阅读说明：本技术 乏氧响应性阳离子聚合物及其制备方法与应用 (Hypoxic responsive cationic polymer and preparation method and application thereof ) 是由 殷黎晨 李旭东 于 2020-06-17 设计创作，主要内容包括：本发明公开了乏氧响应性阳离子聚合物及其制备方法与应用,以4,4’-二羧基偶氮苯和聚乙烯亚胺制备为原料,反应制备乏氧响应性阳离子聚合物；乏氧响应性阳离子聚合物与核酸复合得到纳米药物,或乏氧响应性阳离子聚合物与阴离子聚合物、蛋白复合得到纳米药物。本发明聚合物不仅可以作为核酸载体提供良好的稳定性、乏氧敏感性和生物相容性,并且能够结合负电性材料作为纳米载体用于基因和蛋白的肿瘤靶向递送。(The invention discloses an anaerobic responsive cationic polymer and a preparation method and application thereof, wherein 4,4' -dicarboxy azobenzene and polyethyleneimine are prepared as raw materials and are reacted to prepare the anaerobic responsive cationic polymer; the hypoxic responsive cationic polymer is compounded with nucleic acid to obtain the nano-drug, or the hypoxic responsive cationic polymer is compounded with anionic polymer and protein to obtain the nano-drug. The polymer not only can be used as a nucleic acid carrier to provide good stability, hypoxic sensitivity and biocompatibility, but also can be combined with electronegative materials to be used as a nano carrier for tumor targeted delivery of genes and proteins.)

1. A hypoxic-responsive cationic polymer having the chemical structure shown below:

in the formula: n is 5 to 8, r is 1 to 200, m is 1 to 200, and r + m is 15 to 200.

2. The method for preparing the hypoxic responsive cationic polymer according to claim 1, comprising the step of reacting 4,4' -dicarboxylazobenzene and polyethyleneimine as raw materials to prepare the hypoxic responsive cationic polymer.

3. The method for preparing the hypoxic-responsive cationic polymer according to claim 2, wherein the molar ratio of the 4,4' -dicarboxylazobenzene to the polyethyleneimine is 1: 1-4; 4,4' -dicarboxy azobenzene is prepared by taking 4-nitrobenzoic acid and a reducing agent as raw materials.

4. The method for preparing the hypoxic-responsive cationic polymer according to claim 2, wherein the reaction is carried out in the presence of a dehydrating agent and a catalyst.

5. The method for producing the hypoxic-responsive cationic polymer according to claim 4, wherein the dehydrating agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride or N, N' -dicyclohexylcarbodiimide; the catalyst is N-hydroxysuccinimide or N-hydroxythiosuccinimide.

6. A preparation method of nano-drugs is characterized by comprising the following steps of preparing 4,4' -dicarboxy azobenzene and polyethyleneimine as raw materials, and reacting to prepare a hypoxic responsive cationic polymer; the hypoxic responsive cationic polymer is compounded with nucleic acid to obtain a nano-drug, or the hypoxic responsive cationic polymer is compounded with an anionic polymer and protein to obtain the nano-drug.

7. The method for preparing the nano-drug according to claim 6, wherein the mass ratio of the hypoxic-responsive cationic polymer to the nucleic acid is (0.5-4): 1; the mass ratio of the hypoxic responsive cationic polymer to the anionic polymer is (0.5-8) to 1.

8. The method of claim 6, wherein the anionic polymer comprises hyaluronic acid and mannan.

9. The use of the hypoxic-responsive cationic polymer according to claim 1 for the preparation of a drug carrier or for the preparation of a nano-drug.

10. The use of the nano-drug prepared by the method of claim 6 for the preparation of a gene-or protein-based drug.

Technical Field

The application relates to the field of gene protein loading and delivery, in particular to a hypoxic responsive cation delivery material with high-efficiency gene delivery capacity, a preparation method and application thereof, and can be applied to the field of tumor treatment.

Background

During the growth of cancer cells, nutrients and oxygen are rapidly consumed by the proliferation process of the cells, new blood vessels are not formed in time, and temporary blocking exists in internal tissue structures, so that insufficient perfusion and temporary hypoxia in tumors are caused. As one of the important features of the tumor microenvironment, hypoxia is involved in the growth, invasion and metastasis processes of tumor-associated cells, and has profound effects on hypoxia-targeted drugs in the treatment of tumors, in which bioreductive chemical functional groups (such as nitroimidazole, quinone and azobenzene derivatives) are also being increasingly tried and explored.

Despite the availability of hypoxic activation strategies to deliver drugs to tumor sites, a high degree of selective and real-time release of tumor hypoxia gene delivery is currently not achieved due to tumor hypoxia heterogeneity and the difficulty of gene delivery. In the process of tumor gene delivery, polyethyleneimine is the most widely studied cationic material. While effective in carrying nucleic acids or proteins for use as delivery vehicles, large molecular weight cations inevitably cause strong cytotoxicity.

Disclosure of Invention

The invention provides a cation delivery carrier material which is connected by azobenzene bonds and responds to hypoxia, and the polymer not only can be used as a nucleic acid carrier to provide good stability, hypoxia sensitivity and biocompatibility, but also can be combined with an electronegative material to be used as a nano carrier for tumor targeted delivery of genes and proteins.

The invention adopts the following technical scheme:

a hypoxic-responsive cationic polymer having the chemical structure shown below:

in the formula: n is 5 to 8, r is 1 to 200, m is 1 to 200, and r + m is 15 to 200.

