Injectable organic composite hydrogel combined with photothermal chemotherapy and its preparation method and application

Abstract

The present invention belongs to the field of nano-biomedicine technology, and specifically relates to an injectable organic composite hydrogel combined with photothermal chemotherapy, a preparation method, and an application. The injectable organic composite hydrogel combined with photothermal chemotherapy of the present invention is composed of an orthoester complex and a hydrogel of an aqueous phase. The orthoester complex is uniformly dispersed in the hydrogel after emulsification, and the hydrogel is cross-linked by carboxymethyl chitosan and glutaraldehyde. In the injectable organic composite hydrogel combined with photothermal chemotherapy provided by the present invention, cisplatin prodrug and indocyanine green are first dissolved by oily orthoester to obtain an orthoester complex, and then the orthoester complex is emulsified uniformly and loaded into the hydrogel. The dynamic imine bond of the hydrogel breaks in response to the micro-acidic environment of the tumor, and the orthoester complex successfully escapes. Along with the degradation of the oily orthoester, the cisplatin prodrug and indocyanine green are self-assembled through hydrogen bonds to form nanoparticles, thereby enhancing the enrichment and retention capacity of the two drugs at the tumor site and improving the overall efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomedicine technology, and specifically relates to an injectable organic composite hydrogel combined with photothermal chemotherapy, a preparation method and an application thereof. Background Art

[0002] Although local drug delivery therapies, such as suppositories and emulsions, have achieved certain results, overcoming these obstacles and improving clinical efficacy remain significant challenges. Nanocomposite hydrogels are currently receiving widespread attention due to their dual advantages of both hydrogels and nanomedicines, such as high tissue adhesion, high permeability, and retention of nanoparticles. However, nanoparticles have poor solubility in hydrogels, are prone to precipitation, and are difficult to escape efficiently. After escaping, they are easily cleared by the body and cannot reach tumor tissues to exert their effects, resulting in limited overall therapeutic effects. Therefore, it is particularly important to enhance the efficient escape of drugs from hydrogels and enhance their tumor accumulation.

[0003] Related reports indicate that aqueous hydrogels can encapsulate oily orthoester complexes to form organic composite hydrogels. Because oil and water are immiscible in organic composite hydrogels, the oil phase easily escapes from the aqueous hydrogel, facilitating drug release. The cross-linked components of the aqueous hydrogel exhibit good tissue adhesion, while also reducing interactions with tissues, facilitating the disintegration of the organic composite hydrogel. However, existing organic composite hydrogels suffer from limitations such as difficulty in drug release, poor drug accumulation and retention at the tumor site, and poor therapeutic efficacy. Summary of the Invention

[0004] In response to the deficiencies in the above-mentioned prior art, the present invention provides an injectable organic composite hydrogel combined with photothermal chemotherapy, as well as a preparation method and application. The injectable organic composite hydrogel combined with photothermal chemotherapy of the present invention is composed of an orthoester complex and an aqueous phase hydrogel. The orthoester complex loads and dissolves the chemotherapy drug cisplatin prodrug and the photothermal agent indocyanine green, and the oily orthoester is used to enhance the photothermal efficacy and stability of the photothermal agent. At the same time, due to the oil-water immiscible properties in the injectable organic composite hydrogel combined with photothermal chemotherapy, the effective release of the two drugs, cisplatin prodrug and indocyanine green, is promoted. The oily orthoester assists the two drugs in penetrating the tumor site, and assists the two drugs in self-assembly to form nanoparticles after the degradation of the oily orthoester, thereby comprehensively enhancing the tumor enrichment and retention of the two drugs. The chemotherapy drug cisplatin prodrug responds to the tumor microenvironment and is reduced to exert its effect, reducing systemic toxicity, and achieving a strong tumor inhibition effect of precise synergy between hyperthermia and chemotherapy, thereby overcoming the technical defects of the organic composite hydrogel in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a method for preparing an injectable organic composite hydrogel combined with photothermal chemotherapy, comprising the following steps:

[0007] Under an inert atmosphere, using anhydrous acetonitrile as the reaction solvent, 3-amino-1,2-propanediol and ethyl trifluoroacetate are mixed, and an amidation reaction is carried out in the liquid phase. After the reaction, the solvent is separated by rotary evaporation, and the intermediate product is obtained by extraction, drying, and rotary evaporation. Using anhydrous acetonitrile as the reaction solvent, the intermediate product is mixed with trimethyl orthoformate, and an acetalization reaction is carried out in the liquid phase under the action of a catalyst. After the reaction, the solvent is separated by rotary evaporation, and the oily orthoformate is obtained by extraction, drying, and rotary evaporation. The oily orthoformate can fully dissolve both cisplatin prodrug and indocyanine green, enhance the photothermal efficacy and stability of the photothermal agent, is insoluble in oil and water in the injectable organic composite hydrogel combined with photothermal chemotherapy, and helps accelerate the release of the two drugs. At the same time, the oily orthoformate is lipophilic and acid-sensitive, which can help increase the penetration of the two drugs in the tumor site and degrade in the slightly acidic environment of the tumor, helping the two drugs to self-assemble into nanoparticles through hydrogen bonds.

[0008] Divalent cisplatin is mixed in water, and then H2O2 solution is added dropwise, stirred in the dark and an oxidation reaction is carried out, the solvent is separated, and the product is freeze-dried to obtain a tetravalent cisplatin product; anhydrous N,N-dimethylformamide is used as a reaction solvent, triethylamine is used as a catalyst, the tetravalent cisplatin product, norcantharidin and triethylamine are mixed, the reaction is carried out in the dark under nitrogen protection, and an esterification reaction is carried out in a liquid phase. After the reaction is completed, the solvent is separated by rotary evaporation, and the precipitate is settled, washed, collected and dried to obtain a cisplatin prodrug; the cisplatin prodrug and the preparation method thereof are prior art.

[0009] Cisplatin prodrug and the photothermal agent indocyanine green were ultrasonically dispersed in an oily orthoester to form an orthoester complex. A 2% by mass carboxymethyl chitosan solution was then added and vortexed to uniformly emulsify the orthoester complex. A 5% by mass glutaraldehyde solution was added while vortexing to produce an injectable organic composite hydrogel for combined photothermal chemotherapy. The injectable organic composite hydrogel for combined photothermal chemotherapy, composed of the orthoester complex and an aqueous hydrogel phase, exhibits a compact surface structure and a suitable pore structure. The use of the small molecule crosslinker glutaraldehyde avoids the formation of unnecessary intermolecular forces with biological tissues, facilitating the release of the orthoester complex. The introduction of carboxymethyl chitosan imparts excellent tissue adhesion to the injectable organic composite hydrogel for combined photothermal chemotherapy. The amino groups of the carboxymethyl chitosan crosslink with the aldehyde groups of glutaraldehyde to form dynamic imine bonds, resulting in excellent injectability and self-healing properties for the injectable organic composite hydrogel for combined photothermal chemotherapy.

[0010] Preferably, the amidation reaction is carried out under stirring at room temperature for 10 to 15 hours.

[0011] Preferably, the molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:1-3.

[0012] Preferably, the acetalization reaction is carried out under stirring at room temperature for 10 to 15 hours.

[0013] Preferably, the molar ratio of the intermediate product to trimethyl orthoformate is 1:1-3.

[0014] Preferably, the oxidation reaction is carried out under stirring at 50° C. to 55° C. for 4 to 5 hours.

[0015] Preferably, the mass percentage of the H2O2 solution is 30 wt%, and the volume ratio of the cisplatin concentration to the H2O2 solution is 0.67 mmol:12 mL~14 mL.

[0016] Preferably, the esterification reaction is carried out at 60° C. to 65° C. with stirring for 20 h to 24 h.

[0017] Preferably, the molar ratio of the tetravalent cisplatin product to norcantharidin is 1:2-3.

[0018] Preferably, n in the carboxymethyl chitosan solution -NH2 With n in glutaraldehyde solution -CHO The ratio of carboxymethyl chitosan solution is 1:0.8~2.4. -NH2 With n in glutaraldehyde solution -CHO The ratio is 1:1.6.

[0019] Preferably, the volume ratio of the carboxymethyl chitosan solution to the oily orthoester is 10:1 to 5. Further, the volume ratio of the carboxymethyl chitosan solution to the oily orthoester is 10:3.

[0020] Preferably, the molar ratio of cisplatin prodrug to photothermal agent indocyanine green is 25 to 29: 1. Further, the molar ratio of cisplatin prodrug to photothermal agent indocyanine green is 27:1.

[0021] The present invention also protects the injectable organic composite hydrogel combined with photothermal chemotherapy prepared by the above preparation method.

