy Pyrimidine (PSD).

(2) The product poly-sulfadimethoxine-poly-histidine (PSD-PHIs) is obtained by amidation reaction of poly-sulfadimethoxine (PSD) catalyzed by NHS and DCC.

(3) One end of carboxylated polyethylene glycol (PEG) is connected with PSD-PHIS through amidation reaction to obtain the final product of polyethylene glycol-polyhistidine-polysulfonamide dimethoxy pyrimidine (PEG-PHIS-PSD).

PSD synthesis: dissolving Sulfadimethoxine (SD) and NaOH in acetone/water, slowly adding excessive methyl acryloyl chloride (MC) dropwise, purifying with methanol/water for 3-4h, filtering, and vacuum drying for 48h to obtain SD acylated product (SDM). Dissolving SDM in Dimethylformamide (DMF), introducing nitrogen, adding AIBN, sealing, quickly dropwise adding mercaptoethylamine, stirring, continuously introducing nitrogen at 70-80 ℃ for 48h, aging in water for 3-4h, filtering, and vacuum drying for 48h to obtain PSD.

Synthesis of PHIS-PSD: dissolving Fmoc-NH-PHIs-COOH, NHS and EDC in dimethyl sulfoxide (DMSO), stirring at room temperature for 24h under the protection of nitrogen, adding DMSO solution of PSD, stirring at room temperature for 48h under the protection of nitrogen, precipitating with glacial ethyl ether, filtering, and vacuum drying to obtain Fmoc-NH-PHIs-PSD; removing Fmoc protection: dissolving Fmoc-NH-PHIs-PSD in dimethyl sulfoxide (DMSO), adding diethylamine to react for 2-3h, rotary steaming, adding glacial ethyl ether to precipitate a product, filtering, and vacuum drying to obtain PHIs-PSD powder.

Synthesis of PEG-PHIS-PSD: dissolving PEG, NHS and EDC in dimethyl sulfoxide (DMSO), stirring at room temperature for 24h under the protection of nitrogen, adding PHIS-PSD DMSO solution, stirring at room temperature for 48h under the protection of nitrogen, dialyzing with dialysis bag (MW 3500) for 48-72h, and lyophilizing to obtain PEG-PHIS-PSD powder.

The invention provides a pH sensitive nano-carrier, wherein the pH sensitive nano-carrier is a novel pH sensitive nano-carrier which is obtained by modifying a PEG-PHIS-PSD triblock copolymer on the surface of a cationic liposome, namely the pH sensitive nano-carrier is in adsorption connection with positive and negative charges of the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer by the cationic liposome, and the positive and negative charges of the cationic liposome and the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer are adsorbed in a physiological environment (pH 7.3-7.4).

The cationic liposome at least contains (2, 3-dioleoxypropyl) trimethyl ammonium chloride (DOTAP).

Further, the cationic liposome comprises the following components: (ii) a combination of DOTAP with one or more of: soybean lecithin, cholesterol, egg yolk lecithin, etc.

Preferably a combination of DOTAP, soy lecithin, cholesterol.

Wherein, DOTAP comprises soybean lecithin: cholesterol is 4-8: 2-4: 1.

The cationic liposome is prepared by the following method: dissolving DOTAP, soybean phospholipid and cholesterol in a mixed solvent of chloroform and methanol, wherein the ratio of chloroform: the volume ratio of methanol is 3-4:1, and the organic solvent is removed by reduced pressure rotary evaporation to form a dry lipid film; adding water as water phase, hydrating, ultrasonic treating, and grading with 0.22 μm polycarbonate membrane to obtain cationic liposome (L).

Further, the invention provides a lipid nano-carrier obtained by combining a drug and a pH sensitive nano-carrier through electrostatic interaction, wherein the nano-lipid carrier is prepared through the following steps: in a physiological environment (pH7.3-7.4), the LR is formed by combining the medicament and cationic lipid with positive charge through electrostatic interaction. And then, the PEG-PHIS-PSD triblock copolymer with negative charges is adsorbed with positive and negative charges of LR, thus obtaining the novel drug-containing pH sensitive lipid nano-carrier (PHD/LR).

