CN114369175B

CN114369175B - Low-oxygen-response chitosan polymer and preparation method and application thereof

Info

Publication number: CN114369175B
Application number: CN202210037127.5A
Authority: CN
Inventors: 高瑜; 李旭东; 王玉然; 郑怡琳; 陈海军
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-12-13
Anticipated expiration: 2042-01-13
Also published as: CN114369175A

Abstract

The invention discloses a low-oxygen-response chitosan polymer and a preparation method and application thereof. According to the invention, a click chemistry method is adopted to covalently connect low-oxygen response group azobenzene and BODIPY photosensitizer with chitosan to form a chitosan polymer with low-oxygen response characteristic. The polymer can effectively reduce the phototoxicity of the photosensitizer, improve the stability of the photosensitizer, realize hypoxia response imaging in a tumor hypoxia microenvironment, and can be used as a carrier to encapsulate hydrophobic drugs, so that the drugs are released in a targeted manner at tumor parts, and the aggregation of the drugs at the tumor parts is promoted.

Description

Low-oxygen-response chitosan polymer and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a low-oxygen-response chitosan polymer and a preparation method and application thereof.

Background

Photothermal therapy (PTT for short) is a novel therapeutic means, and a Photothermal agent is excited under excitation of excitation light of a specific wavelength to convert light energy into heat energy and increase the temperature of a tumor site, thereby killing tumor cells (Zhi D, yang T, O' Hagan J, zhang S, donnelly RF. Photothermal therapy, J Control release. 2020. Compared with traditional operations, chemotherapy and radiotherapy, the photothermal therapy has the advantages of small wound, higher specificity, small damage to surrounding tissues and capability of reducing toxic and side effects on normal tissues. However, photothermal therapy has its own drawbacks, and patients receiving photodynamic therapy need to hide in the dark for a long time to avoid phototoxicity, and patient compliance is low.

Hypoxia is a hallmark feature of the tumor microenvironment resulting from aberrant angiogenesis, vascular damage and disorders of lymphatic drainage in solid tumors. The oxygen concentration in hypoxic tumor tissue is about 5mm Hg, which is significantly lower than the hypoxic level (70 mm Hg) in normal tissue. The fluorescent imaging molecules are released through hypoxia response, so that the hypoxic tumor tissue can be distinguished, lesion tissues and normal tissues can be distinguished, and a reference is provided for treatment.

The surface of the chitosan is rich in modified amino, and can be connected with other functional groups through structural modification to prepare a multifunctional drug carrier, and meanwhile, the chitosan has good biocompatibility and biodegradability and great clinical application potential. The invention discloses a novel chitosan nano material, which is characterized in that azobenzene and BODIPY molecules are connected to chitosan through click chemical reaction to obtain a nano carrier with functions of low-oxygen response release and photothermal therapy.

Disclosure of Invention

The invention aims to provide a chitosan polymer which can realize controllable release and hypoxia response imaging and drug release and a preparation method thereof, so as to solve the problem of high phototoxicity of the traditional photosensitizer, and enable the drug to be accumulated at a tumor part so as to improve the curative effect of the drug.

In order to realize the purpose, the invention adopts the following technical scheme:

a hypoxia-responsive chitosan polymer (CsB) of the formula:

，

wherein n and m are the number of repeating units. The resulting chitosan polymer has a heavy molecular weight of 10-1000 kilodaltons.

The preparation process of the chitosan polymer comprises the following steps:

；

which comprises the following steps:

i) 4-hydroxybenzaldehyde, 1,2-dibromoethane and K ₂ CO ₃ The mixture was added to DMF in a molar ratio of 1 ₂ CO ₃ Then dissolving the reaction product in DCM, washing with saturated brine and drying over anhydrous sodium sulfate, removing the solvent by evaporation under reduced pressure, and purifying by silica gel column chromatography with PE/EA =3:1 (v/v) as an eluent to obtain light yellow 4- (2-bromomethoxy) benzaldehyde (compound 1);

ii) dissolving the obtained 4- (2-bromomethoxy) benzaldehyde and sodium azide in a molar ratio of 1:1 in DMF, heating to react at 100 ℃ for 2 hours, dissolving a reaction product in EA, washing with saturated saline solution, drying by anhydrous sodium sulfate, and evaporating under reduced pressure to remove the solution to obtain 4- (2-azidoethoxy) benzaldehyde (compound 2);

iii) 4- (2-azidoethoxy) benzaldehyde and 2,4-dimethylpyrrole were dissolved in dry DCM at a molar ratio of 1:2, then a drop of trifluoroacetic acid (TFA) was added to the stirred solution, and the reaction was stirred at room temperature overnight; after TLC detection of aldehyde disappearance, adding 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) of 1EP, continuing stirring for reaction for 30min, then carrying out ice bath on the reactant, adding excessive triethylamine and boron trifluoride diethyl ether, continuing stirring for reaction for 2h, extracting the reaction mixture for several times with water to remove unreacted triethylamine, then extracting with DCM, collecting an organic phase, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, using DCM as an eluent, and purifying the residue by silica gel column chromatography to obtain a boron dipyrromethene azide compound (compound 3, abbreviated as BDP 1);

iv) adding BDP1 and p-hydroxybenzaldehyde into dry acetonitrile according to a molar ratio of 1:6, adding piperidine with the volume of 1/10 of the reaction liquid, heating the mixture at 80 ℃ for reaction for 36 hours until the solution changes from wine red to blue-green, stopping the reaction, pouring the reaction product into saturated saline solution, extracting the mixture by dichloromethane, collecting an organic phase, drying the organic phase by anhydrous sodium sulfate, performing reduced pressure spin drying, and then performing reaction on the mixture by dichloromethane/methanol =30:1 (v/v) is used as eluent, and a near-infrared nitrine boron dipyrromethene compound (compound 4, abbreviated as BDP 2) is obtained by silica gel column chromatography separation;

v) adding 4,4' -dicarboxylazobenzene, propynylamine, benzotriazole-N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HBTU) and N, N-Diisopropylethylamine (DIPEA) into DMF according to the molar ratio of 1;

vi) refluxing chitosan and 4-bromobenzene anhydride in a molar ratio of 1:3 in 1.0vol% acetic acid aqueous solution for 48 h, then cooling to room temperature, collecting precipitate by centrifugation, dispersing the precipitate in methanol, filtering, washing with methanol, and vacuum drying at 100 ℃ to obtain brominated chitosan (compound 6, abbreviated as Cs-Br);

vii) putting the obtained Cs-Br and sodium azide in a molar ratio of 1:7 in N-methyl-2-pyrrolidone, stirring and reacting at 80 ℃ under the protection of argon gas for 24 hours, filtering, pulping the filtrate in 80-100vol% ethanol solution, filtering the precipitate, washing with absolute ethanol and acetone, and drying in vacuum at 100 ℃ for 8 hours to obtain azide chitosan (compound 7, abbreviated as Cs-N3);

viii) adding the obtained compounds Aza, BDP2, cs-N3 and hexynic acid into a mixed solution consisting of dimethyl sulfoxide and water according to a molar ratio of 1.

Step vi) the chitosan used has a heavy molecular weight of 6 to 1000 kilodaltons.

The obtained chitosan polymer can be used for hypoxia-responsive imaging and preparation of nano-drugs with hypoxia-responsive release properties. The application method of the chitosan polymer in the preparation of the nano-drug specifically comprises the steps of taking the chitosan polymer as a carrier to entrap the hydrophobic drug; the hydrophobic drugs include, but are not limited to, doxorubicin, paclitaxel, and ocitinib.

The invention relates to the following principle:

firstly, the reduction stress is increased by the special pathological environment of tumor cell hypoxia, so that the over-expression of nitroimidazole enzyme, azo reductase and quinone reductase is caused, and the azobenzene group in the chitosan polymer obtained by the invention can be reduced by the azo reductase in tumor tissues, so as to achieve the purpose of hypoxia response release.