[0022] Preferably, the injectable organic composite hydrogel combined with photothermal chemotherapy is composed of an orthoester complex and an aqueous phase hydrogel, the orthoester complex is emulsified and uniformly dispersed in the hydrogel, and the hydrogel is cross-linked by carboxymethyl chitosan and glutaraldehyde; wherein the drug loaded on the orthoester complex is composed of a chemotherapy drug cisplatin prodrug and a photothermal agent indocyanine green.

[0023] The present invention also protects the use of injectable organic composite hydrogel combined with photothermal chemotherapy in the preparation of drug carriers, photothermal therapy drugs or anticancer drugs.

[0024] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0025] 1. The present invention provides an injectable organic composite hydrogel for combined photothermal chemotherapy. The hydrogel comprises an aqueous phase and an oily orthoester in which the chemotherapy drug cisplatin prodrug and the photothermal agent indocyanine green are dissolved. The orthoester complex is uniformly dispersed in the hydrogel after emulsification. The hydrogel is formed by cross-linking amino groups on carboxymethyl chitosan with aldehyde groups on the small molecule glutaraldehyde, forming dynamic Schiff base imine bonds. The injectable organic composite hydrogel for combined photothermal chemotherapy provided by the present invention can dissolve the two drugs in the oily orthoester and then uniformly load them into the hydrogel through emulsification. The dynamic imine bonds in the hydrogel break in response to the slightly acidic environment of the tumor, allowing the orthoester complex to escape successfully. Due to the low specific heat capacity, lipophilicity, and pH responsiveness of the oily orthoester, the oily orthoester not only enhances the photothermal therapeutic effect of the photothermal agent indocyanine green but also enhances the penetration of both drugs into tumor tissue. As the oily orthoester degrades, the two drugs self-assemble into nanoparticles through hydrogen bonding, enhancing drug accumulation and retention at the tumor site and overall improving efficacy.

[0026] 2. The injectable organic composite hydrogel combined with photothermal chemotherapy of the present invention has good tissue adhesion, injectability, biodegradability, biosafety and photothermal performance. It can be smoothly passed through the syringe and used for long-term combined chemotherapy and photothermal therapy of tumors in situ in a minimally invasive manner, and significantly improves the therapeutic efficacy.

[0027] 3. The introduction of the oily orthoester of the present invention not only improves the photothermal conversion efficiency and photothermal stability of the photothermal agent, but also its fat solubility and acid sensitivity enhance the penetration of the two drugs into the tumor site, respond to the tumor's slightly acidic environment and degrade, while assisting the two drugs to self-assemble into nanoparticles through hydrogen bonds.

[0028] 4. The present invention utilizes combined photothermal therapy and chemotherapy to achieve synergistic anti-tumor effects, reduce systemic toxicity, and avoid multidrug resistance. It exhibits low toxicity, high retention, and high efficacy, overcoming the limitations of single treatments. In summary, the injectable organic composite hydrogel combined with photothermal chemotherapy addresses the challenges of existing organic composite hydrogels, including difficulty in drug release, poor drug accumulation and retention at the tumor site, strong toxic side effects, and poor efficacy.

[0029] In the present invention, Gel represents an injectable hydrogel, FOE represents an oily orthoester, Pt (IV) -1 represents a cisplatin prodrug, ICG represents indocyanine green, IP represents a mixture of cisplatin prodrug and indocyanine green, CMCS represents carboxymethyl chitosan, GA represents glutaraldehyde, Gel / FOE represents an injectable organic composite hydrogel, FOE / IP represents an orthoester complex, Gel / FOE / IP represents an injectable organic composite hydrogel combined with photothermal chemotherapy, Gel / IP represents a drug-loaded injectable hydrogel, Gel / FOE / Pt (IV) -1 represents an injectable organic composite hydrogel loaded with a cisplatin prodrug, Gel / FOE / ICG represents an injectable organic composite hydrogel loaded with indocyanine green, and NIR is near-infrared light of 808 nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 In the figure, (a) is the SEM image of Gel-1, Gel and Gel-2, and the inset is the corresponding macroscopic gelation image; (b) is the single frequency scanning image of Gel-1, Gel and Gel-2; (c) is the cross-linking degree image of Gel-1, Gel and Gel-2.

[0031] Figure 2 SEM images of Gel / FOE-1, Gel / FOE, and Gel / FOE-2, where the inset in the upper left corner is the corresponding macroscopic state image, and the inset in the lower left corner is the microscopic section image.

