阅读说明：本技术 一种智能响应性壳-核式聚电解质纳米凝胶的制备方法与应用 (Preparation method and application of intelligent responsive shell-core polyelectrolyte nanogel ) 是由 钟伊南 张媛媛 张俊梅 黄德春 陈维 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种智能响应性壳-核式聚电解质纳米凝胶的制备方法与应用,该纳米凝胶由透明质酸或其衍生物、类似物的壳层和由谷胱甘肽敏感或酸敏感的交联剂交联而成的配体修饰的超支化聚乙烯亚胺的核层构成,透明质酸外壳和超支化聚乙烯亚胺内核再由肿瘤微环境敏感的交联剂通过点击化学反应连接形成智能响应性壳-核式纳米凝胶。该纳米凝胶由于其带电性可包载多种含电性的亲疏水药物或蛋白质,并且含有多种肿瘤微环境响应的交联剂,可在肿瘤部位响应并快速释放其包载的药物,达到高效的抗肿瘤效果。(The invention discloses a preparation method and application of an intelligent responsive shell-core type polyelectrolyte nanogel. The nanogel can wrap various hydrophilic and hydrophobic drugs or proteins with electric property due to the electric property of the nanogel, and contains various cross-linking agents responding to the tumor microenvironment, so that the nanogel can respond to the tumor part and quickly release the wrapped drugs, and the high-efficiency anti-tumor effect is achieved.)

1. A preparation method of intelligent responsive shell-core polyelectrolyte nanogel is characterized by comprising the following steps: the hyaluronic acid core-shell type nano-gel is composed of a hyaluronic acid or a derivative thereof, a shell layer of an analogue and a core layer of ligand-modified hyperbranched polyethyleneimine formed by crosslinking of a glutathione-sensitive or acid-sensitive crosslinking agent, wherein a hyaluronic acid shell and a hyperbranched polyethyleneimine core are connected by a tumor microenvironment-sensitive crosslinking agent through a click chemical reaction to form the intelligent responsive shell-core type nano-gel.

2. The method of preparing a smart responsive shell-core polyelectrolyte nanogel according to claim 1, wherein: the molecular weight of the hyaluronic acid or the derivative or the analogue thereof is 7-100 kDa; the molecular weight of the hyperbranched polyethyleneimine is 0.6-70 kDa.

3. The method of preparing a smart responsive shell-core polyelectrolyte nanogel according to claim 1, wherein: the crosslinking agent containing glutathione sensitivity or acid sensitivity is selected from the following components:

wherein R is₁Is selected from H or CH₃。

4. The method of preparing a smart responsive shell-core polyelectrolyte nanogel according to claim 1, wherein: the preparation method of the hyperbranched polyethyleneimine comprises the following steps:

dissolving branched polyethyleneimine and a glutathione-sensitive or acid-sensitive cross-linking agent in methanol, adding triethylamine as a catalyst, and reacting to obtain the compound.

5. The method of preparing a smart responsive shell-core polyelectrolyte nanogel according to claim 1, wherein: the ligand-modified hyperbranched polyethyleneimine is selected from azido-modified hyperbranched polyethyleneimine.

6. The method of preparing a smart responsive shell-core polyelectrolyte nanogel according to claim 1, wherein: the crosslinking agent sensitive to the tumor microenvironment comprises an active oxygen sensitive crosslinking agent and a Matrix Metalloproteinase (MMP) sensitive crosslinking agent, and the active oxygen sensitive crosslinking agent is selected from compounds with the following structures:

the Matrix Metalloproteinase (MMP) -sensitive cross-linking agent is selected from bismaleimide-MMP 9 polypeptide.

7. The method of preparing a smart responsive shell-core polyelectrolyte nanogel according to claim 1, wherein: the hyaluronic acid or the derivative and the analogue thereof are selected from hyaluronic acid modified by azide group.

8. Use of the smart responsive shell-core polyelectrolyte nanogel prepared according to any one of claims 1 to 7 for the preparation of a pharmaceutical carrier.

9. Use of the smart responsive shell-core polyelectrolyte nanogel prepared according to any one of claims 1 to 7 in the preparation of antitumor drugs.

