An injectable hydrogel with self-healing, degradable, environmentally responsive and biocompatible properties and a method for preparing the same

Abstract

The application discloses an injectable hydrogel with self-repairing, degradable, environmental responsiveness and biocompatibility and a preparation method thereof, and the method comprises the following steps: synthesizing an aldehyde group functionalized camptothecin prodrug; synthesizing a camptothecin modified polyethyleneimine polymer; synthesizing an amino functionalized IR780 monomer; synthesizing a photothermal agent IR780 modified polysaccharide compound and a derivative polymer thereof; synthesizing an acid-sensitive injectable hydrogel; dissolving the camptothecin modified polyethyleneimine polymer and the photothermal agent IR780 modified polysaccharide compound and the derivative polymer thereof in water respectively, and then mixing the two solutions to react, so that the injectable hydrogel with self-repairing, degradable, environmental responsiveness and biocompatibility is formed. The injectable hydrogel with self-repairing, degradable, environmental responsiveness and biocompatibility can be injected into a tumor site through a syringe, and long-distance delivery of drugs is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical preparations, in particular to an injectable hydrogel with self-repairing, degradable, environmental responsiveness and biocompatibility and a preparation method thereof. BACKGROUND

[0002] In recent decades, chemotherapy for cancer has been a research hotspot and has made great progress. Local drug delivery systems are a focus of attention in the direction of chemotherapy because of their advantages over traditional drug delivery methods. In the development of local drug delivery systems, hydrogels have attracted great interest due to their unique properties. Hydrogels are three-dimensional polymer networks formed by chemical and physical crosslinking of polymers in aqueous solutions, which can be prepared from a variety of polymers or crosslinking agents, enabling them to absorb and retain large amounts of water or physiological fluids. In addition, most of the materials used in hydrogels are biopolymer materials, and after appropriate modification of their molecular structure, hydrogels can exhibit certain properties such as biodegradability, biocompatibility, tissue adhesion, self-healing, stimulus responsiveness, and injectability.

[0003] Among the various properties of hydrogels, one of the most important criteria for achieving minimally invasive local drug delivery is injectability. This property is crucial for more precise implantation of drugs into difficult-to-reach tissue sites and specific sites within tissues. They can be delivered to the target site in the body or tissue through a catheter or syringe. Injectable hydrogels are a surgical-free method of hydrogel implantation that undergoes a sol-gel transition at the desired site after injection in the body, attracting attention in biomedical applications. Injectable hydrogels have been proven to be the best tool for intratumoral drug delivery.

[0004] Biodegradability is an important consideration in the design of biomedical injectable hydrogels, which is closely related to the amount of hydrolysable groups (such as ester groups, carbonate groups, polypeptide groups, etc.). In polymer chains, synthetic polymers have greater flexibility and possibility in terms of chemical structure design and physical property control, which can endow injectable hydrogels with self-repairing properties. For example, redox-responsive disulfide bonds, Diels-Alder cycloaddition reactions, and hydrazide bond formation are all dynamic chemical reactions used to prepare self-repairing hydrogels. In addition, Schiff base bonds are also a dynamic bond that can be formed efficiently and orthogonally between amino and aldehyde groups under physiological conditions without the need for any external additives. This dynamic property supports self-repairing, endowing the gel with injectability and degradability. For example, using sodium periodate to cleave vicinal diols and generate open-loop dialdehydes, then forming Schiff base bonds with amino-containing polymers, the injectable hydrogel formed after modification of natural polymers endows the injectable hydrogel with better biocompatibility. This is one of the reasons why injectable hydrogels are the best candidate carriers for the delivery of anticancer drugs in local regional drug delivery.

[0005] At present, there are many anticancer drugs on the market, including camptothecin, doxorubicin, paclitaxel, etc., among which camptothecin is a common one in experimental research and clinical practice. Camptothecin (CPT) is an alkaloid extracted from camptotheca acuminata for the first time, and its molecular formula is C20H16N2O4. It is a DNA topoisomerase I inhibitor that prevents DNA replication and causes irreversible DNA strand damage, and has a strong inhibitory effect on various cancers and has significant antitumor activity in preclinical studies. Since Wall et al. discovered the drug in 1966, people have been trying to apply the drug to current cancer treatment. However, due to its strong hydrophobicity, the drug has strong toxicity to cells and tissues during drug transport, which limits its application. When the drug is used in combination with hydrogel, the current common method is to physically mix it in the hydrogel, which cannot achieve precise drug release. Secondly, most of the hydrogels on the market are generated by a large number of chemical bonds, and the problem that follows is that it is difficult to place them completely in a specific location by means other than surgery. After the hydrogel reaches the specific location, some hydrogels cannot be effectively degraded and cannot be released on demand, hindering the normal growth of tissues. These are the problems that hydrogels often face at present. SUMMARY

[0006] The present application aims to provide a self-repairable, degradable, environmentally responsive and biocompatible injectable hydrogel and a preparation method thereof, which can be injected into the tumor site by a syringe, reducing the long-distance transport of drugs.

