CN114369175A

CN114369175A - Low-oxygen-response chitosan polymer and preparation method and application thereof

Info

Publication number: CN114369175A
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: 2022-04-19
Anticipated expiration: 2042-01-13
Also published as: CN114369175B

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 tumor site in a targeted manner, and the aggregation of the drugs in the tumor site 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 under the excitation of specific wavelength exciting light, Photothermal agent is excited to convert light energy into heat energy, and the temperature of tumor part is raised, so that tumor cells are killed (Zhi D, Yang T, O' Hagan J, Zhang S, Donnelly RF. Photothermal therapy, J Control Release. 2020;325: 52-71). 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 released through hypoxia response can be used for distinguishing hypoxic tumor positions, distinguishing lesion tissues from normal tissues and providing a reference for treatment.

The surface of the chitosan is rich in modified amino, and the chitosan 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 a 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 be controlled to release and realize low-oxygen 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 drugs to be accumulated at tumor positions so as to improve the curative effect of the drugs.

In order to achieve 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 K₂CO₃Adding into DMF at a molar ratio of 1:10:2, stirring at 70 deg.C for reaction for 4 hr, and filtering unreacted K₂CO₃Then 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 at 100 ℃ for reaction for 2 hours, dissolving a reaction product in EA, washing with saturated saline solution, drying with anhydrous sodium sulfate, and removing the solution by evaporation under reduced pressure to obtain 4- (2-azidoethoxy) benzaldehyde (compound 2);

iii) 4- (2-azidoethoxy) benzaldehyde and 2, 4-dimethylpyrrole are dissolved in a molar ratio of 1:2 in dry DCM, then one drop of trifluoroacetic acid (TFA) is added to the stirred solution, and the reaction is stirred at room temperature overnight; after TLC detection of aldehyde disappearance, adding 1EP 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ), continuing stirring for reaction for 30min, then carrying out ice bath on the reaction product, adding excessive triethylamine and boron trifluoride diethyl ether, continuing stirring for reaction for 2h, extracting the reaction mixture for several times by using water to remove unreacted triethylamine, then extracting by using DCM, collecting an organic phase, drying by using anhydrous sodium sulfate, evaporating the solvent under reduced pressure, and purifying the residue by using DCM as an eluent through 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 the mol ratio of 1:6, adding 1/10 volume of piperidine into reaction liquid, heating at 80 ℃ for reaction for 36h until the solution changes from wine red to blue-green, pouring the reaction product into saturated saline, extracting with dichloromethane, collecting organic phase, drying with anhydrous sodium sulfate, performing reduced pressure spin drying, and performing reaction at the ratio of dichloromethane/methanol =30:1 (v/v) is used as eluent, and the near infrared azido BODIPY 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 1:8:8:16, reacting at normal temperature for 24-36h until the reaction liquid becomes clear from turbid, then pulping the reaction liquid in 8 times volume of water, and performing suction filtration and drying to obtain an azophenyl compound (compound 5, abbreviated as Azo);

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 drying at 100 ℃ in vacuum to obtain brominated chitosan (compound 6, abbreviated as Cs-Br);

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

viii) adding the obtained compounds Azo, BDP2, Cs-N3 and hexynic acid into a mixed solution of dimethyl sulfoxide and water according to the molar ratio of 1:1:1:1, adding 0.1-1 equivalent of sodium sulfate pentahydrate and 0.2-2 equivalent of ascorbic acid, vigorously stirring at room temperature for 24h, filtering out insoluble cross-linking impurities, collecting filtrate, pulping the filtrate in 80-100vol% ethanol solution, filtering, and drying to obtain the product CsB.