Second, BODIPY photosensitizers can perform imaging and photothermal therapy functions under the irradiation of excitation light with a certain wavelength. Therefore, the chitosan polymer containing the photosensitizer can be used for judging the position of the tumor and effectively killing the tumor, and can improve the photosensitivity of BODIPY and reduce the phototoxicity of the BODIPY by polymerizing with the chitosan.

Thirdly, the chitosan polymer of the invention can improve the accumulation of hydrophobic antitumor drugs in tumor parts.

The invention has the advantages that:

(1) The invention adopts the click chemistry method to prepare the polymer, the raw materials are easy to obtain, the cost is low, the preparation method is relatively simple, most products can be obtained by a pulping method, the side reaction is less, and the reaction efficiency is high.

(2) Azobenzene is a sensitive hypoxia response group, is sensitive to a tumor hypoxia microenvironment, and has strong response release specificity.

(3) The maximum absorption and the maximum emission of the modified BODIPY derivative are in a near infrared region, the tissue penetrability is good, the imaging effect can be improved, and the modified BODIPY derivative is an ideal probe.

Drawings

FIG. 1 is an infrared spectrum (A) of BDP2, azo, cs-N3 and CsB and a Raman spectrum (B) of CsB prepared in example 1.

FIG. 2 is a graph showing drug release of CsB under hypoxic conditions in example 7.

FIG. 3 is the in vitro toxicity test of CsB and nanoparticle 1 on H1975 cells in example 8.

FIG. 4 is an in vitro imaging experiment of CSB and BDP2 on H1975 cells in example 9.

Detailed Description

The present invention is further described below in conjunction with specific examples to assist those of ordinary skill in the art in further understanding the present invention, but are not intended to limit the invention in any way.

Example 1

(1) 4-hydroxybenzaldehyde (2.9g, 24mmol), 1,2-dibromoethane (24ml, 240mmol) and K ₂ CO ₃ (6.6 g, 48mmol) was added to DMF (20 mL) and the reaction was stirred at 70 ℃ for 4h, and the unreacted K was filtered ₂ CO ₃ (ii) a The reaction product was extracted with DCM and brine, the organic phase was dried over anhydrous sodium sulfate and the solvent was removed by evaporation under reduced pressure; the crude product was purified by silica gel column chromatography using PE/EA =3:1 (v/v) as eluent to give 4- (2-bromomethoxy) benzaldehyde (2.19 g, 40% yield) as a pale yellow solid, which was identified as follows:

¹ H NMR (500 MHz, Chloroform-d) δ 9.82 (s, 1H), 7.77 (s, 2H), 6.96 (s, 2H), 4.32 (s, 2H), 3.61 (s, 2H)； ¹³ C NMR (126 MHz, CHLOROFORM-D) δ 190.85, 163.09, 132.13, 130.54, 114.96, 68.03, 28.59. m/z calculate in 228.9859, found in 228.9852。

(2) 4- (2-bromomethoxy) benzaldehyde (3g, 13mmol) and sodium azide (0.85g, 13mmol) are dissolved in DMF (15 mL) and heated to react for 2h at 100 ℃; after the reaction, the product was dissolved in EA, extracted with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to remove the solvent, to obtain 4- (2-azidoethoxy) benzaldehyde (2.48 g, yield 99%) which was identified as follows:

¹ H NMR (500 MHz, Chloroform-d) δ 9.84 (s, 1H), 7.80 (s, 2H), 6.98 (s, 2H), 4.18 (s, 2H), 3.60 (s, 2H)； ¹³ C NMR (126 MHz, CHLOROFORM-D) δ 190.86, 163.22, 132.08, 130.48, 114.89, 67.30, 50.03. Calculated at 192.0768, found in 192.0762。