Technical Field

The invention relates to a preparation method and application of a high polymer material, in particular to a preparation method and application of an intelligent responsive shell-core polyelectrolyte nanogel.

Background

In the past decades, in order to solve the precise treatment of chemotherapeutic drugs, various biocompatible nano drug delivery systems such as polymer prodrugs, nano micelles, vesicles, liposomes, etc. have been developed to improve the stability and solubility of drugs and prolong the circulation time in vivo. Such as(polyethylene glycol-polylactic acid paclitaxel loaded micelle drugs), which have been applied to clinical treatment of non-small cell lung cancer, breast cancer, ovarian cancer;(Albumin-binding paclitaxel nano-drugs) have been used in the treatment of metastatic breast cancer, non-small cell lung cancer (in combination with carboplatin), advanced pancreatic cancer (in combination with gemcitabine), etc. However, with the research of people, the nano-drug still has some problems which are difficult to overcome in clinical treatment, such as premature ejaculation, low endocytosis efficiency of cells, and incapability of quick release at focus parts.

With the research of people on the tumor microenvironment, the multifunctional intelligent responsive nano-drug is researched and developed in large quantity to achieve the controlled release of the drug and improve the bioavailability of the drug by utilizing the lower pH value of the tumor tissue, the higher active oxygen level and the overexpression of various enzymes. People design the decomposable nano-carrier by utilizing the characteristics of the tumor microenvironment. The nano carrier can keep a stable structure and a proper nano size (20-100nm) in blood circulation, can permeate from blood vessels to tumor tissues through an enhanced permeation and interception (EPR) effect, and can change the structure and the size of the nano particles irreversibly from a large size to a small size (less than 10nm) or reverse the surface charge to be beneficial to the endocytosis of cells to nano drugs due to the change of the environment in the tumor tissues. For example, Fukumura (proc.natl.acad.sci.u.s.a.2011, 108(6), 2426-2431.) et al report that gelatin nanoparticles are degraded from the surface under the action of tumor microenvironment metalloproteinase MMP-2 to release nanoparticles of about 10nm, and enhance osmotic diffusion inside fibrosarcoma HT-1080 tumor tissues. Chen (Drug Deliv.2019, 26(1), 1125-1139.) et al reported that 3, 4-dihydroxyphenylpropionic acid-chitooligosaccharide-dithiodipropionic acid-berberine (DHPA-CDB) with a mitochondrial targeting polymer self-assembles to form cationic micelles, and negatively charged oligomeric hyaluronic acid-3-carboxyphenylboronic acid (oHA-PBA) was further added to the surface of the preformed DHPA-CDB core as a ligand for sialic acid and CD44 to shield the positive charge and prolong blood persistence. The weak acidic tumor environment causes the degradation of borate bonds, the exposure of cationic micelles is realized, the charge is transferred from-19.47 to +12.01mV, and the internalization of cells and the positioning of mitochondria are promoted. Therefore, the construction of the intelligent detachable nano-carrier mediated by the tumor microenvironment has great research significance for further overcoming the problems encountered in tumor treatment.

Nanogels are nano-sized cross-linked network-like polymer particles capable of absorbing large amounts of water. The nanogel combines the performances of hydrogel and nano material, has very high water content, adjustable chemical and physical structures, and good mechanical property and biocompatibility. In the current research, nanogels have good stability due to the cross-linked network structure, can achieve long circulation in vivo, reduce early drug release and improve pharmacokinetic parameters. Under the action of a tumor/inflammation microenvironment, the intelligent nanogel can be degraded into a prodrug with a smaller size, so that the penetration of the drug in tumor tissues is enhanced, and meanwhile, active macromolecules encapsulated by the nanogel can play a role in the tumor microenvironment. At present, intelligent nanogels with multiple functions and novel properties and good biocompatibility have been reported. In future research, the dissociable type shell-core nanogel has great significance for treating tumors.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a preparation method of an intelligent responsive shell-core polyelectrolyte nanogel.

It is another object of the present invention to provide use of the smart responsive shell-core polyelectrolyte nanogel.

