CN111607078B

CN111607078B - Hypoxic responsive cationic polymer and preparation method and application thereof

Info

Publication number: CN111607078B
Application number: CN202010556577.6A
Authority: CN
Inventors: 殷黎晨; 李旭东
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2022-06-03
Anticipated expiration: 2040-06-17
Also published as: CN111607078A

Abstract

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.

Description

Hypoxic responsive cationic polymer and preparation method and application thereof

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: in the order of 4,4' -dicarboxy azobenzene and polyethyleneimine are prepared as raw materials and react to prepare an oxygen-deficient 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 acidifying the obtained crude product by hydrochloric acid; 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.

In the examples:

polyethyleneimine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 4-nitrobenzoic acid, biphenyldicarboxylic acid, and the like are purchased from an avastin reagent.

All were purchased from synerville using a glass instrument.

Analytical balances were purchased from Sartorius (model: BSA 224S).

The centrifuge was purchased from Thermo SCIENTIFIC (model: MULTIPUGE X1R).

A magnetic stirrer was purchased from IKA (model: RH digital).

A rotary evaporator was purchased from IKA (model: RV 10).

PrimeScript RT kit and SYBR Premix Ex Taq kit were purchased from Baozi (Beijing, China).

XIAP siRNA (siXIAP), negative control siRNA (sinc) were purchased from the gimar gene (shanghai, china).

Oxygen from moist O₂（20%）、CO₂(5%) and N₂Mixed atmosphere, and hypoxic by humid O₂（1%）、CO₂(5%) and N₂Forming a mixed atmosphere; in particular, the conditions of the test are conventional in the art.

Hypoxic treatment conditions: HEPES buffer (containing rat liver microsomes (0.5 mg/mL) and NADPH (100. mu.M)) was incubated with the material for 12 hours under hypoxic conditions.

Example one

4-Nitrobenzoic acid (10.0 g, 59.9 mmol) and water (50 mL) were added to a three-neck round bottom flask, after which sodium hydroxide solution (5.6M, 100 mL) was added and heated to 50%^oC, then an aqueous glucose solution (1.7M, 200 mL) was added and air was continuously bubbled through the solution, which initially precipitated an orange color, after which the color gradually changed to brown. 50^oAnd C, reacting for 18 hours, and filtering to obtain a crude product. The crude product was redissolved in water (300 mL) and acidified with hydrochloric acid (1M) to precipitate an orange precipitate. Filtered again and the solid washed with water (100 mL. times.5), 60^oDrying under C to obtain 4,4' -dicarboxylazobenzene (4.26 g, 43% yield) as an orange solid, and the nuclear magnetic spectrum of deuterated dimethyl sulfoxide is shown in figure 1.

Example two

4,4' -dicarboxylazobenzene (50 mg, 0.19 mmol) was dissolved in dimethyl sulfoxide (3 mL), followed by addition of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (210 mg, 1.10 mmol) and N-hydroxysuccinimide (80 mg, 0.70 mmol). After stirring at room temperature for 4 hours, a solution of polyethyleneimine in dimethyl sulfoxide (0.37M, 1 mL) having a molecular weight of 600 was added dropwise. And (3) reacting for 24 hours in a dark place, dialyzing with deionized water (MWCO is 3.5 kDa) for 2 days, and freeze-drying at the temperature of-80 ℃ to obtain the cationic polymer (AO) in an orange foam shape, wherein the nuclear magnetism is performed by deuterium-substituted heavy water, and the nuclear magnetism spectrogram is shown in the attached figure 2.

The specific reaction described above can be represented as follows:

based on the method, the dimethyl sulfoxide solution of polyethyleneimine is replaced by 0.5 mL, the rest is unchanged, the obtained cationic polymer has no water solubility, when deionized water is added to prepare a solution with the concentration of 1 mg/mL, a turbid solution is formed, and 10 times of deionized water is added to form the turbid solution.

