阅读说明：本技术 仿细胞膜聚合物的制备方法、及其修饰壳聚糖-金自组装载药纳米粒子的制备方法和应用 (Preparation method of cell membrane-imitated polymer, and preparation method and application of chitosan-gold self-assembled drug-loaded nanoparticle modified by same ) 是由 贾鹏翔 马可 卫欣冉 陈代君 于 2019-09-27 设计创作，主要内容包括：本发明公开一种仿细胞膜聚合物的制备方法,包括以下步骤：(1)制备2-溴-2-甲基丙酸-4-甲酰基苯酯；(2)制备仿细胞膜聚合物；同时,本发明还公开所述仿细胞膜聚合物修饰壳聚糖-金自组装载药纳米粒子的制备方法及其应用,制备方法包括以下步骤：将壳聚糖溶于醋酸-氨水缓冲溶液中,然后向其中加入氯金酸和药物,混合搅拌；在匀速搅拌状态下,将其中滴加交联剂,搅拌；再加入所述仿细胞膜聚合物,搅拌；离心,将离心得到的沉淀用超纯水洗涤,然后超声分散,冷冻干燥,得到仿细胞膜聚合物修饰壳聚糖-金自组装载药纳米粒子。本发明提供的仿细胞膜聚合物,能修饰制备壳聚糖-金自组装载药纳米粒子,兼具诊断、治疗肿瘤的双功能。(The invention discloses a preparation method of a cell membrane imitating polymer, which comprises the following steps: (1) preparing 4-formylphenyl 2-bromo-2-methylpropionate; (2) preparing a cell membrane imitating polymer; meanwhile, the invention also discloses a preparation method and application of the cell membrane-imitated polymer modified chitosan-gold self-assembled drug-loaded nanoparticle, wherein the preparation method comprises the following steps: dissolving chitosan in acetic acid-ammonia water buffer solution, adding chloroauric acid and medicine, mixing and stirring; under the state of uniform stirring, the cross-linking agent is dripped into the mixture and stirred; then adding the cell membrane imitating polymer and stirring; centrifuging, washing the precipitate obtained by centrifuging with ultrapure water, then performing ultrasonic dispersion, and freeze-drying to obtain the cell membrane polymer-simulated modified chitosan-gold self-assembled drug-loaded nanoparticles. The cell membrane-imitated polymer provided by the invention can be modified to prepare chitosan-gold self-assembled drug-loaded nanoparticles, and has the double functions of diagnosing and treating tumors.)

1. The preparation method of the cell membrane imitating polymer is characterized in that: the method comprises the following steps:

(1) preparation of 4-formylphenyl 2-bromo-2-methylpropionate: bromo isobutyryl bromideIs precooled to 0 ℃ in N₂Under the environment, dropwise adding a dichloromethane solution containing 0.8mmol/mL hydroxybenzaldehyde and 1.6mmol/mL triethylamine, reacting at room temperature for 24 hours after dropwise adding, filtering, washing the filtrate with a 1 mol/L hydrochloric acid solution and a saturated sodium carbonate solution, then drying with anhydrous magnesium sulfate, vacuum concentrating, recrystallizing the obtained solid with n-hexane to obtain white microcrystals, and vacuum drying;

(2) preparation of cell membrane-mimicking polymer: adding 2-methacryloyloxyethyl phosphorylcholine, a ligand 2, 2' -bipyridine, an initiator 2-bromo-2-methylpropanoic acid-4-formylphenyl ester and methanol into a Schlenk reaction bottle, freezing and pumping air for 3 times, then opening an inflation valve under the liquid nitrogen environment, adding CuBr into the reaction bottle, closing the reaction bottle, freezing and pumping air for three times, reacting for 16 hours under the condition of water bath at 25 ℃, then precipitating with acetone, dissolving the precipitate with ultrapure water, then placing the precipitate into a dialysis bag with the molecular weight cutoff of 1000Da for dialysis, and freezing and drying the retentate obtained by dialysis to obtain the benzaldehyde end-capped cell membrane-imitating polymer.