The technical scheme is as follows: the invention relates to a preparation method of an intelligent responsive shell-core polyelectrolyte nanogel, which comprises the following steps:

the hyaluronic acid nano-gel is composed of a shell layer of hyaluronic acid or a derivative thereof and an analogue thereof and a core layer of hyperbranched polyethyleneimine formed by crosslinking a glutathione-sensitive or acid-sensitive crosslinking agent, wherein a hyaluronic acid shell and a hyperbranched polyethyleneimine core are connected through a tumor microenvironment-sensitive crosslinking agent to form the intelligent responsive shell-core nano-gel.

Further, the molecular weight of the hyaluronic acid or the derivative or the analogue thereof is 7-100 kDa; the molecular weight of the branched polyethyleneimine is 0.6-70 kDa.

Further, the crosslinking agent containing glutathione sensitivity or acid sensitivity is selected from compounds with the structures shown as follows:

wherein R is₁Is selected from H or CH₃。

Further, the hyperbranched polyethyleneimine is synthesized by the following method:

dissolving branched polyethyleneimine and a glutathione-sensitive or acid-sensitive cross-linking agent in methanol according to the molar ratio of amino groups to double bonds of 3: 1, adding triethylamine as a catalyst, and reacting to obtain the compound.

Further, the ligand-modified hyperbranched polyethyleneimine is selected from azido-modified hyperbranched polyethyleneimines.

Further, the tumor microenvironment-sensitive cross-linking agents, such as: the active oxygen-sensitive cross-linking agent is selected from compounds of the structure shown below:

the Matrix Metalloproteinase (MMP) -sensitive cross-linking agent is selected from bismaleimide-MMP 9 polypeptide.

Further, the hyaluronic acid or the derivative or the analogue thereof is selected from the group consisting of azido-modified hyaluronic acid.

Further, the click chemistry reaction is to perform the click chemistry reaction of azide and alkynyl on hyaluronic acid and hyperbranched polyethyleneimine which are respectively modified by azide and a cross-linking agent which is responsive to a tumor microenvironment with alkynyl at two ends under the condition of not needing a catalyst.

Further, the mass ratio of the hyperbranched polyethyleneimine to the hyaluronic acid is 1: 2-4.

The intelligent responsive shell-core type polyelectrolyte nanogel is composed of a core layer of hyperbranched polyethyleneimine and a hyaluronic acid shell layer coated on the outer layer, wherein the core layer is connected with the core layer through a cross-linking agent, and the core layer is the cross-linked hyperbranched polyethyleneimine and has positive electricity, so that the whole nanoparticle is an electrified nanogel with a net-shaped space structure.

The method for preparing the intelligent responsive shell-core polyelectrolyte nanogel comprises the following steps: (1) connecting the prepared azido modified hyperbranched polyethyleneimine with one end of a cross-linking agent with alkynyl at two ends and responding to a tumor microenvironment, wherein the molar ratio of azido to alkynyl is preferably 1: 2.

(2) And (2) dispersing the alkynyl-modified hyperbranched polyethyleneimine obtained in the step (1) in water, dropwise adding the dispersed alkynyl-modified hyperbranched polyethyleneimine into an azido-modified hyaluronic acid aqueous solution, and reacting to obtain the intelligent responsive shell-core type polyelectrolyte nanogel.

The invention also provides application of the intelligent responsive shell-core polyelectrolyte nanogel as a drug carrier.

The reticular space structure of the intelligent responsive shell-core polyelectrolyte nanogel can be used for loading charged hydrophilic and hydrophobic drugs or proteins, and the stability of the drug-loaded nanogel is improved through crosslinking.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the nanogel can load charged hydrophilic and hydrophobic drugs or proteins through a cross-linked reticular space structure and physical action, and the stability of the nanogel is increased through cross-linking. The multiple responsiveness remarkably improves the application of the compound in tumor treatment, so that the compound can quickly release the drug at the tumor part, thereby generating high-efficiency treatment effect and having great application prospect in the fields of intelligent responsiveness and controlled release of the drug.