Comparative example 1

Biphenyldicarboxylic acid (46 mg, 0.19 mmol) was dissolved in dimethyl sulfoxide (3 mL), after which 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (210 mg, 1.10 mmol) and N-hydroxysuccinimide (80 mg, 0.70 mmol) were added. After stirring at room temperature for 4 hours, a solution of polyethyleneimine in dimethyl sulfoxide (0.37M, 1 mL) having a molecular weight of 600 was added dropwise. The reaction was carried out for 24 hours under dark conditions, dialyzed with deionized water (MWCO ═ 3.5 kDa) for 2 days, and lyophilized to obtain biphenyl cationic polymer (BO) as a white foam:

EXAMPLE III

The cationic material was evaluated for hypoxia responsiveness by incubating HEPES buffer (containing rat liver microsomes (0.5 mg/mL) and NADPH (100 μ M)) with the material for 12 hours under hypoxic conditions. The UV absorption spectrum of the cationic material under hypoxic or normoxic conditions was measured with a UV-Vis spectrophotometer (LAMBDA 750, PerkinElmer, USA) and the change in MW of the polymer before and after the hypoxic treatment was determined by MALDI-TOF.

FIG. 3 is a UV chart before and after hypoxic treatment, and the chart shows that the absorption peak of 450 nm azobenzene in the cationic polymer disappears after the treatment, thereby proving the hypoxic sensitivity of AO.

FIG. 4 is a graph of the change in Molecular Weight (MW) measured by MALDI-TOF before and after hypoxic treatment, and analysis of the data shows that the MW of polyethyleneimine increases from 665 to 4382 Da for AO; the MW of AO also decreased to 785 Da after the hypoxic treatment, indicating the successful synthesis of the hypoxic-responsive cationic polymer AO.

EXAMPLE four preparation, characterization and Performance of siRNA Nanoparticulates

The hypoxic responsive cationic polymer of example two was dissolved inPreparing a solution (clear solution) with the concentration of 1 mg/mL by deionized water, and preparing a DEPC aqueous solution with the concentration of 0.1 mg/mL by XIAP siRNA (siXIAP, purchased from Jima gene (Shanghai, China)); the siRNA and the hypoxic responsive cationic polymer were then mixed at different mass ratios (1/0.25, 1/0.5, 1/1, 1/2, 1/4, 1/6), the mixture was vortexed for 10s, then at 37^oAnd C, incubating for 30 minutes to form the hypoxia-responsive cationic polymer/siRNA compound nano-drug. Loading 3% agarose gel electrophoresis, running at 100V for 40 min, ethidium bromide display, imaging with a gel imaging system, and determining the loading efficiency of siRNA. Parallel experimental comparisons were performed with BO instead of AO.

siRNA and the cationic polymer of the anaerobic responsive azobenzene are mixed according to different mass ratios (1/0.25, 1/0.5, 1/1, 1/2, 1/4 and 1/6), the mixture is vortexed for 10s, and then the vortexed mixture is vortexed at 37^oAnd C, incubating for 30 minutes to form an azobenzene cationic polymer/siRNA complex. The azobenzene cationic polymer/siRNA complex was treated with rat liver microsomes (0.5 mg/mL) and NADPH (100. mu.M) for 12 hours, and the change of the loading capacity of the azobenzene cationic polymer to siRNA before and after hypoxic treatment was evaluated by gel electrophoresis analysis.

Dynamic Light Scattering (DLS) was used to evaluate the particle size and potential of azobenzene cationic polymer/siRNA complexes mixed at different mass ratios.

Skov-3 cells at 5X 10⁵The cells/well density were plated on 6-well plates and cultured for 24 hours. The medium was replaced with serum-free DMEM, and BO/siXIAP and AO/siXIAP (w/w = 1/1) binary complex (2. mu.g siXIAP/well) were added, respectively. At 37^oC, incubation under hypoxic or normoxic conditions for 6 hours, the medium was changed to DMEM containing 10% FBS, and incubation was continued for 24 hours. Total RNA was isolated from the cells, cDNA was synthesized using a cDNA reverse transcription kit, and the level of XIAP gene silencing was determined by real-time PCR. Untreated cells were used as controls to evaluate the efficiency of XIAP gene silencing under hypoxic conditions.

Skov-3 cells at 1X 10⁴The cells/well were plated on 96-well plates and cultured for 24 hours. Adding azobenzene cationic polymer with different concentrations, and incubating for 6 hrThen (c) is performed. The cells were incubated for 72 hours in the presence of oxygen or normoxic conditions after changing the medium, and the cytotoxicity was measured by MTT assay.

FIG. 5 is an agarose gel electrophoresis comparing the siRNA entrapment capacity of AO versus BO before and after hypoxic treatment. Due to the positive charge density of the amino group and the hydrophobicity of azobenzene, the cationic polymer AO exhibits an excellent nucleic acid-entrapping ability at a mass ratio of 1. After hypoxic treatment, AO promotes release of siRNA, while BO has no change in entrapment capacity before and after treatment, indicating that breakage of azobenzene bonds under hypoxic results in degradation of cationic polymers and promotion of gene release.