[0032] Figure 3 In the figure, (a) is the Fourier infrared spectra of GA, CMCS, Gel, FOE and Gel / FOE; (b) is the X-ray diffraction pattern of GA, CMCS, Gel, Gel / FOE, FOE / IP and Gel / FOE / IP of Example 1.

[0033] Figure 4 In the figure, (a) is a time scan diagram of Gel and Gel / FOE; (b) is a shear rate scan diagram of Gel / FOE, where the inset is a diagram of the permeability of Gel / FOE; (c) is a diagram of the storage modulus and loss modulus of Gel / FOE under amplitude scanning; (d) is a diagram of the storage modulus and loss modulus of Gel / FOE under alternating high and low strain scanning, where the inset is a diagram of the self-healing process.

[0034] Figure 5 Figure (a) shows the H2O / ICG, Gel / ICG, FOE / ICG, and Gel / FOE / ICG at a power density of 0.5 W / cm 2(a) The temperature change curve of H2O / ICG, Gel / ICG, FOE / ICG, and Gel / FOE / ICG during the heating process; (b) The infrared imaging of H2O / ICG, Gel / ICG, FOE / ICG, and Gel / FOE / ICG during the heating process; (c) The infrared imaging of H2O / ICG, Gel / ICG, FOE / ICG, and Gel / FOE / ICG during the heating process at a power density of 0.5 W / cm 2 (d) The temperature change curves in 4 cycles after continuous laser irradiation for 5 minutes; (d) The fluorescence intensity change curves of H2O / ICG and FOE / ICG.

[0035] Figure 6 In the figure, (a) shows the in vitro degradation of Gel / FOE at different pH values; (b) shows the in vitro degradation of Gel, Gel / FOE, and Gel / FOE / ICG+NIR in a 0.2 mol / L, pH 6.8 PBS environment.

[0036] Figure 7 This is a diagram of the escape process of FOE in Gel / FOE at different times.

[0037] Figure 8 In the figure, (a) is a verification diagram of FOE degradation after Gel / FOE is placed for 3h, 6h, and 12h; (b) is a verification diagram of FOE escaping from Gel in Gel / FOE.

[0038] Figure 9 In the figure, (a) is the particle size diagram of the nanoparticles, in which the inset is a TEM image; (b) is the particle size stability diagram of the nanoparticles at different pH values; (c) is the potential diagram of the nanoparticles at different pH values.

[0039] Figure 10 The graphs show the drug release of Pt in Gel / IP and Gel / FOE / IP of Example 1 at different pH values.

[0040] Figure 11 The toxicity graphs are as follows: Gel / IP containing different concentration gradients of cisplatin, Gel / FOE / IP of Example 1, Gel / IP+NIR, and Gel / FOE / IP+NIR were co-incubated with HepG2 cells for 48 hours.

[0041] Figure 12 In the figure, (a) is a qualitative uptake graph of HepG2 cells co-incubated with Gel / IP and Gel / FOE / IP of Example 1 for 4 hours, and (b) is a quantitative uptake graph of HepG2 cells co-incubated with Gel / IP and Gel / FOE / IP of Example 1 for 4 hours.

[0042] Figure 13In the figure, (a) is the tissue adhesion diagram of Gel / FOE to pig skin, pork, and pig liver; (b) is the adhesion diagram of Gel / FOE to glass, plastic, rubber, and metal; (c) is the adhesion diagram of Gel / FOE / ICG in mice.

[0043] Figure 14 Schematic diagram of hemolysis of Gel / FOE / Pt(IV)-1.

[0044] Figure 15 In the figure, (a) is a graph showing the changes in tumor volume of mice in FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP in Example 1, Gel / FOE / IP+NIR, and blank control group; (b) is a graph showing the tumor weight of mice in FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP in Example 1, Gel / FOE / IP+NIR, and blank control group; (c) is a graph showing the tumors of mice in FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP in Example 1, Gel / FOE / IP+NIR, and blank control group; (d) is a graph showing the changes in weight of mice in FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP in Example 1, Gel / FOE / IP+NIR, and blank control group. DETAILED DESCRIPTION

[0045] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] The 2% CMCS solution was mixed with the 5% GA solution. -NH2 n with GA -CHO The ratio was 1:0.8, and an injectable hydrogel was obtained, which was recorded as Gel-1.

[0047] The 2% CMCS solution was mixed with the 5% GA solution. -NH2 n with GA -CHO The ratio was 1:1.6, and an injectable hydrogel was obtained, which was recorded as Gel.