Drawings

FIG. 1 is a hydrogen nuclear magnetic spectrum of the reactive oxygen species-sensitive crosslinker RBCN of example 2;

FIG. 2 is hyaluronic acid azide (HA-N) of example 3₃) Hydrogen nuclear magnetic spectrum diagram of (1);

FIG. 3 is a particle size diagram of a smart responsive shell-core polyelectrolyte nanogel (HA-D-PEI) in example 4;

FIG. 4 shows that the nanogel obtained in example 6 is at 100. mu. M H₂O₂Particle size change under 10mM GSH (pH 7.4);

FIG. 5 is a graph showing the cytotoxicity results of nanogels obtained in example 7 on mouse breast cancer 4T1 cells.

Detailed Description

EXAMPLE 1 Synthesis of Azide hyperbranched polyethyleneimine (D-PEI-N)₃)

(1) Synthesis of hyperbranched polyethyleneimine (D-PEI)

Polyethyleneimine PEI (600mg, 1mmol) and N, N' -bis (acryloyl) cystamine (CBA, 260mg, 1mmol) were dissolved in 5mL of methanol (MeOH), respectively, mixed, added with 20. mu.L of Triethylamine (TEA), and stirred at room temperature for reaction. And after the reaction is finished, collecting the reaction solution in MeOH by using a dialysis bag for dialysis, replacing a dialysis medium with high-purity water for dialysis, and carrying out freeze vacuum drying to obtain a product.

(2) Synthesis of azido hyperbranched polyethyleneimine (D-PEI-N)₃)

Azidoacetic acid (AATA, 12mg, 116 mu mol) is dissolved in water, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM, 25mg, 92.8 mu mol) is added, after stirring evenly at room temperature, the hyperbranched polyethyleneimine (D-PEI, 50mg, 58 mu mol) is added, the pH is adjusted to be neutral, stirring is carried out overnight at room temperature, after the reaction is finished, reaction liquid is collected by a dialysis bag, dialyzed in water, and frozen and dried in vacuum to obtain the product.

EXAMPLE 2 Synthesis of active oxygen sensitive crosslinker RBCN

Bicyclo [6.1.0] non-4-yn-9-ylmethanol (BCN, 100mg, 0.67mmol) was dissolved in DCM, 92. mu.L of LTEA was added, nitrogen blanketed, and a solution of oxalyl chloride (37. mu.L, 0.44mmol) in DCM was slowly added dropwise under ice-bath conditions, and the reaction was stirred at room temperature. And after the reaction is finished, concentrating the reaction solution under reduced pressure, separating the RBCN by adopting a column chromatography, collecting the product, concentrating under reduced pressure, and drying in vacuum to obtain a white solid RBCN. The hydrogen nuclear magnetic spectrum is shown in FIG. 1.

EXAMPLE 3 Synthesis of hyaluronic acid Azide (HA-N)₃)

Hyaluronic acid (HA, 1g, 0.17. mu. mol) was dissolved in 20mL of water, DMTMM (110mg, 0.4mmol) was added thereto, and after stirring at room temperature, 2- [2- (2-azidoethoxy) ethoxy ] was added]Ethylamine (NH)₂-PEG₂-N₃) (69mg, 0.4mmol), adjusting pH to neutral, stirring at room temperature for 2 days, reactingAfter the reaction solution is finished, collecting the reaction solution in water by using a dialysis bag for dialysis, and carrying out freeze vacuum drying to obtain a product. The hydrogen nuclear magnetic spectrum is shown in FIG. 2.

Example 4 preparation of a Smart responsive Shell-core polyelectrolyte Nanogel (HA-D-PEI)

Adding D-PEI-N₃(15mg, 14. mu. mol) was dissolved in 1.5mL of DMSO, RBCN (14.8mg, 42. mu. mol) was added thereto and stirred at room temperature overnight, after completion of the reaction, the reaction mixture was dialyzed in DMSO using a dialysis bag, and the dialysis medium was replaced with high-purity water for dialysis, followed by lyophilization to obtain the product. Dissolving 1mg of D-PEI-RBCN in 1mL of DMSO, and dropwise adding the solution to HA-N under the condition of stirring₃To 5mM PB solution (pH 7.4, 1mg/mL, 1mL), stirring was continued for 5-8h after completion of the dropwise addition, and after completion, the solution was dialyzed for 4h against 5mM PB solution (pH 7.4) collected by a dialysis bag to remove the organic solvent. The average particle size of the micelles was 188nm and the particle size distribution index was 0.16 as measured by dynamic light scattering, as shown in FIG. 4.