(3) 2,4-dimethylpyrrole (2.48g, 0.026 mol) and 4- (2-azidoethoxy) benzaldehyde (2.5g, 0.013mol) were dissolved in dry DCM (250 mL), then a drop of trifluoroacetic acid (TFA) was added to the stirred solution and the reaction was stirred at room temperature overnight; after TLC detection of aldehyde disappearance, DDQ (3.2g, 0.013 mmol) was added and the reaction was continued with stirring for 30min, then triethylamine (10 mL) was added to the mixture, followed by stirring for 15min and dropwise addition of BF at 0 deg.C ₃ ·OEt ₂ (10 mL) and stirring was continued for 2h and the reaction mixture was extracted several times with water to remove unreacted triethylamine, then extracted with DCM, the organic phase was collected, dried over anhydrous sodium sulfate, and after evaporation of the solvent under reduced pressure, the residue was purified by silica gel column chromatography using DCM as eluent to obtain compound BDP1 as a red solid (1.00 g, yield 18.8%) whose identification data are as follows:

¹ H NMR (400 MHz, Chloroform-d) δ 7.20 (d, J = 8.7 Hz, 1H), 7.05 (d, J= 8.7 Hz, 1H), 6.00 (s, 1H), 4.22 (t, J = 4.9 Hz, 1H), 3.67 (t, J = 4.9 Hz, 1H), 2.57 (s, 3H), 1.45 (s, 3H)； ¹³ C NMR (101 MHz, CDCl ₃ ) δ 158.80, 155.37, 143.11, 141.53, 131.79, 129.38, 127.82, 121.15, 115.16, 77.44, 67.01, 50.24, 14.58. HRMS (ESI): calculated at 410.1958, Found in 410.1943。

(4) BDP1 (1.23g, 3 mmol) was dissolved in 50mL acetonitrile, p-hydroxybenzaldehyde (2.20g, 18mmol) and piperidine (5.76 mL) were added, the reaction was stirred at 80 ℃ for 36 hours until the solution turned from wine red to blue-green, the resulting mixture was poured into saturated saline (30 mL) and extracted with dichloromethane (50 mL), and the organic phase was collected with anhydrous sulfurAfter drying the sodium salt, it is concentrated by evaporation under reduced pressure and the crude product is taken up in DCM with CH ₃ OH =30 as eluent, purification by column chromatography on silica gel afforded compound BDP2 as a dark blue solid (1.01 g, 58% yield), whose identification data are as follows:

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.18 (s, 1H), 7.34 (d, J = 8.8 Hz, 3H), 7.21 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.4 Hz, 1H), 6.78 (s, 1H), 6.73 (d, J= 8.6 Hz, 2H), 4.14 – 4.11 (m, 1H), 3.59 – 3.57 (m, 1H), 1.33 (s, 3H). HRMS (ESI): m/z: calculated at 618.2483, Found in 618.2463。

(5) To a solution of 4,4' -dicarboxylazobenzene (135mg, 0.5 mmol) in DMF (4 ml) was added propynylamine (274 μ L,4 mmol), HBTU (1496 mg,4 mmol) and DIPEA (1390.4 μ L,8 mmol), the reaction was stirred at room temperature for 24h until the reaction solution became clear from turbidity, and then the resulting mixture was slurried in water (50 mL), suction filtered and dried to obtain compound Azo as an orange solid (110 mg, yield 64.0%) with the following identification data:

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 9.12 (t, J = 5.5 Hz, 2H), 8.06 – 8.03 (m, 4H), 7.98 – 7.94 (m, 4H), 4.06 (dd, J = 5.5, 2.5 Hz, 4H), 3.11 (t, J = 2.5 Hz, 2H). ¹³ C NMR (101 MHz, DMSO) δ 165.61, 153.80, 136.77, 129.15, 123.11, 81.58, 73.51, 29.12. HRMS (ESI): m/z Calculated at 345.1346 found in 345.1351。

(6) Refluxing fully deacetylated chitosan (2.05 g,12.8 mmol) with 4-bromobenzoic anhydride (8.71 g,38.4 mmol) in 1.0vol% aqueous acetic acid (100 mL) 48 h; after the reaction, the mixture was cooled to room temperature, and the precipitate was collected by centrifugation, dispersed in methanol, filtered, washed with methanol, and dried under vacuum at 100 ℃ to give Cs — Br (3.46 g, 99% yield).