2. The method of claim 1, wherein: the molar ratio of the hydroxybenzaldehyde, the bromoisobutyryl bromide and the triethylamine in the step (1) is 1:1.1: 2.

3. The method of claim 1, wherein: the molar ratio of the 2-methacryloyloxyethyl phosphorylcholine, the 2, 2' -bipyridine, the 2-bromo-2-methylpropanoic acid-4-formylphenyl ester and the CuBr added in the step (2) is 30:2:1: 1.

4. The preparation method of the cell membrane polymer-simulated modified chitosan-gold self-assembled drug-loaded nanoparticle is characterized by comprising the following steps: the method comprises the following steps:

dissolving chitosan in acetic acid-ammonia water buffer solution with pH =7.4, then adding chloroauric acid and a medicine, and mixing and stirring for 1-2 h;

secondly, under the state of uniform stirring, dropwise adding a cross-linking agent into the mixed solution obtained in the step one, and stirring at room temperature for 12-24 h;

(III) adding the cell membrane imitating polymer into the mixed solution obtained in the step (II), and stirring at room temperature for 12-24 h;

fourthly, centrifuging, washing the precipitate obtained by centrifuging with ultrapure water, then performing ultrasonic dispersion and freeze drying to obtain the cell membrane polymer-simulated modified chitosan-gold self-assembled drug-loaded nanoparticles;

wherein the cell membrane-imitated polymer is prepared by the preparation method of any one of claims 1, 2 or 3;

the chitosan is a low molecular weight water-soluble chitosan with a molecular weight of less than 6000.

5. The method according to claim 4, wherein: the drug is a hydrophobic drug.

6. The method according to claim 5, wherein: the medicine is adriamycin or paclitaxel.

7. The method according to claim 4, wherein: the cross-linking agent is at least one of glutaraldehyde, sodium tripolyphosphate and genipin.

8. The method according to claim 4, wherein: the molar ratio of the chitosan to the chloroauric acid is 10: (1-5), wherein the mass ratio of the chitosan to the medicament to the cell membrane imitating polymer is 10: (1-4): (5-30), wherein the mass ratio of the chitosan to the cross-linking agent is 10: (0.5-2), the adding proportion of the chitosan to the buffer solution is 2 mg: 1 mL.

9. The method according to claim 4, wherein: the rotation speed of the centrifugation is 4000-.

10. The application of the chitosan-gold self-assembled drug-loaded nano-particle modified by the cell membrane-like polymer in biomedicine is characterized in that: the application is the preparation of medicines for diagnosing and treating tumors or cell imaging reagents; the chitosan-gold self-assembled drug-loaded nanoparticle modified by the cell membrane-like polymer is prepared by the preparation method of claim 4.

Technical Field

The invention belongs to the technical field of polymer biomedical materials, and particularly relates to a preparation method of a cell membrane-like polymer, and a preparation method and application of a chitosan-gold self-assembled drug-loaded nanoparticle modified by the cell membrane-like polymer.

Background

Chitosan is a straight-chain macromolecular compound formed by connecting N-acetyl-D-glucosamine monomers through beta-l, 4-glycosidic bonds, and an amino group and two hydroxyl groups are respectively arranged at the C-2, C-3 and C-6 positions in the molecule. The macromolecule can be linked with other anionic macromolecules through ionic bonds to form a network structure, and if the medicament is embedded in the macromolecule or is linked on the macromolecule through covalent bonds, a complex system of the carrier and the medicament can be formed. Compared with other high molecular substances, the chitosan serving as a carrier material also has the following advantages: (1) good biocompatibility, degradability and excellent cell membrane penetrability, and degradation products are non-toxic; (2) the modified amphiphilic compound can be modified into amphiphilic molecules through hydroxyl and amino in the molecules, so that the affinity to hydrophobic or hydrophilic drugs can be improved, and the solubility of the drugs in body fluid can be promoted; (3) the drug can pass through the cell membrane and enter the cytoplasm through the endocytosis of the cell; (4) by enhancing the osmotic and retention Effects (EPR), the tissue selectivity of the medicament is improved, and the toxic and side effects of the medicament are reduced; (5) after the medicament (containing the targeting factor) in the carrier enters the body, the medicament can accurately act on target tissues through the targeting factor. Due to the advantages, more and more researchers have conducted extensive research on the nano drug delivery system using chitosan as a carrier. The chitosan is a cationic polysaccharide material, and usually tumor cells have more negative charges than normal cell surfaces, so the chitosan microspheres have selective adsorption and electric neutralization effects on the tumor cell surfaces and certain effects of directly inhibiting the tumor cells, show anticancer activity by activating an immune system, and can enhance the anticancer effect of the medicine when being used together with the existing anticancer medicine. Therefore, the chitosan microspheres are ideal carriers of anticancer drugs and have attracted much attention in recent years.