FIG. 6 is a graph of particle size and potential of siRNA and AO in nanocomplexes at different ratios. When the mass ratio is 1, the stable particle diameter of about 100 nm can be formed by the siRNA and the azobenzene cationic polymer, and the potential is always maintained at about 30 mV, so that the cationic material has good loading capacity on nucleic acid.

Figure 7 is a graph of the determination of whether cationic polymer AO has the effect of promoting xixiap release and reducing XIAP gene levels under hypoxic conditions. XIAP mRNA expression decreased by about 50% after incubation with AO/siXIAP complex. Under hypoxic conditions, an additional 20-30% reduction in gene levels was achieved with the AO/siXIAP complex 6 hours after cell treatment. The control group did not change before and after treatment, indicating that hypoxia induced degradation of the azobenzene polymer, which prompted siXIAP to function intracellularly, unlike the prior art in which cleavage occurs extracellularly.

FIG. 8 is a graph of AO toxicity levels in HeLa cells under normoxic and hypoxic conditions. AO showed strong concentration dependence and cytotoxicity after 48 hours of incubation with cells under normal oxygen. And the cell survival rate of the maximum concentration (35 mu g/mL) under the AO hypoxia condition is still higher than 90%, and the toxicity is obviously reduced.

EXAMPLE preparation, characterization and Properties of protein Nanoparticulates

The water-soluble azobenzene cationic polymer AO (1 mg/mL) and hyaluronic acid HA (5 mg/mL) of example two were dissolved in deionized water, respectively. AO was mixed with HA in different mass ratios (1/0.5, 1/1, 1/2, 1/4, 1/8), the mixture was vortexed for 10s and then at 37^oIncubation for 30 min at C, shapeForming HA/AO complex. Dynamic Light Scattering (DLS) was used to evaluate HA/AO for complex particle size and potential at different mass ratio mixtures.

The glucose oxidase GOx (0.1 mg/mL) was then dissolved in deionized water, HA was added to the solution of GOx (HA/GOx = 80/1), vortexed for 10 seconds, 37 seconds^oIncubate for 10 min at C. Thereafter, AO was added to the mixture (AO/HA = 2/1) and incubation was continued for 1 hour at room temperature to form HA/AO/GOx protein nanocomplexes (HAG NCs). In the same manner, GOx was replaced with FITC-fluorescently labeled GOx (FITC-GOx), and HAG NCs encapsulating FITC-GOx were prepared.

HAG NCs (200. mu.L) loaded with FITC-GOx were placed in dialysis bags (MWCO = 10 kDa) at 37^oC, PBS solution (pH 7.4, 30 mL) or Na₂S₂O₄Was incubated in PBS solution (155 mM) to investigate the release behavior of FITC-GOx. At predetermined time intervals, the release medium (1 mL) was collected and analyzed by fluorescence spectrophotometry (. lamda.)_ex = 490 nm，λ_em= 525 nm) the concentration of GOx was quantified and the cumulative drug release amount was calculated. The solution outside the dialysis bag was supplemented with fresh PBS (1 mL) keeping its volume constant.

To determine the catalytic ability of GOx in HAG NCs, HAG NCs or free GOx (10. mu.g/mL) were incubated with different concentrations of glucose solution under continuous oxygen supply. The solutions were collected at different time points and tested for H using ROS detection kit (Biyunyan, China)₂O₂And (4) concentration. HAG NCs or GOx (10. mu.g/mL) were directly mixed with a fixed concentration of glucose solution (1 mg/mL), and the real-time pH change of the solution was measured using a pH meter. To further investigate the ability of hypoxia-induced nanocomposites to release GOx, the material was taken with Na₂S₂O₄After 4 hours of treatment (155 mM), HAG NCs were purified by ultrafiltration (MWCO = 3.5 kDa) and lyophilized for future use to exclude Na₂S₂O₄The treatment produces an effect of ions on the pH change.