[0007] In one aspect of the present application, a preparation method of a self-repairable, degradable, environmentally responsive and biocompatible injectable hydrogel is provided. According to an embodiment of the present application, the preparation method comprises the following steps:

[0008] (1) Synthesis of aldehyde-functionalized camptothecin prodrug: first, bromoisobutyryl bromide reacts with disulfide in tetrahydrofuran solution, and the reaction product reacts with sodium azide in N,N'-dimethylformamide solution, and finally the reaction product reacts with camptothecin in dry dichloromethane. After the reaction, the reaction solution is first filtered, washed with deionized water, separated by silica gel column, and finally a light yellow reaction product I is obtained, which is an aldehyde-functionalized camptothecin prodrug, and its structural formula is as follows:

[0009]

[0010] (2) Synthesis of camptothecin-modified polyethyleneimine polymer: polyethyleneimine and aldehyde-functionalized camptothecin prodrug undergo Schiff base reaction in a mixed solvent of methanol and N,N'-dimethylformamide, and the reaction is carried out overnight at room temperature. After the reaction is completed, precipitation is carried out, and finally an orange yellow product II is obtained by drying, and its structural formula is as follows:

[0011]

[0012] (3) Synthesis of amino-functionalized IR780 monomer: IR780 and p-aminophenylthiol undergo substitution reaction in N,N'-dimethylformamide solvent, and the reaction is carried out at room temperature in the dark overnight. After the reaction is completed, the reaction solvent is spin-dried, and the dark green product III is obtained by silica gel column separation, and the structure of the product is as follows:

[0013]

[0014] (4) Synthesis of polysaccharide compound and its derivative polymer modified by photothermal agent IR780: The polysaccharide compound and its derivative polymer are dissolved in a mixed solvent of methanol and water to carry out Schiff base reaction with the dark green product III, and the reaction is carried out at room temperature in the dark overnight. The green product V is obtained by sedimentation in ethanol and then drying.

[0015] (6) Synthesis of acid-sensitive injectable hydrogel: The orange-yellow product II and the green product V are respectively dissolved in water, and then the two solutions are mixed to form the injectable hydrogel with self-repairing, degradable, environmentally responsive and biocompatible properties.

[0016] In addition, the preparation method of the injectable hydrogel with self-repairing, degradable, environmentally responsive and biocompatible properties according to the above-mentioned embodiments of the present application can also have the following additional technical features:

[0017] In some embodiments of the present application, the step (1) specifically comprises the following steps:

[0018] a. Synthesis of bromine-containing disulfide monomer:

[0019] The bromoisobutyryl bromide and the dihydroxyethyl disulfide are subjected to substitution reaction, and after separation, washing and drying, the bromine-containing disulfide monomer molecule is obtained, and the structural formula is as follows:

[0020]

[0021] b. Synthesis of azide-functionalized disulfide monomer:

[0022] The sodium azide and the bromine-containing disulfide monomer molecule are subjected to substitution reaction, and after separation, washing and drying, the azide-functionalized disulfide monomer is obtained, and the structural formula is as follows:

[0023]

[0024] c. Synthesis of azide-functionalized camptothecin monomer:

[0025] After substitution reaction, separation, washing and drying of azide disulfide monomer and camptothecin, azide functionalized camptothecin is obtained, and its structural formula is as follows:

[0026]

[0027] d. Synthesis of aldehyde functionalized camptothecin prodrug:

[0028] After click reaction, separation, washing and drying of azide functionalized camptothecin and alkynyl benzaldehyde, aldehyde functionalized camptothecin prodrug is obtained, and its structural formula is as follows:

[0029]

[0030] In some embodiments of the present application, in step a, the molar ratio of bromoisobutyryl bromide and dihydroxyethyl disulfide is 3:2; in step b, sodium azide and disulfide monomer with one end of bromine are subjected to substitution reaction at a molar ratio of 3:1; in step c, azide disulfide monomer and camptothecin are subjected to substitution reaction at a ratio of 1.5:1; and in step d, azide functionalized camptothecin and alkynyl benzaldehyde are subjected to click reaction at a molar ratio of 1:1.2.

[0031] In some embodiments of the present application, in step (2), after the reaction is completed, precipitation is carried out in cold ether, and the molar ratio of polyethyleneimine and aldehyde functionalized camptothecin prodrug is 1:5-10.

[0032] In some embodiments of the present application, in step (3), the molar ratio of IR780 and p-aminophenylthiol is 1:3.