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 certain wavelength exciting light. 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 an 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 (2.9 g, 24 mmol), 1, 2-dibromoethane (24 ml, 240 mmol) and K₂CO₃(6.6 g, 48 mmol) was added to DMF (20 mL) and the reaction stirred at 70 ℃ for 4h, the unreacted K was filtered₂CO₃(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:

¹H 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)； ¹³C 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 (3 g, 13 mmol) and sodium azide (0.85 g, 13 mmol) were dissolved in DMF (15 mL) and the reaction was heated at 100 ℃ for 2 h; 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:

¹H 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)；¹³C 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 (2.48 g, 0.026 mol) and 4- (2-azidoethoxy) benzaldehyde (2.5 g, 0.013 mol) were dissolved in dry DCM (250 mL), then one 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 (3.2 g, 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.C₃·OEt₂(10 mL), the reaction mixture was extracted several times with water after stirring for a further 2h to remove unreacted triethylamine, then extracted with DCM, the organic phase collected was dried over anhydrous sodium sulphate and the solvent evaporated under reduced pressure and the residue purified by column chromatography on silica gel using DCM as eluent to give compound BDP1 as a red solid (1.00 g, 18.8% yield) which was identified as follows:

¹H 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)； ¹³C NMR (101 MHz, CDCl₃) 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 (1.23 g, 3 mmol) was dissolved in 50mL acetonitrile and p-hydroxybenzaldehyde (2.20 g, 18 mmol) 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 brine (30 mL) and extracted with dichloromethane (50 mL), the organic phase was dried over anhydrous sodium sulfate, evaporated under reduced pressure and concentrated, and the crude product was taken up in DCM: CH₃OH =30:1 as eluent, and purification by silica gel column chromatography gave compound BDP2 as a dark blue solid (1.01 g, 58% yield) with the following identification data:

¹H 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) propiolamine (274. mu.L, 4 mmol), HBTU (1496 mg, 4 mmol) and DIPEA (1390.4. mu.L, 8 mmol) were added to a DMF (4 mL) solution containing 4,4' -dicarboxylazobenzene (135 mg, 0.5 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), filtered with suction, and dried to obtain the compound Azo as an orange solid (110 mg, 64.0% yield) with the following identification data:

¹H 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). ¹³C 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) for 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) was dissolved in N-methyl-2-pyrrolidone (340 mL), and sodium azide (1.37 g, 21 mmol) was added, and the reaction was stirred at 80 ℃ under argon protection for 24h and then filtered to remove salts, the filtrate was slurried in 95% ethanol solution, the precipitate was filtered, washed with absolute ethanol and acetone, and vacuum dried at 100 ℃ for 8h to obtain the product Cs-N3 (0.42 g, 56% yield).

(8) Azo (34.4 mg, 0.1 mmol), Cs-N3 (33.4 mg, 0.1 mmol), copper sulfate pentahydrate (6.4 mg, 0.04 mmol) and sodium ascorbate (14.08 mg, 0.8 mmol) were added to DMSO/H containing BDP2 (61.7 mg, 0.1 mmol)₂O (4 mL:1 mL) solution, after vigorously stirring at room temperature for 24 hours, hexynoic acid (11.2 mg, 0.1 mmol) was added in combination with unreacted azide groups, the resulting mixture was filtered of insoluble cross-linking impurities, the collected filtrate was poured into anhydrous ethanol (30 mL) and slurried and filtered to obtain a crude product, which was washed three times with anhydrous ethanol to obtain CsB as a black solid (50.3 mg, yield 38.8%), grafting rate 24%.

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 (68.9 mg, 0.2 mmol), Cs-N3 (66.8 mg, 0.2 mmol), copper sulfate pentahydrate (12.8 mg, 0.08 mmol) and sodium ascorbate (28.16 mg, 1.6 mmol) were added to DMSO/H containing BDP2 (123.5 mg, 0.2 mmol)₂O (8 mL:2 mL) solution, vigorously stirred at room temperature for 24 hours, the resulting mixture was filtered of insoluble cross-linking impurities, and the residue was collectedThe collected filtrate was poured into absolute ethanol (50 mL) and filtered to obtain a crude product, which was further washed three times with absolute ethanol to obtain CsB (110 mg, yield 42.5%) as a black solid with a grafting rate of 41%.

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. + -. 2.4 nm.