To examine the cellular uptake capacity of HAG NCs, HeLa cells were used at 1X 10⁵Cell/well Density seeded in 24-well platesAnd culturing for 24 hours. DMEM medium was changed and GOx and HAG NCs (FITC-GOx, 2. mu.g/mL) were added separately. 37^oAfter 4 hours incubation at C, cells were washed 3 times with PBS and trypsinized (without EDTA). The suspension was centrifuged at 1000 g for 5 min, washed 2 times with PBS and finally resuspended in PBS (300. mu.L). Cell uptake levels were measured by flow cytometry (Beckton Dickinson, usa) and the data analyzed using Cell Quest software. The cells were pretreated with free HA (final concentration of 10 mg/mL) for 6 hours and washed 3 times with PBS before adding HAG NCs, and the level of cellular uptake was determined in the same manner.

To determine the cytotoxicity of the nanocomposites, HeLa cells were used at 1X 10⁴Cells/well density were seeded in 96-well plates and cultured for 24 hours. The cells were incubated for 48 hours under hypoxic or normoxic conditions with different concentrations of HA/AO/GOx (w/w = 4/1/0.05) (HAG) NCs and HA/BO/GOx (w/w = 4/1/0.05) (HBG) NCs added, respectively, and the cell viability was calculated by MTT.

To measure intracellular H by GOx₂O₂Concentration, HeLa cells at 1X 10⁶Cells/well density were seeded in 12-well plates and cultured for 24 hours. The medium was changed to DMEM without glucose (1 mL/well) and different concentrations of HAG NCs were added. At 37^oC incubation, cell lysis at different assay time points, and H in cell lysate using hydrogen peroxide assay kit (Biyun, China)₂O₂The content is measured.

Figure 9 is a graph of particle size and potential of HA and AO electrostatically bound to form a nanocomposite at different scales. When the HA/AO mass ratio was increased from 0.5 to 4, NCs having a particle size of 150-200 nm could be obtained, and the Zeta potential was continuously decreased from +28.9 mV to-23.1 mV. When the mass ratio is larger than 4, no change in potential is continued. Thus, AO can effectively entrap proteins to form HA/AO/GOx NCs (HAG NCs, HA/AO/GOx = 4/1/0.05, w/w/w) having a particle size of about 140 nm and a surface charge of-18.1 mV.

FIG. 10 is a graph showing the release amount of GOx protein in HAG NCs before and after hypoxic treatment. Under untreated conditions, release from HAG NCs within 24 hoursThe GOx of (A) is only 12.2%, indicating the ability of HARPG NCs to stably entrap proteins. And through Na₂S₂O₄After treatment, the release of GOx from HAG NCs increased to 73.8% within 24 hours. The above results demonstrate that hypoxia-mediated AO degradation can achieve controlled release of proteins in NCs.

FIG. 11 is a graph showing the uptake ability of HAG NCs in HeLa cells. NCs showed superior cellular uptake levels compared to low GOx uptake. In order to prove that HAG NCs surface HA mediated targeting HAs a positive effect on the uptake behavior of tumor cells, HeLa cells are pretreated by HA, and the influence of the tumor cells on the endocytosis level of NCs is detected. The uptake was greatly reduced in the pretreatment group compared to the untreated group, suggesting that HA contributes to the promotion of uptake of NCs by tumor cells.

FIG. 12 is a graph showing the difference in cytotoxicity of HAG NCs and HBG NCs against HeLa under normoxic and hypoxic conditions. In the process of catalyzing the conversion of glucose, GOx can consume a large amount of oxygen and simultaneously generate hydrogen peroxide. Under the normoxic condition, HAG NCs can enhance the proliferation inhibition of GOx on cancer cells, while HBG NCs have no obvious toxicity, so that the HAG NCs can promote AO degradation in the process that GOx causes hypoxia, thereby rapidly releasing a large amount of GOx, and finally completing the killing effect on HeLa cells.

FIG. 13 is the time-varying H in HeLa cells following delivery of HAG NCs₂O₂Graph of concentration change. HAG NCs can produce H intracellularly in the presence of oxygen₂O₂Successful delivery of GOx in HeLa cells was demonstrated and the function of GOx to catalyze glucose substrates was also demonstrated.

The invention provides a cationic polymer with hypoxic responsiveness, which is characterized in that azobenzene micromolecules are used as connecting groups, polyethyleneimine with different molecular weights is used as a monomer, and a delivery carrier material with a cross-linked structure is prepared together. The degradable cationic material synthesized by the invention overcomes the defects of the existing cationic carrier materials, and improves the delivery efficiency of genes or proteins while degrading the toxicity of the materials.

Claims

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

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 a nano-drug is characterized by comprising the following steps of preparing 4,4' -dicarboxylazobenzene 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.