[0033] In some embodiments of the present application, in step (4), the polysaccharide compound and its derivative include sodium alginate, chitosan, dextran, hyaluronic acid, cellulose and agarose, and the polysaccharide compound and its derivative polymer are mixed with the olive green product III at a molar ratio of oxidized repeating unit: olive green product monomer of 20:1.

[0034] In some embodiments of the present application, the synthesis method of the polysaccharide compound and its derivative polymer comprises the following steps: reacting the polysaccharide compound and its derivative with sodium periodate, dialyzing after the reaction is completed, freeze-drying, and finally obtaining the polysaccharide compound and its derivative polymer.

[0035] In some embodiments of the present application, in step (5), the mass ratio of the orange yellow product II and the green product V is 1-3:1.

[0036] In another aspect of the present application, the present application provides an injectable hydrogel with self-repairing, degradable, environmental responsiveness and biocompatibility.

[0037] The beneficial effects of the present application compared with the prior art are:

[0038] (1) In the present application, the hydrophobic drug camptothecin is connected with the hydrophilic polymer polyethyleneimine to improve the water solubility of the hydrophobic drug, solubilize the drug, and on the other hand, the hydrophobic photothermal agent IR780 is connected with oxidized sodium alginate or similar oxidized polysaccharide compounds such as chitosan, dextran cellulose, etc. to solubilize the photothermal agent into water. The two parts are mixed to form an injectable hydrogel. The advantage of the injectable hydrogel is that it can be injected into the tumor site through a syringe, reducing the long-distance delivery of the drug.

[0039] (2) In the present application, the Schiff base bond formed by the reaction of oxidized sodium alginate and polyethyleneimine is a dynamic bond, which gives the hydrogel injectability and self-repairing performance. At the same time, the bond is also an acid-responsive bond. In the acidic microenvironment of tumor cells, the Schiff base bonds between oxidized sodium alginate and polyethyleneimine, IR780 and oxidized sodium alginate, and camptothecin and polyethyleneimine have pH responsiveness, and break to release camptothecin prodrug and glutathione to break the disulfide bond to release camptothecin.

[0040] (3) Under the irradiation of 808 nm near-infrared laser, the photothermal agent IR780 generates photothermal effect, which simultaneously performs chemotherapy and photothermal therapy on the tumor, thereby inhibiting the drug resistance of the tumor and killing the tumor, avoiding the emergence of single chemotherapy drug resistance, which also shows the environmental responsiveness and degradability of the hydrogel.

[0041] (4) The reactions in the present application are safe and reliable, easy to operate, and the conversion rate of each step is relatively high.

[0042] (5) In the present application, the polysaccharide compounds such as sodium alginate, chitosan, dextran, hyaluronic acid, cellulose and agarose selected in the hydrogel have wide sources and good biocompatibility, viscosity and stability. This gives the hydrogel good biocompatibility. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the bromine-functionalized disulfide monomer in Example 1 of the present application;

[0044] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of the azide-functionalized camptothecin monomer, aldehyde-functionalized camptothecin monomer and camptothecin-modified polyethyleneimine polymer in Example 1 of the present application;

[0045] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum of IR780 and amino-functionalized IR780 in Example 1 of the present application;

[0046] Figure 4 The nuclear magnetic resonance hydrogen spectrum of sodium alginate, sodium alginate oxide and IR780 modified sodium alginate oxide polymer in Example 1 of the present application;

[0047] Figure 5 The infrared spectrum of disulfide, bromine-containing disulfide and azide functionalized disulfide in Example 1 of the present application, in which BHD is disulfide, HO-ss-iBuBr is bromine-containing disulfide, and HO-ss-N3 is azide functionalized disulfide;

[0048] Figure 6 The infrared spectrum of camptothecin, azide functionalized camptothecin monomer, aldehyde functionalized camptothecin monomer, polyethyleneimine and camptothecin modified polyethyleneimine polymer in Example 1 of the present application, in which CPT is camptothecin, CPT-N3 is azide functionalized camptothecin monomer, CPT-CHO is aldehyde functionalized camptothecin monomer, PEI is polyethyleneimine, and CPT-PEI is camptothecin prodrug modified polyethyleneimine polymer;

[0049] Figure 7 The infrared spectrum of sodium alginate, sodium alginate oxide polymer, amino functionalized IR780 monomer and IR780 modified sodium alginate oxide polymer in Example 1 of the present application, in which SA is sodium alginate, OSA is sodium alginate oxide polymer, NH2-780 is amino functionalized IR780 monomer, and OSA-780 is IR780 modified sodium alginate oxide polymer;

[0050] Figure 8 The ring-opening rate test of sodium alginate oxide in Example 1 of the present application, the simulated curve graph of hydroxylamine hydrochloride titration, the left graph is the curve graph of pH change with NaOH dosage in OSA solution, and the right graph is the differential graph of the plotted curve;