(7) Cs-Br (0.80 g,3.0 mmol) is dissolved in N-methyl-2-pyrrolidone (340 mL), sodium azide (1.37 g,21 mmol) is added, stirring reaction is carried out at 80 ℃ for 24 hours under the protection of argon, then filtration is carried out to remove salts, filtrate is pulped in 95% ethanol solution, precipitate is filtered, after washing by absolute ethanol and acetone, vacuum drying is carried out at 100 ℃ for 8 hours, and the product Cs-N3 (0.42 g, yield 56%) is obtained.

(8) Azo (34.4mg, 0.1mmol), cs-N3 (33.4mg, 0.1mmol), copper sulfate pentahydrate (6.4mg, 0.04mmol) and sodium ascorbate (14.08mg, 0.8mmol) were added to DMSO/H containing BDP2 (61.7mg, 0.1mmol) ₂ O (4 ml.

FIG. 1 is an infrared spectrum (A) of the prepared BDP2, azo, cs-N3 and CsB and a Raman spectrum (B) of the CsB. As can be seen from the infrared spectrogram, the absorption peaks at 2158nm and 2163nm representing azide groups on CsB disappear, and the peak at 1601nm representing azo groups appears on Raman spectrum, which indicates that the original azide groups on chitosan are consumed, and azobenzene is connected to chitosan.

Example 2

Azo (68.9mg, 0.2mmol), cs-N3 (66.8mg, 0.2mmol), copper sulfate pentahydrate (12.8mg, 0.08mmol), and sodium ascorbate (28.16mg, 1.6mmol) were added to DMSO/H containing BDP2 (123.5mg, 0.2mmol) ₂ O (8ml.

Example 3

1mg of CsB prepared in example 2 was dissolved in 1mL of DMSO, and was added dropwise to 10mL of water, and after stirring at room temperature in the dark for 12 hours, the reaction solution was added to a dialysis bag for dialysis, followed by lyophilization, to obtain CsB nanoparticles (CsBNs) having a particle size of 109.5. + -. 2.4nm.

Example 4

Dissolving 1mg of molecular targeted drug oxcininib in 1mL of DMSO solution, dispersing the oxcininib in 10mL of water, dropwise adding 1mL of DMSO solution containing 1mg of CsB, stirring the mixture at room temperature in a dark place for 12 hours, adding the reaction solution into a dialysis bag for dialysis, and freeze-drying the mixture to obtain the nanoparticles 1 with the particle size of 127.5 +/-5.1 nm and the encapsulation rate of 27 +/-5%.

Example 5

Dissolving 1mg of chemotherapy drug adriamycin into 1mL of DMSO solution, dispersing the DMSO solution into 10mL of water, then dropwise adding 1mL of DMSO solution containing 1mg of CsB, stirring the mixture at room temperature in a dark place for 12 hours, adding the reaction solution into a dialysis bag for dialysis, and freeze-drying the mixture to obtain the nanoparticles 2, wherein the particle size of the nanoparticles is 134.5 +/-1.4 nm, and the encapsulation rate is 35 +/-4%.

Example 6

Dissolving 1mg of chemotherapeutic drug paclitaxel in 1mL of DMSO solution, dispersing the solution in 10mL of water, then dropwise adding 1mL of DMSO solution containing 1mg of CsB, stirring the solution at room temperature in a dark place for 12 hours, adding the reaction solution into a dialysis bag for dialysis, and freeze-drying the solution to obtain nanoparticles 3, wherein the particle size of the nanoparticles is 113.5 +/-5.4 nm, and the encapsulation rate is 29 +/-4%.