[0048] The 2% CMCS solution was mixed with the 5% GA solution. -NH2 n with GA-CHO The ratio was 1:2.4, and an injectable hydrogel was obtained, which was recorded as Gel-2.

[0049] The present invention first uses Gel-1, Gel and Gel-2 to screen injectable hydrogels. The results show that Gel has the best performance. Then, Gel is used to prepare Gel / FOE-1, Gel / FOE and Gel / FOE-2 respectively to screen injectable organic composite hydrogels. Gel / FOE-1, Gel / FOE and Gel / FOE-2 are prepared according to the following steps:

[0050] Add 2% CMCS solution to FOE, vortex to emulsify FOE, and add 5% GA solution while vortexing. -NH2 and n in GA -CHO The ratio is 1:1.6, V CMCS :V FOE The ratio of FOE to Gel was 10:1, and an injectable organic composite hydrogel was obtained, which was recorded as Gel / FOE-1.

[0051] Add 2% CMCS solution to FOE, vortex to emulsify FOE, and add 5% GA solution while vortexing. -NH2 and n in GA -CHO The ratio is 1:1.6, V CMCS :V FOE The ratio of the organic composite hydrogel to the FOE was 10:3, and an injectable organic composite hydrogel was obtained, which was recorded as Gel / FOE.

[0052] Add 2% CMCS solution to FOE, vortex to emulsify FOE, and add 5% GA solution while vortexing. -NH2 and n in GA -CHO The ratio is 1:1.6, V CMCS :V FOE The ratio of FOE to Gel was 2:1, and an injectable organic composite hydrogel was obtained, which was recorded as Gel / FOE-2.

[0053] The following examples are used to further study the technical solution of the present invention. The specific research methods and results are as follows:

[0054] Example 1

[0055] A method for preparing an injectable organic composite hydrogel combined with photothermal chemotherapy comprises the following steps:

[0056] S1. Under nitrogen environment, 3-amino-1,2-propanediol and ethyl trifluoroacetate were added to a dry 500mL three-necked round-bottom reaction flask containing a magnetic rotor. The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate was 1:2. Then, 150mL of anhydrous acetonitrile was added as the reaction solvent, and the reaction was stirred for 12 hours under nitrogen. After the reaction, the anhydrous acetonitrile solvent was removed by rotary evaporation, and the mixture was dissolved with 150mL of ethyl acetate solution containing 100μL of triethylamine, and then extracted once with 0.5mol / L potassium bisulfate solution and saturated sodium chloride solution alternately. The mixture was dried over anhydrous magnesium sulfate for 6 hours, and the ethyl acetate was removed by rotary evaporation to collect the intermediate product.

[0057] The intermediate product and trimethyl orthoformate were placed together in a 500 mL dry reaction bottle with a molar ratio of the intermediate product to trimethyl orthoformate of 1:2. 150 mL of anhydrous acetonitrile was added as a reaction solvent. A catalyst, p-toluenesulfonic acid monohydrate, was added. After stirring and reacting for 12 hours under nitrogen, triethylamine was added dropwise to terminate the reaction. The anhydrous acetonitrile was removed by rotary evaporation, and the mixture was dissolved with ethyl acetate. The mixture was then extracted once with saturated sodium bicarbonate and once with saturated sodium chloride. The organic phase was collected and the organic solvent, ethyl acetate, was removed by rotary evaporation to obtain FOE.

[0058] S2. Add 200 mg, 0.67 mmol of cisplatin to a 100 mL round-bottom flask, then add 12 mL of deionized water to dissolve the cisplatin, add 13 mL of 30 wt% H2O2 solution dropwise through a separatory funnel, and stir the reaction at 55 °C in the dark for 5 h. After the reaction is completed, remove most of the water and H2O2 solution by rotary evaporation, and then lyophilize to obtain a yellow tetravalent cisplatin product.

[0059] 100 mg, 0.3 mmol of tetravalent cisplatin product and 151 mg, 0.9 mmol of norcantharidin were mixed, then dissolved in 5 mL of anhydrous N,N-dimethylformamide and added to a round-bottom reaction flask. 121.16 mg, 1.2 mmol of catalyst triethylamine were added, and the mixture was reacted at 65°C in the dark under nitrogen protection for 24 h. After the reaction, N,N-dimethylformamide was removed by vacuum distillation, and the mixture was precipitated with anhydrous ether under an ice bath. The precipitate was washed with acetone and finally dried to obtain a light yellow Pt(IV)-1 powder.