The medicine is gene medicine and nucleic acid medicine selected from: siRNA, miRNA and plasmid DNA, wherein the nucleic acid drug has negative charges and is combined with the pH sensitive nano-carrier with positive charges to form the drug-containing pH sensitive lipid nano-carrier under the adsorption effect of the positive charges and the negative charges.

The weight ratio of the gene medicine to the pH sensitive nano-carrier is as follows: 1-10: 1.

the structure of the liposome has higher similarity with a normal biological membrane, so that the liposome is easily degraded and metabolized by various substances in the body circulation so as to reduce the circulation time in the body. In order to solve the problem, the polyethylene glycol-polyhistidine-polysulfonamide dimethoxy pyrimidine (PEG-PHIs-PSD) triblock copolymer achieves the long circulation effect; the prepared liposome has smaller particle size (about 150nm-200nm), and can be specifically enriched in tumor tissues through an EPR effect so as to embody targeting; the gene medicine is delivered into tumor cells and phagocytized by lysosomes, and the escape effect of the lysosomes is achieved due to the proton sponge effect of the PHIs on the surfaces of the gene medicine, so that the gene medicine is released into cytoplasm; meanwhile, under the acidic environment of lysosome, the pH-sensitive PSD is changed from negative charge to neutral charge, and is separated from the surface of the cationic liposome to quickly release gene drugs, thereby playing a therapeutic role.

Compared with the prior art, the invention has the following advantages:

1) in the drug-containing pH sensitive lipid nano-carrier, the cationic lipid in the pH sensitive nano-carrier is combined with the gene drug through electrostatic interaction, so that the encapsulation rate of the gene drug in the pH sensitive nano-carrier is improved, the preparation method is simple and easy to implement, the reproducibility is good, and the stability is high;

2) in the invention, the prepared liposome has smaller particle size (about 150-200nm), and can be specifically enriched in tumor tissues through an EPR effect so as to have targeting property;

3) according to the invention, the PEG of the pH sensitive triblock copolymer can increase the circulation time of the pH sensitive triblock copolymer in blood, and the rapid lysosome escape function of PHIs can be realized.

4) Compared with blank cationic liposome, the invention modifies PSD-PHIs-PEG on the surface of the cationic liposome through electrostatic adsorption, thereby increasing the stability of the preparation, improving the specificity and enhancing the targeting property.

Drawings

FIG. 1 is a drawing showing a copolymer of example 1 of the present invention¹HNMR mapping:

(A) process for preparing Polysulfonamidodimethoxine (PSD)¹A HNMR map; (B) method for preparing polysulfonamide dimethoxy pyrimidine-polyhistidine (PSD-PHIS)¹A HNMR map; (C) method for preparing polysulfonamide dimethoxy pyrimidine-polyhistidine-polyethylene glycol (PSD-PHIS-PEG)¹A HNMR map;

FIG. 2 is an FTIR spectrum of the copolymer of example 1 of the present invention:

(A) FTIR spectrum of Polysulfonamidodimethoxine (PSD); (B) FTIR spectrum of Polysulfadimethoxine-polyhistidine (PSD-PHIs); (C) FTIR spectrum of Polysulfadimethoxine-polyhistidine-polyethylene glycol (PSD-PHIs-PEG);

FIG. 3 is a pH-sensitive pH-sensitivity study of pH-sensitive PHD/LR of example 3 of the present invention.

FIG. 4 is a TEM image of pH sensitive PHD/LR of example 4 of the present invention.

FIG. 5 is an atomic force microscope photograph of pH sensitive PHD/LR of example 4 of the present invention.

FIG. 6 is an in vitro release assay of PHD/LR of example 5 of the present invention in simulated media of different pH.

FIG. 7 is a photograph of in vitro heparin-decomplexation assay of PHD/LR of example 6 of the present invention.

FIG. 8 is a photograph of an in vitro serum stability assay of PHD/LR of example 7 of the present invention.

FIG. 9 is a graph of the change in tumor volume of PHD/LR in vivo anti-tumor experiments according to example 8 of the present invention.

FIG. 10 is a graph of the weight change of PHD/LR in mice tested for in vivo anti-tumor effect according to example 8 of the present invention.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended only to specifically describe the present invention and should not be construed as limiting the present invention.