Example 7

Adding 1mL of 5mg/mL CsBNs into dialysis bags containing a mixture of mouse liver microsomes and NADPH (used for simulating in-vivo hypoxia reduction conditions) with different concentrations (10, 30 and 50 mu M), putting the dialysis bags into 20mL of PBS solution, stirring at 37 ℃, taking 1mL for detection at a set time, and simultaneously adding 1mL of the buffer solution; the results are shown in FIG. 2, with the control group treated without the mixture of mouse liver microsomes and NADPH.

As shown in FIG. 2, the cumulative amount of drug released in the experimental group gradually increased with the increase in the concentration of the mixture of liver microsomes and NADPH added to the mice, as compared to the control group.

Example 8

Human lung cancer cell line H1975 cells (cells purchased from the cell resource center of Shanghai Life sciences institute of Chinese academy of sciences) were used as the test cell line.

The cell culture method comprises the following steps: taking out the frozen cells from the liquid nitrogen tank, quickly placing the cells in a water bath at 37 ℃, continuously shaking the cells to quickly melt the cells, and centrifuging the cells at the room temperature of 1000 rpm; discarding the frozen stock solution, beating with 1ml culture solution to obtain cell suspension, transferring the cell suspension into culture flask, supplementing 3ml culture solution, and placing the flask at 37 deg.C and 5% CO ₂ After culturing for 24 hours in the incubator, the old culture solution is discarded, the new culture solution is replaced, and the culture is continued.

Cytotoxicity test: selecting H1975 cells with logarithmic phase growth and good state, digesting with trypsin, and making into cell suspension (0.5-1 × 10) ⁵ one/mL). Inoculating the suspension into a 96-well plate according to the amount of 100 muL cell suspension per well, and placing the plate in a container containing 5% CO ₂ After incubation in a 37 ℃ incubator at 24h, different amounts of CsBNs and nanoparticles 1 were added. After 4h incubation, the irradiated group was given laser light (1.0W/cm) ² 2 min). After 24h of drug action, the wells were washed twice with PBS, 100. Mu.l of MTT solution (5 mg/ml, i.e., 0.5% MTT) was added to each well, the culture was terminated after continuing the culture for 4h, and the culture medium in the wells was carefully aspirated. Add 100. Mu.l DMSO into each well, and shake on a shaker for 10min at low speed to dissolve the crystals sufficiently. The absorbance of each well was measured at OD570 nm in an ELISA detector. And the survival rate of the cells was calculated as follows.

Survival (%) = (experimental absorbance-solvent control absorbance)/(blank absorbance-solvent control absorbance).

The cytotoxicity results are shown in fig. 3. As can be seen from fig. 3, under the normoxic environment, csBNs and nanoparticles 1 showed almost no significant toxicity, while both showed some degree of cytotoxicity after light irradiation. Under the hypoxia environment, the cytotoxicity of CsBNs is not obviously changed, but the cytotoxicity of the nanoparticle 1 is obviously enhanced, and after the illumination effect, the cytotoxicity is further enhanced, which proves that the drug encapsulated by the nanoparticle 1 can be released under the hypoxia condition, and the drug and the photosensitizer synergistically enhance the cytotoxicity.

Example 9

Cell imaging experiments: selecting H1975 cells with logarithmic phase growth and good state, digesting with trypsin, and preparing into cellsSuspension (0.5-1X 10) ⁵ one/mL). Inoculating the suspension into a 96-well plate according to the amount of 100 muL cell suspension per well, and placing the plate in a container containing 5% CO ₂ After 24h is incubated in an incubator at 37 ℃, 10 muM of compound 4 and CsBNs containing the compound 4 at the same concentration are added, the incubator is placed for 4 hours, 1mL of 10 muM hoechst is added for nuclear staining for 15min, and after paraformaldehyde is fixed for 15min, confocal shooting is carried out, and the result is shown in fig. 4.