Nanogold (Au NPs) refer to gold microparticles, 1-100 nm in diameter, that are typical of plasma nanoparticles. Among many inorganic nanoparticles, gold nanoparticles stand out because of some of their advantages over other materials, and have been widely used in the biomedical field: (I) the synthesis method of the Au NPs is simple and efficient, and the Au NPs with different shapes and different particle sizes can be synthesized in a large amount; in addition, water is generally used as a medium in the preparation process, so that the Au NPs are more convenient to apply in the biomedical field; (II) compared with other metal nanoparticles, Au NPs have the advantages of chemical inertness and biocompatibility, and are more suitable for organisms. Research shows that in the dosage concentration range allowed by clinical test, Au NPs do not show obvious biological toxicity or biological immune response to various cells; (III) the shape, the particle size and the surface chemistry of the Au NPs are easy to regulate and control, so that the endocytosis rate of target cells to the Au NPs is improved to the maximum extent; (IV) Au NPs are easy to chemically modify, and can be bonded with other functional molecules (such as Au-S) on the surface, including therapeutic drugs, targeting molecules or biological agents with good biocompatibility. Based on the advantages, the Au NPs are widely concerned in the fields of biological imaging, drug loading, biosensing, tumor treatment and the like.

Disclosure of Invention

In order to solve the problem of single function of a nano transport carrier in the prior art and realize simultaneous diagnosis and treatment of tumors by the nano drug transport carrier, the invention provides a preparation method of a cell membrane imitating polymer, and a preparation method and application of chitosan-gold self-assembled drug-loaded nanoparticles modified by the cell membrane imitating polymer.

The preparation method of the cell membrane imitating polymer comprises the following steps:

(1) preparation of 4-formylphenyl 2-bromo-2-methylpropionate: precooling the bromoisobutyryl bromide in dichloromethane to 0 ℃ and reacting the solution in N₂Under the environment, dropwise adding a dichloromethane solution containing 0.8mmol/mL hydroxybenzaldehyde and 1.6mmol/mL triethylamine, reacting at room temperature for 24 hours after dropwise adding, filtering, washing the filtrate with a 1 mol/L hydrochloric acid solution and a saturated sodium carbonate solution, then drying with anhydrous magnesium sulfate, vacuum concentrating, recrystallizing the obtained solid with n-hexane to obtain white microcrystals, and vacuum drying;

(2) preparation of cell membrane-mimicking polymer: adding 2-methacryloyloxyethyl phosphorylcholine, a ligand 2, 2' -bipyridine, an initiator 2-bromo-2-methylpropanoic acid-4-formylphenyl ester and methanol into a Schlenk reaction bottle, freezing and pumping air for 3 times, then opening an inflation valve under the liquid nitrogen environment, adding CuBr into the reaction bottle, closing the reaction bottle, freezing and pumping air for three times, reacting for 16 hours under the condition of water bath at 25 ℃, then precipitating with acetone, dissolving the precipitate with ultrapure water, then placing the precipitate into a dialysis bag with the molecular weight cutoff of 1000Da for dialysis, and freezing and drying the retentate obtained by dialysis to obtain the benzaldehyde end-capped cell membrane-imitating polymer.