[0051] Figure 9 The simulated curve graph of hydroxylamine hydrochloride titration of aldehyde group of IR780 modified sodium alginate oxide in Example 1 of the present application, the left graph is the curve graph of pH change with NaOH dosage in OSA-780 solution, and the right graph is the differential graph of the plotted curve;

[0052] Figure 10 The ultraviolet spectrum of aldehyde functionalized camptothecin and camptothecin modified polyethyleneimine polymer in Example 1 of the present application, in which CPT-CHO is aldehyde functionalized camptothecin, and CPT-PEI is camptothecin modified polyethyleneimine polymer;

[0053] Figure 11The figure is a fluorescence spectrum diagram of the aldehyde group functionalized camptothecin and the camptothecin modified polyethyleneimine polymer in the embodiment 1 of the present application, in which CPT-CHO is the aldehyde group functionalized camptothecin, and CPT-PEI is the camptothecin modified polyethyleneimine polymer;

[0054] Figure 12 The figure is a UV spectrum diagram of the amino functionalized IR780 and the IR780 modified oxidized sodium alginate polymer in the embodiment 1 of the present application, in which NH2-780 is the amino functionalized IR780 monomer, and OSA-780 is the IR780 modified oxidized sodium alginate polymer;

[0055] Figure 13 The figure is a scanning electron microscope diagram of the injectable hydrogel in the embodiment 1 of the present application;

[0056] Figure 14 The figure is a photothermal curve diagram of the injectable hydrogel in the embodiment 1 of the present application under 808 nm laser irradiation, in which gel is the injectable hydrogel;

[0057] Figure 15 The figure is a modulus diagram of the injectable hydrogel in the embodiment 1 of the present application, in which figure a is a test diagram about frequency, figure b is a test diagram about time, and figure c is a test diagram about strain;

[0058] Figure 16 The figure is a diagram about self-healing of the injectable hydrogel in the embodiment 1 of the present application, in which figure a is a self-healing photo of the injectable hydrogel taken macroscopically, and figure b is a self-healing picture tested by a rheometer;

[0059] Figure 17 The figure is a diagram about injectability of the injectable hydrogel in the embodiment 1 of the present application, in which figure a is a photo of the hydrogel extruded through a syringe, and figure b is a viscosity test diagram of the injectable hydrogel;

[0060] Figure 18 The figure is a degradation picture of the injectable hydrogel in the embodiment 1 of the present application under pH 5.5 and pH 7.4 conditions in a macroscopic gel;

[0061] Figure 19 The figure is a drug release curve of the injectable hydrogel in the embodiment 1 of the present application under acidic conditions, in which figure a is a release curve of camptothecin, and figure b is a release curve of IR780;

[0062] Figure 20 The figure is a drug release curve of the injectable hydrogel in the embodiment 1 of the present application under acidic conditions and DTT (10 mmol / L) conditions, in which figure a is a release curve of camptothecin, and figure b is a release curve of IR780, and the release effect is better after adding DTT;

[0063] Figure 21The three pictures are respectively the results of the cells cultured in PBS solution, OSA-PEI extract solution and OSA-780+PEI-CPT extract solution. DETAILED DESCRIPTION

[0064] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0065] Embodiment 1

[0066] A preparation method of an injectable hydrogel with self-repairing, degradable, environmental responsiveness and biocompatibility, comprising the following steps:

[0067] (1) Synthesis of aldehyde-functionalized camptothecin prodrug

[0068] a. Synthesis of disulfide monomer with bromine at one end:

[0069] Dihydroxyethyl disulfide (2.2 g, 14.26 mmol) and anhydrous triethylamine (1.2 g, 11.86 mmol) were dissolved in anhydrous THF, and bromoisobutyryl bromide (2.19 g, 9.53 mmol) was dissolved in anhydrous THF, and slowly added in an ice bath, and the reaction solution was stirred at room temperature for 24 h. The completion of the reaction was determined by TLC plate. After a new point was generated, the reaction solution was filtered, concentrated, redissolved in dichloromethane, washed with deionized water twice, saturated sodium chloride solution twice, and the organic phase was collected, dried over anhydrous sodium sulfate for 3 h, filtered and removed anhydrous sodium sulfate, rotary evaporated to remove dichloromethane, and purified by silica gel column (eluent: petroleum ether and ethyl acetate, volume ratio of petroleum ether to ethyl acetate is 4:1), collected the product, vacuum dried, to obtain a compound, and its structural formula is:

[0070]

[0071] b. Synthesis of azide-functionalized disulfide monomer:

[0072] The bromine-functionalized disulfide monomer (1 g, 3.3 mmol) and sodium azide (0.69 g, 10.53 mmol) were dissolved in anhydrous DMF, replaced with nitrogen, stirred at 40 °C for 48 h, after the reaction was completed, directly filtered, rotary evaporated to remove the reaction solvent, redissolved with dichloromethane, washed with deionized water twice, washed with saturated sodium chloride solution twice, collected the organic phase, dried with anhydrous sodium sulfate for 3 h, removed the anhydrous sodium sulfate with a suction filter funnel, rotary evaporated to remove the organic solvent, purified with a silica gel column (eluent: petroleum ether and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is 3:1), collected the product, vacuum dried, to obtain compound 2, the structural formula of which is as follows:

[0073]

[0074] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the bromine-functionalized disulfide and the azide-functionalized disulfide monomer illustrates the successful reaction of the disulfide and the synthesis of the product. Figure 5 The infrared spectrum of the disulfide, the bromine-functionalized disulfide and the azide-functionalized disulfide, through the formation of the carbonyl peak and the azide peak, assists in proving the successful modification of the disulfide.

[0075] c. Synthesis of azide-functionalized camptothecin monomer:

[0076] After weighing camptothecin (0.72 g, 2.07 mmol) and DMAP (0.75 g, 6.1 mmol) and placing them in a two-neck flask, dry dichloromethane was added, and then triphosgene dissolved in dichloromethane was added dropwise into the two-neck flask, and activated for 2 h. Then the azide-functionalized disulfide dissolved in dry dichloromethane was added, and the reaction was carried out at room temperature overnight. After the reaction was completed, suction filtration was carried out, and the product was washed with saturated sodium chloride solution three times, deionized water once, the organic phase was collected, dried with anhydrous sodium sulfate for 3 h, removed the anhydrous sodium sulfate with a suction filter funnel, rotary evaporated to remove the dichloromethane, purified with a silica gel column (eluent: dichloromethane and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is 4:1), collected the product, vacuum dried, to obtain product 4, the structural formula of which is as follows:

[0077]

[0078] d. Synthesis of aldehyde-functionalized camptothecin monomer:

[0079] The azido-functionalized camptothecin (0.2 g, 0.313 mmol), alkyne benzaldehyde (0.06 g, 0.375 mmol), PMDETA (0.027 g, 0.156 mmol) were dissolved in anhydrous chloroform. The solution was chilled in liquid nitrogen, the air was pumped out and replaced with nitrogen three times, then CuBr was added. The reaction was carried out at 40 °C overnight. After the formation of a new spot on the TLC plate, the copper salt was removed by passing the solution through a neutral alumina column. The solvent was collected and evaporated, then dissolved in dichloromethane and purified by silica gel column (eluent: dichloromethane and ethyl acetate, 4:1 by volume). The product was collected and dried under vacuum. The structure of the product is:

[0080]

[0081] The synthetic route of the aldehyde-functionalized camptothecin prodrug is as follows:

[0082]

[0083] (2) Synthesis of camptothecin-modified polyethyleneimine polymer

[0084] The polyethyleneimine (0.25 g) was dissolved in deionized water, and the aldehyde-functionalized camptothecin prodrug (0.1 g) was dissolved in methanol, then the two were mixed in a 10 mL polymerization bottle. The reaction was stirred at room temperature under nitrogen for 18 h. After the reaction was completed, it was precipitated with ether, centrifuged, and the supernatant was removed and repeated three times, and finally dried to obtain the product. The structure of the product is:

[0085]

[0086] The synthetic route of the camptothecin-modified polyethyleneimine polymer is as follows:

[0087]

[0088] Figure 2 The proton nuclear magnetic resonance spectrum of the camptothecin prodrug and the camptothecin prodrug-modified polyethyleneimine polymer shows that the camptothecin prodrug and the polymer reacted successfully. Figure 6 The infrared spectrum of camptothecin, camptothecin prodrug, and camptothecin prodrug-modified polyethyleneimine shows that the synthesis of the camptothecin prodrug is assisted by the formation of the aldehyde group peak, and the characteristic absorption peak of camptothecin in the polymer shows that the camptothecin prodrug successfully modified the polyethyleneimine. Figure 10 The ultraviolet spectrum of the aldehyde-functionalized camptothecin and the camptothecin-modified polyethyleneimine shows that the peak shape and chemical shift of the polymer change compared to the monomer, which assists in showing that camptothecin is successfully modified. Figure 11The fluorescence spectrum of aldehyde-functionalized camptothecin and camptothecin-modified polyethyleneimine, assisted by the red shift of the polymer peak relative to the monomer, helps to illustrate the successful modification of camptothecin.