The invention provides a preparation method of the hypoxic responsive cationic polymer, which takes 4,4' -dicarboxy azobenzene and polyethyleneimine as raw materials to prepare the hypoxic responsive cationic polymer through reaction; specifically, 4' -dicarboxy azobenzene, polyethyleneimine, a dehydrating agent and a catalyst are dissolved in an organic solvent dimethyl sulfoxide according to the molar ratio of 1: 1-4: 5: 3.6, and the mixture is reacted at room temperature for 45-50 hours to obtain a hypoxic responsive cationic polymer; preferably, 4' -dicarboxylazobenzene is prepared by using 4-nitrobenzoic acid and a reducing agent as raw materials.

Preferably, the polyethyleneimine is selected from polyethyleneimines having a weight average molecular weight of 600 to 25000, such as 600, 1800, 1000 and 25000.

Preferably, the dehydrating agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride or N, N' -dicyclohexylcarbodiimide.

Preferably, the catalyst is N-hydroxysuccinimide or N-hydroxythiosuccinimide.

The invention discloses a preparation method of a nano-drug, which comprises the following steps: 4,4' -dicarboxy azobenzene and polyethyleneimine are prepared as raw materials and react to prepare a hypoxic responsive cationic polymer; the hypoxic responsive cationic polymer is compounded with nucleic acid to obtain a nano-drug, or the hypoxic responsive cationic polymer is compounded with an anionic polymer and protein to obtain a nano-drug; specifically, for the binary complex, the hypoxic responsive cationic polymer is dissolved in deionized water, the nucleic acid solution is added, and then 37^oC, incubation to obtain the nano-drug; for the ternary complex, the hypoxic responsive cationic polymer and the anionic polymer are respectively dissolved in deionized water, the protein solution is firstly mixed with the anionic solution, then the hypoxic responsive cationic polymer is added, and then 37^oAnd C, incubation to finally obtain the nano-drug.

Specifically, the preparation method of the hypoxic responsive cationic polymer comprises the following steps:

(1) 4,4' -dicarboxy azobenzene is prepared by taking 4-nitrobenzoic acid, glucose and sodium hydroxide as raw materials through reaction;

(2) 4,4' -dicarboxy azobenzene and polyethyleneimine are used as raw materials to react to prepare the cationic polymer with the hypoxic response.

In the reaction in the step (1), the reaction solvent is water, 50%^oC, reacting for 18 hours in the presence of oxygen, and passing the obtained crude product through hydrochloric acidAcidifying; the chemical structural formula of the 4,4' -dicarboxy azobenzene is as follows:

the specific reaction described above can be represented as follows:

in the present invention, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA) and DNA.

In the invention, the protein is selected from therapeutic proteins such as ribonuclease A, glucose oxidase and the like.

In the present invention, the mass ratio of the hypoxic-responsive cationic polymer to the nucleic acid is (0.5-4): 1, and the preferred mass ratio is (1-2): 1.

In the present invention, the mass ratio of the hypoxic-responsive cationic polymer to the anionic polymer is (0.5-8) to 1, and the preferred mass ratio is (1-4) to 1.

The invention discloses an application of a hypoxic responsive cationic polymer in preparation of a drug carrier or an application in preparation of a nano-drug; or the application of the nano-drug in the preparation of gene and protein drugs.

The cationic polymer has abundant positive charges, can well compound nucleic acid drugs to form stable nano compounds, and realizes polymer degradation under the condition of oxygen deficiency, so that the toxicity of materials is reduced, and the transfection efficiency is remarkably improved. On the other hand, the delivery efficiency of the carrier to the protein drug is improved by combining the anionic polymer and the protein drug to form the nano-composite.

Drawings

FIG. 1 is a nuclear magnetic spectrum of 4,4' -dicarboxylazobenzene in example one.

FIG. 2 is a nuclear magnetic spectrum of the hypoxia-responsive cationic polymer of example two.

FIG. 3 is a graph of the UV absorption of AO cationic polymers before and after the hypoxic treatment in example three.

FIG. 4 is a MALDI-TOF measurement Molecular Weight (MW) chart before and after hypoxic treatment in example three.

FIG. 5 is an agarose gel electrophoresis of the siRNA entrapment capacity of AO and BO before and after hypoxic treatment in example four.

FIG. 6 is a graph of particle size and potential of the siRNA and AO at different ratios to form nanocomplexes for example four.

FIG. 7 is a graph of the expression levels of mRNA XIAP in Skov-3 cells of the AO/siXIAP complex under normoxia and hypoxia in example four.

FIG. 8 is a graph of the toxicity test of AO in Skov-3 cells under normoxia and hypoxia in example four.

FIG. 9 is a graph of particle size and potential for the nanocomposite formation of HA and AO at different ratios in example V.

FIG. 10 is a graph showing the cumulative release of GOx protein from HAG nanocomplexes before and after the hypoxic treatment in example.

FIG. 11 is a graph showing the uptake content of the five HAG nanocomplexes in HeLa cells in example.

FIG. 12 is a graph of the toxicity level of HAG and HBG nanocomplexes in HeLa cells under normoxia and hypoxia in example five.

FIG. 13 shows HAG nanocomplexes in HeLa cells H in example V₂O₂To generate a concentration map.

Detailed Description

For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the following examples, which are intended to further illustrate features and advantages of the invention, but are not intended to limit the claims of the invention.