Example 5 encapsulation of a Smart responsive Shell-core polyelectrolyte Nanogel (HA-D-PEI) on glucose oxidase (Gox) and a carboxyl-containing Small molecule drug UK-5099

0.5mg of UK-5099 was added to a DMSO solution of D-PEI-RBCN (0.5mg/mL, 50. mu.L), dissolved by vortexing, and allowed to stand. The solution was added dropwise slowly to a mixed pH 7.4 aqueous solution of HA and Gox (HA: 1mg/mL, Gox: 0.05mg/mL, 1mL) under stirring, and after stirring for 3 to 5 hours, the organic solvent was removed by dialysis.

The drug loading (wt.%) is (drug mass in nanoparticles/total mass of polymer and drug in nanoparticles) × 100%

Encapsulation efficiency (%) - (drug mass in nanoparticle/drug mass charged) × 100%

TABLE 1 characterization of nanogels encapsulating Gox and UK-5099

^aThe final micelle concentration was 1 mg/mL.

^bAverage particle diameter (nm) and particle diameter distribution of 2Measured by a dynamic light scattering instrument at 5 ℃ and pH 7.4.

Example 6 Smart responsive Shell-core polyelectrolyte Nanogels (HA-D-PEI) at 100. mu. M H₂O₂10mM GSH (pH 7.4) change in particle size

Taking the prepared nanogel (0.5mg/mL, 1mL), and adding a certain amount of high-concentration H₂O₂And mixing the GSH and the mixed solution to be adjusted to the required buffer environment. The sample was placed in a 37 ℃ constant temperature shaker (200 rpm). The change in particle size was measured at the indicated time points using a dynamic light scattering instrument.

FIG. 4 shows the signal at 100. mu. M H₂O₂Particle size distribution of 10mM GSH (pH 7.4) over time. The particle size of the micelles did not change significantly after one day. However, at 100 μ M H₂O₂The micelle particle size can be obviously enlarged to 300nm after 6 hours under the condition of 10mM GSH (pH 7.4), which indicates that the nanogel is obviously swelled; by 16 hours, the particle size began to be disordered, indicating that the nanogel had disintegrated.

Example 7 Smart responsive Shell-core polyelectrolyte Nanogels (HA-D-PEI) for the cytotoxicity test 4T1 (MTT)

Toxicity of nanogels (HA-D-PEI) in 4T1 cells was determined by the MTT method. Firstly, 100 mu L of DMEM suspension of cells (10% fetal calf serum, 100IU/mL penicillin and 100 mu g/mL streptomycin contained in DMEM culture medium) is paved in a 96-well culture plate and is placed under the conditions of 37 ℃ and 5% carbon dioxide for culture h, so that the coverage rate of the monolayer cells reaches 70-80%. Then 10. mu.L of a PB solution of nanogel (G/HA-D-PEI/U) at different concentrations was added to each well. After further incubation for 24h, 10. mu.L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) in PBS (5mg/mL) was added to each well, and the mixture was placed in an incubator for further incubation for 4h to allow the MTT to react with living cells. The MTT-containing culture solution was then removed, 150 μ L DMSO was added to each well to dissolve living cells and MTT-produced purple formazan crystals, and the absorbance at 570nm of each well was measured using a plate reader (SpectraMax i3 x). Cell relative viability was obtained by comparing the absorbance at 570nm of control wells with only blank cells. The experimental data were performed in three parallel groups.

Cell viability (%) - (OD570 samples/OD 570 control) × 100%

FIG. 5 is a graph showing the cytotoxicity results of a smart responsive shell-core polyelectrolyte nanogel (HA-D-PEI) on 4T1 cells. The results show that: free Gox and blank nanogels were almost non-toxic to cells at the set concentrations, while the cytotoxicity of the Gox-loaded nanogels increased with the increase in Gox concentration, indicating that the nanogels contribute to some extent to the endocytosis of Gox by 4T1 cells to generate cytotoxicity.