[0089] (3) Synthesis of amino-functionalized IR780 monomer

[0090] P-aminobenzenethiol (0.113 g, 0.903 mmol) and IR780 (0.2 g, 0.3 mmol) were dissolved in DMF (5 times). The reaction was stirred at room temperature overnight in the dark. After determining the completion of the reaction by TLC plate, the solvent was removed by oil pump rotary evaporation, and then precipitated with ether, centrifuged, removed the supernatant, further removed DMF, dissolved in dichloromethane, purified by silica gel column separation (eluent was anhydrous methanol and dichloromethane, with a volume ratio of 1:10 of anhydrous methanol and ethyl acetate), then rotary evaporation, precipitated in cold ether, vacuum dried at room temperature, to obtain amino-functionalized IR780, whose structural formula is as follows:

[0091]

[0092] The synthesis route of amino-functionalized IR780 monomer is as follows:

[0093]

[0094] Figure 3 The nuclear magnetic resonance hydrogen spectrum of IR780 and amino-functionalized IR780 proves the successful amino-functionalization of IR780.

[0095] (4) Synthesis of oxidized sodium alginate polymer

[0096] Sodium alginate (5 g) was dissolved in deionized water (166 mL), and sodium periodate (2.969 g, 13.88 mmol) was dissolved in deionized water and slowly added to the sodium alginate aqueous solution with a constant pressure dropping funnel, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction was completed, 5 mL of ethylene glycol was injected into the reaction solution with a syringe, and the reaction was stopped after stirring for half an hour. Then it was dialyzed with a 3500 kDa dialysis bag, and the water was changed every 4 h. After two days, it was taken out and dried with a freeze dryer for two days to obtain oxidized sodium alginate, whose structural formula is as follows:

[0097]

[0098] The synthesis route of oxidized sodium alginate polymer is as follows:

[0099]

[0100] Figure 8For the open ring rate test of oxidized sodium alginate, the generation of aldehyde group was proved, and the simulated curve of hydroxylamine titration was used. The open ring rate of sodium alginate was measured by differentiating the simulated curve and corresponding calculation formula, and the value was 63.11%; the calculation formula is:

[0101] Alginate-(CHO) n +nH2N-OH-HCl=Alginate-(CH=N-OH) n +nH2O+nHCl (1)

[0102] HCl+NaOH=NaCl+H2O (2)

[0103] ΔV*0.001*n NaOH =n CHO (3)

[0104] [CHO]=n CHO / WSA (4)

[0105] OD=(n CHO / 2) / (WSA / 198.11) (5)

[0106] Wherein, Alginate is alginate, ΔV is the volume of NaOH consumed during titration, unit mL; nNaOH is the molar concentration of NaOH, unit mol / L; [CHO] is the aldehyde group concentration, unit mol / g; nCHO is the molar number of aldehyde group on sodium alginate; WSA is the mass of sodium alginate, unit g. OD is the degree of oxidation, and 198.11 is the molar mass of sodium alginate monomer unit.

[0107] (5) Synthesis of photothermal agent IR780 modified oxidized sodium alginate polymer

[0108] Oxidized sodium alginate (0.26 g) was dissolved in deionized water, and amino-functionalized IR780 (0.026 g, 0.035 mmol) was dissolved in methanol solution. Then the two were mixed in a 10 mL reaction bottle, and reacted at 35℃ in the dark for 18 h. After the reaction was completed, the solution was precipitated with ethanol, centrifuged, and the precipitate was washed with ethanol repeatedly for three times, and then freeze-dried to obtain IR780 modified oxidized sodium alginate polymer, and its structural formula is:

[0109]

[0110] The synthesis route of photothermal agent IR780 modified oxidized sodium alginate polymer is as follows:

[0111]

[0112] Figure 4 The nuclear magnetic resonance hydrogen spectrum of sodium alginate, oxidized sodium alginate and IR780 modified oxidized sodium alginate polymer proves the success of sodium alginate oxidation and the success of IR780 modification of oxidized sodium alginate. Figure 7 The infrared spectrum of sodium alginate, oxidized sodium alginate and IR780 modified oxidized sodium alginate polymer, through the formation of aldehyde group peak, auxiliary explains the success of sodium alginate ring opening, the appearance of the characteristic absorption peak of IR780 in the polymer, explains the success of IR780 modification of oxidized sodium alginate.

[0113] Figure 9 The simulated curve of IR780 modified oxidized sodium alginate titrating aldehyde group with hydroxylamine hydrochloride, according to the curve, it can be proved that the polymer still has residual aldehyde group, which can further react with amino group, and according to the previous formula, the amount of residual aldehyde group can be calculated as 61.08%;

[0114] Figure 12 The ultraviolet spectrum of amino functionalized IR780 and IR780 modified oxidized sodium alginate, through the red shift of polymer peak relative to monomer, auxiliary explains the success of IR780 modification.