Preferably, the molar ratio of the hydroxybenzaldehyde, the bromoisobutyryl bromide and the triethylamine in the step (1) is 1:1.1: 2.

Preferably, the molar ratio of the 2-methacryloyloxyethyl phosphorylcholine, the 2, 2' -bipyridine, the 4-formylphenyl 2-bromo-2-methylpropionate and the CuBr added in the step (2) is 30:2:1: 1.

The preparation method of the chitosan-gold self-assembly drug-loaded nanoparticle modified by the cell membrane-like polymer comprises the following steps:

dissolving chitosan in acetic acid-ammonia water buffer solution with pH =7.4, then adding chloroauric acid and a medicine, and mixing and stirring for 1-2 h;

secondly, under the state of uniform stirring, dropwise adding a cross-linking agent into the mixed solution obtained in the step one, and stirring at room temperature for 12-24 h;

(III) adding the cell membrane imitating polymer into the mixed solution obtained in the step (II), and stirring at room temperature for 12-24 h;

fourthly, centrifuging, washing the precipitate obtained by centrifuging with ultrapure water, then performing ultrasonic dispersion and freeze drying to obtain the cell membrane polymer-simulated modified chitosan-gold self-assembled drug-loaded nanoparticles;

wherein, the cell membrane imitating polymer is prepared by the preparation method;

the chitosan is a low molecular weight water-soluble chitosan with a molecular weight of less than 6000.

Preferably, the drug is a hydrophobic drug;

more preferably, the drug is doxorubicin or paclitaxel.

Preferably, the cross-linking agent is at least one of glutaraldehyde, sodium tripolyphosphate and genipin.

Preferably, the molar ratio of the chitosan to the chloroauric acid is 10: (1-5), wherein the mass ratio of the chitosan to the medicament to the cell membrane imitating polymer is 10: (1-4): (5-30), wherein the mass ratio of the chitosan to the cross-linking agent is 10: (0.5-2), the adding proportion of the chitosan to the buffer solution is 2 mg: 1 mL.

Preferably, the rotation speed of the centrifugation is 4000-.

Preferably, the dropping speed of the dropping in the step (two) is 40 ~ 160 μ L/min.

The application of the chitosan-gold self-assembled drug-loaded nanoparticle modified by the cell membrane-imitated polymer in biomedicine is to prepare a drug for diagnosing and treating tumors or a cell imaging reagent; the cell membrane-imitated polymer modified chitosan-gold self-assembled drug-loaded nanoparticle is prepared by the preparation method.

The cell membrane imitating polymer modified chitosan-gold self-assembled drug-loaded nanoparticle prepared by the invention is of a core-shell structure, wherein the core layer is a drug-loaded nanoparticle which takes cross-linked chitosan as a carrier to wrap drugs and gold nanoparticles, and the shell layer is a hydrophilic layer wrapped by the cell membrane imitating polymer.

The invention has the advantages that:

the benzaldehyde-terminated cell membrane-imitating polymer provided by the invention can modify and prepare chitosan-gold self-assembled drug-loaded nanoparticles, the nanoparticles carry drugs, chitosan corresponds to a low-pH environment of tumors and further controls drug release behaviors of the tumors, and meanwhile, the fluorescence characteristic of nanogold enables the nanoparticles to image cells, so that the prepared cell membrane-imitating polymer modified chitosan-gold self-assembled drug-loaded nanoparticles have the double functions of diagnosing and treating tumors.

Drawings

FIG. 1 is the NMR spectrum of the cell membrane-imitated polymer.

FIG. 2 is a transmission electron microscope image of chitosan-gold self-assembled drug-loaded nanoparticles modified by cell membrane-like polymer.

Fig. 3 is an in vitro drug release curve of the chitosan-gold self-assembled drug-loaded nanoparticle modified by the cell membrane-imitated polymer.

FIG. 4 is a confocal fluorescence image excited after the chitosan-gold self-assembled drug-loaded nanoparticles modified by the cell membrane-like polymer and cervical cancer cells (Hela) are incubated for 12 hours.

Detailed Description