As can be seen from fig. 4, csBNs showed little fluorescence under normoxic conditions, but showed fluorescence similar to that of the free drug under hypoxic conditions, demonstrating that it can be released under hypoxic conditions, indicating a hypoxic region.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A hypoxia-responsive chitosan polymer, characterized in that: the structural formula is as follows:

，

wherein n and m are the number of repeating units; the preparation method comprises the following steps:

i) 4-hydroxybenzaldehyde, 1,2-dibromoethane and K ₂ CO ₃ Adding the mixture into N, N-dimethylformamide according to a molar ratio of 1;

ii) dissolving the obtained 4- (2-bromomethoxy) benzaldehyde and sodium azide in a molar ratio of 1:1 in N, N-dimethylformamide, heating at 100 ℃ for reaction for 2 hours, dissolving a reaction product in ethyl acetate, washing with saturated saline solution, drying by anhydrous sodium sulfate, and removing the solution by evaporation under reduced pressure to obtain 4- (2-azidoethoxy) benzaldehyde;

iii) Dissolving 4- (2-azidoethoxy) benzaldehyde and 2,4-dimethylpyrrole in dry dichloromethane according to a molar ratio of 1:2, then adding a drop of trifluoroacetic acid to the stirred solution, and stirring at room temperature for reaction overnight; after the aldehyde reaction is finished, adding 2,3-dichloro-5,6-dicyano-1,4-benzoquinone of 1EP, continuously stirring and reacting for 30min, then carrying out ice bath on the reactant, adding excessive triethylamine and boron trifluoride diethyl etherate, continuously stirring and reacting for 2h, sequentially extracting with water and dichloromethane, collecting an organic phase, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, and purifying the residue through silica gel column chromatography to obtain the BODIPY compound;

iv) adding the BODIPY compound and p-hydroxybenzaldehyde into dry acetonitrile according to a molar ratio of 1:6, adding piperidine with the volume of 1/10 of the reaction liquid, heating at 80 ℃ for reaction for 36 hours until the solution changes from wine red to blue-green, stopping the reaction, pouring the reactant into saturated saline solution, extracting with dichloromethane, collecting an organic phase, drying with anhydrous sodium sulfate, performing reduced pressure spin drying, and performing silica gel column chromatography separation to obtain the near-infrared BODIPY compound;

v) adding 4,4' -dicarboxylazobenzene, propynylamine, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine into N, N-dimethylformamide according to a molar ratio of 1;

vi) refluxing chitosan and 4-bromobenzene anhydride in a molar ratio of 1:3 in 1.0vol% acetic acid aqueous solution for 48 h, cooling to room temperature, collecting precipitate by centrifugation, dispersing the precipitate in methanol, filtering, washing and drying to obtain brominated chitosan;

vii) dissolving the obtained chitosan bromide and sodium azide in a molar ratio of 1:7 in N-methyl-2-pyrrolidone, stirring and reacting at 80 ℃ under the protection of argon for 24 hours, filtering, pulping filtrate in 80-100vol% ethanol solution, filtering precipitate, washing with absolute ethyl alcohol and acetone, and drying in vacuum at 100 ℃ for 8 hours to obtain chitosan azide;

viii) adding the obtained compound azophenyl compound, near-infrared nitrine boron dipyrromethene compound, nitrine chitosan and hexynoic acid into a mixed solution consisting of dimethyl sulfoxide and water according to the mol ratio of 1.

2. The chitosan polymer of claim 1, wherein: the weight average molecular weight of the obtained chitosan polymer is 10-1000 kilodaltons.

3. The chitosan polymer of claim 1, wherein: the chitosan used in step vi has a weight average molecular weight of 6 to 1000 kilodaltons.

4. The chitosan polymer of claim 1, wherein: and viii, the volume ratio of the dimethyl sulfoxide to the water in the mixed solution is 4:1.

5. Use of the chitosan polymer of claim 1 in hypoxia responsive imaging.

6. Use of the chitosan polymer of claim 1 in the preparation of a nano-drug, wherein: the chitosan polymer is used as a carrier to entrap hydrophobic drugs to prepare the nano-drug with tumor hypoxia response release performance.