[0115] (6) Synthesis of acid-sensitive injectable hydrogel

[0116] The camptothecin prodrug modified polyethyleneimine polymer (0.15g) is dissolved in a certain amount of deionized water, and the photothermal agent modified oxidized sodium alginate polymer (0.15g) is dissolved in a certain amount of deionized water, and the two are mixed together. The remaining amino group of polyethyleneimine and the remaining aldehyde group of oxidized sodium alginate will form Schiff base bond in a short time, so as to rapidly form an injectable hydrogel.

[0117] The synthesis route of the acid-sensitive injectable hydrogel is as follows:

[0118]

[0119] Figure 13 The scanning electron microscope image of the injectable hydrogel, through the electron microscope image, it can be clearly seen that the hydrogel is a reticular structure; Figure 14 The photothermal curve of the injectable hydrogel under 808nm laser irradiation with time is shown in the figure. With the increase of irradiation time, the temperature of the whole system also increases, and at 175s, the temperature reaches a balance of 53℃.

[0120] Figure 15 The modulus diagram of the injectable hydrogel, figure a is the test diagram about frequency, figure b is the test diagram about time, figure c is the test diagram about strain, with the continuous increase of frequency or time, the test curve does not fluctuate up and down, which shows that the hydrogel has good stability;Figure 16 For injectable hydrogel about self-healing, Figure a is a self-healing photo of injectable hydrogel taken macroscopically, Figure b is a self-healing picture of rheometer test, by fixing the frequency, the strain is repeatedly switched from 100% to 1%, the two pictures show that the hydrogel has good self-healing ability. Figure 17 For injectable hydrogel about injectability, Figure a is a photo of hydrogel extruded by a syringe, Figure b is a viscosity test picture of injectable hydrogel, by increasing the shear force, the viscosity decreases continuously, the two pictures show that the hydrogel has injectability.

[0121] Figure 18 For injectable hydrogel degradation picture in acidic conditions, the fastest degradation can be completed in 12 days. Figure 19 For injectable hydrogel drug release curve in acidic conditions, Figure a is the release curve of camptothecin, Figure b is the release curve of IR780, the two release curves show that the release of camptothecin and IR780 can achieve good results; Figure 20 For injectable hydrogel drug release curve in acidic conditions and DTT (10 mmol / L) conditions, Figure a is the release curve of camptothecin, Figure b is the release curve of IR780, the release effect is better after adding DTT.

[0122] Figure 21 For injectable hydrogel biocompatibility test picture, from the picture it can be seen that the cell activity cultured in the presence of gel and PBS solution has little change, which shows that the gel has good biocompatibility.

[0123] Example 2

[0124] The difference between this embodiment and Example 1 is that the polysaccharide compound and its derivative used is chitosan.

[0125] A preparation method of an injectable hydrogel with self-repairing, degradable, environment-responsive and biocompatibility, the first three steps are the same as Example 1, and further comprising the following steps:

[0126] (4) Synthesis of oxidized chitosan polymer

[0127] Dissolve chitosan in certain deionized water, take sodium periodate dissolved in deionized water, slowly add it to the sodium alginate aqueous solution with a constant pressure dropping funnel, react at room temperature in dark environment for 4 h, after the reaction is completed, inject ethylene glycol into the reaction solution with a syringe, continue to stir for half an hour to stop the reaction, then dialyze with a 3500 kDa dialysis bag, change the water every 4 h, take out after two days, and dry with a freeze dryer for two days to obtain oxidized chitosan polymer, and its structural formula is:

[0128]

[0129] The synthesis route of the oxidized chitosan polymer is as follows:

[0130]

[0131] (5) Synthesis of the photo-thermal agent IR780 modified oxidized chitosan polymer

[0132] The oxidized sodium alginate is dissolved in a certain amount of deionized water, and the amino-functionalized IR780 is dissolved in a methanol solution. Then the two are mixed in a two-necked flask and reacted at 35°C for 18h in the dark. After the reaction is completed, the solution is precipitated with ethanol, centrifuged, and the precipitate is washed repeatedly with ethanol for three times, and then freeze-dried to obtain the photo-thermal agent IR780 modified oxidized chitosan polymer, whose structural formula is as follows:

[0133]

[0134] The synthesis route of the photo-thermal agent IR780 modified oxidized chitosan polymer is as follows:

[0135]

[0136] (6) Synthesis of the acid-sensitive injectable hydrogel

[0137] The camptothecin prodrug modified polyethyleneimine polymer (0.15g) is dissolved in 1mL deionized water, and the photo-thermal agent IR780 modified oxidized chitosan polymer (0.15g) is dissolved in 1mL deionized water. The two are mixed together, and the remaining amino groups of the polyethyleneimine and the remaining aldehyde groups of the oxidized sodium alginate will form Schiff base bonds in a short time, thereby rapidly forming an injectable hydrogel.