Example 4

Dissolving 1mg of molecular targeted drug oxcetinic acid in 1mL of DMSO solution, dispersing the oxcetinic acid in 10mL of water, then dropwise adding 1mL of DMSO solution containing 1mg of CsB, stirring the oxcetinic acid in the dark at room temperature for 12 hours, adding the reaction solution into a dialysis bag for dialysis, and freeze-drying the reaction solution 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 chemotherapeutic drug adriamycin in 1mL DMSO solution, dispersing the solution in 10mL water, then dropwise adding 1mL DMSO solution containing 1mg CsB, stirring at room temperature in the dark for 12 hours, adding the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain the nanoparticles 2 with the particle size of 134.5 +/-1.4 nm and the encapsulation rate of 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 a dialysis bag 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 bag 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% CO₂After 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 experiments: selecting H1975 cells with logarithmic phase growth and good state, digesting with trypsin, and making into cell suspension (0.5-1 × 10)⁵one/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% CO₂After incubation for 24h in an incubator at 37 ℃, different amounts of CsBNs and nanoparticles 1 were added. After 4h incubation, the irradiated groups were given a laser (1.0W/cm)²2 min). After 24h of drug action, the cells were washed twice with PBS, 100. mu.l of MTT solution (5 mg/ml, i.e., 0.5% MTT) was added to each well, and after further incubation for 4h, the incubation was terminated and the culture medium was carefully aspirated from the wells. Add 100. mu.l DMSO into each well, 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 making into cell suspension (0.5-1 × 10)⁵one/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% CO₂And after incubation for 24 hours in an incubator at 37 ℃, adding 10 mu M of compound 4 and CsBNs containing the compound 4 with the same concentration, placing the mixture in the incubator for 4 hours, adding 1mL of 10 mu M hoechst to stain nuclei for 15min, and after fixation of paraformaldehyde for 15min, carrying out confocal shooting, wherein 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.

2. The chitosan polymer of claim 1, wherein: the resulting chitosan polymer has a heavy molecular weight of 10-1000 kilodaltons.

3. A method of preparing the chitosan polymer of claim 1, wherein: the method comprises the following steps:

i) 4-hydroxybenzaldehyde, 1, 2-dibromoethane and K₂CO₃Adding the mixture into DMF according to the molar ratio of 1:10:2, stirring and reacting for 4 hours at 70 ℃, dissolving a reaction product in DCM, washing with saturated saline solution, drying by anhydrous sodium sulfate, removing the solvent by reduced pressure evaporation, and purifying by silica gel column chromatography to obtain 4- (2-bromomethoxy) benzaldehyde;

ii) dissolving the obtained 4- (2-bromomethoxy) benzaldehyde and sodium azide in a molar ratio of 1:1 in DMF, heating at 100 ℃ for reaction for 2 hours, dissolving a reaction product in EA, washing with saturated saline solution, drying with 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 a molar ratio of 1:2 in dry DCM, 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 DDQ 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 DCM, 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 a BODIPY compound;

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

v) adding 4,4' -dicarboxy azobenzene, propynylamine, HBTU and DIPEA into DMF according to the mol ratio of 1:8:8:16, reacting at normal temperature for 24-36h until the reaction solution becomes clear from turbidity, stopping the reaction, pulping the reaction solution into water with the volume of 8 times of that of the reaction solution, and performing suction filtration and drying to obtain an azophenyl compound;

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 and drying to obtain brominated chitosan;

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

viii) adding the obtained compound azophenyl compound, near-infrared nitrine boron dipyrrole compound, nitrine chitosan and hexynoic acid into a mixed solution composed of dimethyl sulfoxide and water according to the molar ratio of 1:1:1:1, then adding 0.1-1 equivalent of sodium sulfate pentahydrate and 0.2-2 equivalent of ascorbic acid, violently stirring for 24 hours at room temperature, filtering, collecting filtrate, pulping in 80-100vol% ethanol solution, filtering, and drying to obtain the final product.

4. The method for preparing a chitosan polymer according to claim 3, wherein: step vi the chitosan used has a heavy molecular weight of 6-1000 kilodaltons.

5. The method for preparing a chitosan polymer according to claim 3, wherein: and viii, the volume ratio of the dimethyl sulfoxide to the water in the mixed solution is 4: 1.

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

7. 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.