[0138] The synthesis route of the acid-sensitive injectable hydrogel is as follows:

[0139]

[0140] The above is only an example and description of the structure of the present application. Those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace, as long as they do not deviate from the structure of the present application or exceed the scope defined by the present claims, which shall belong to the protection scope of the present application.

Claims

1. A method for preparing an injectable hydrogel with self-healing, biodegradable, environmentally responsive, and biocompatible properties, characterized in that, Includes the following steps: (1) Synthesis of aldehyde-functionalized camptothecin prodrug: a. Synthesis of disulfide monomers with a bromine end: Bromoisobutyryl bromide and dihydroxyethyl disulfide were subjected to a monosubstitution reaction, separated, washed and dried to obtain a disulfide monomer molecule with bromine at one end. b. Synthesis of azide-functionalized disulfide monomers: Sodium azide and a disulfide monomer molecule with bromine at one end were subjected to a substitution reaction. After separation, washing and drying, the azide-functionalized disulfide monomer was obtained. c. Synthesis of azide-functionalized camptothecin monomers: A zisto-functionalized disulfide monomer and camptothecin were subjected to a substitution reaction, and the mixture was separated, washed and dried to obtain zisto-functionalized camptothecin. d. Synthesis of aldehyde-functionalized camptothecin prodrug: Azid-functionalized camptothecin and alkynylbenzaldehyde were subjected to a click reaction, separated, washed and dried to obtain aldehyde-functionalized camptothecin prodrug. (2) Synthesis of camptothecin-modified polyethyleneimine polymer: Polyethyleneimine and aldehyde-functionalized camptothecin prodrug undergo Schiff base reaction in a mixed solvent of methanol and N,N'-dimethylformamide. The reaction is allowed to proceed overnight at room temperature. After the reaction is completed, precipitation is carried out and finally dried to obtain orange-yellow product II. (3) Synthesis of amino-functionalized IR780 monomer: IR780 and p-aminothiophenol undergo a substitution reaction in N,N'-dimethylformamide solvent. The reaction is carried out overnight at room temperature in the dark. After the reaction is completed, the reaction solvent is evaporated and separated by silica gel column chromatography to obtain dark green product III. (4) Synthesis of sodium alginate polymer modified by photothermal agent IR780: Sodium alginate polymer and dark green product III were dissolved in a mixed solvent of methanol and water for Schiff base reaction. The reaction was carried out overnight at room temperature in the dark. The product was then precipitated in ethanol and dried to obtain green product V. The method for synthesizing the oxidized sodium alginate polymer includes the following steps: reacting sodium alginate with sodium periodate, dialysis after the reaction is completed, freeze-drying, and finally obtaining the oxidized sodium alginate polymer. (5) Synthesis of acid-sensitive injectable hydrogel: Dissolve orange-yellow product II and green product V in water respectively, and then mix the two solutions to form the self-healing, degradable, environmentally responsive and biocompatible injectable hydrogel.

2. The method for preparing an injectable hydrogel with self-healing, degradable, environmentally responsive, and biocompatible properties according to claim 1, characterized in that: In step a, the molar ratio of bromoisobutyryl bromide to dihydroxyethyl disulfide is 3:2; in step b, sodium azide and a disulfide monomer with a bromine end undergo a substitution reaction in a molar ratio of 3:1; in step c, the azide-functionalized disulfide monomer and camptothecin undergo a substitution reaction in a molar ratio of 1.5:1; in step d, the azide-functionalized camptothecin and alkynylbenzaldehyde undergo a click reaction in a molar ratio of 1:1.

2.

3. The method for preparing an injectable hydrogel with self-healing, degradable, environmentally responsive, and biocompatible properties according to claim 1, characterized in that: In step (2), after the reaction is complete, the product is precipitated in cold diethyl ether.

4. The method for preparing an injectable hydrogel with self-healing, degradable, environmentally responsive, and biocompatible properties according to claim 1, characterized in that: In step (3), the molar ratio of IR780 to p-aminothiophenol is 1:

3.

5. The method for preparing an injectable hydrogel with self-healing, degradable, environmentally responsive, and biocompatible properties according to claim 1, characterized in that: In step (4), the oxidized sodium alginate polymer and the dark green product III are mixed in a molar ratio of 20:1, with the oxidized repeating unit being the dark green product III.

6. The method for preparing an injectable hydrogel with self-healing, degradable, environmentally responsive, and biocompatible properties according to claim 1, characterized in that: In step (5), the mass ratio of orange-yellow product II to green product V is 1:

1.

7. An injectable hydrogel with self-healing, degradable, environmentally responsive and biocompatible properties prepared by a method according to any one of claims 1-6.

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