[0060] S3, 7.3 mg of Pt (IV) -1 and 0.33 mg of ICG were placed in 300 μL of FOE, and the solid was completely dissolved by ultrasound for 30 min to obtain an orthoester complex, which was recorded as FOE / IP; 2% by mass of CMCS solution was added to FOE / IP, and vortexed to emulsify the orthoester complex uniformly, and 5% by mass of GA solution was added while vortexing. -NH2 and n in GA -CHO The ratio is 1:1.6, V CMCS:V FOE The ratio of the organic composite hydrogel to photothermal chemotherapy was 10:3, and an injectable organic composite hydrogel was obtained, which was recorded as Gel / FOE / IP.

[0061] Example 2

[0062] A method for preparing an injectable organic composite hydrogel combined with photothermal chemotherapy comprises the following steps:

[0063] S1. Under nitrogen environment, 3-amino-1,2-propanediol and ethyl trifluoroacetate were added to a dry 500mL three-necked round-bottom reaction flask containing a magnetic rotor. The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate was 1:1, and then 150mL of anhydrous acetonitrile was added as the reaction solvent. The reaction was stirred and reacted for 10 hours under nitrogen. After the reaction, the anhydrous acetonitrile solvent was removed by rotary evaporation, and the mixture was dissolved with 150mL of ethyl acetate solution containing 80μL of triethylamine, and then extracted once with 0.5mol / L potassium bisulfate solution and saturated sodium chloride solution alternately. The mixture was dried over anhydrous magnesium sulfate for 6 hours, and the ethyl acetate was removed by rotary evaporation to collect the intermediate product.

[0064] The intermediate product and trimethyl orthoformate were placed together in a 500 mL dry reaction bottle with a molar ratio of the intermediate product to trimethyl orthoformate of 1:3. 150 mL of anhydrous acetonitrile was added as a reaction solvent. A catalyst, p-toluenesulfonic acid monohydrate, was added. After stirring and reacting for 15 hours under nitrogen, triethylamine was added dropwise to terminate the reaction. The anhydrous acetonitrile was removed by rotary evaporation, and the mixture was dissolved with ethyl acetate. The mixture was then extracted once with saturated sodium bicarbonate and once with saturated sodium chloride. The organic phase was collected and the organic solvent, ethyl acetate, was removed by rotary evaporation to obtain FOE.

[0065] S2. Add 200 mg, 0.67 mmol of cisplatin to a 100 mL round-bottom flask, then add 12 mL of deionized water to dissolve the cisplatin, add 12 mL of 30 wt% H2O2 solution dropwise through a separatory funnel, and stir the reaction at 50 °C in the dark for 4.5 h. After the reaction is completed, remove most of the water and H2O2 solution by rotary evaporation, wash with deionized water, and lyophilize to obtain a yellow tetravalent cisplatin product.

[0066] 100 mg, 0.3 mmol of tetravalent cisplatin product and 181.2 mg, 0.6 mmol of norcantharidin were mixed, then dissolved in 5 mL of anhydrous N,N-dimethylformamide and added to a round-bottom reaction flask. 121.16 mg, 1.2 mmol of catalyst triethylamine were added, and the mixture was reacted at 60°C in the dark under nitrogen protection for 22 h. After the reaction, N,N-dimethylformamide was removed by vacuum distillation, and the mixture was precipitated with anhydrous ether under an ice bath. The precipitate was washed with acetone and finally dried to obtain a light yellow Pt(IV)-1 powder.

[0067] S3, 6.76 mg of Pt (IV) -1 and 0.33 mg of ICG were placed in 300 μL of FOE, and the solid was completely dissolved by ultrasound for 30 min to obtain an orthoester complex; 2% by mass of CMCS solution was added to the orthoester complex, and vortexed to emulsify the orthoester complex uniformly, and 5% by mass of GA solution was added while vortexing. -NH2 and n in GA -CHO The ratio is 1:0.8, V CMCS :V FOE The ratio of the organic composite hydrogel to the photothermal chemotherapy was 10:5, and an injectable organic composite hydrogel combined with photothermal chemotherapy was obtained.

[0068] Example 3

[0069] A method for preparing an injectable organic composite hydrogel combined with photothermal chemotherapy comprises the following steps:

[0070] S1. Under nitrogen, add 3-amino-1,2-propanediol and ethyl trifluoroacetate to a dry 500 mL three-necked round-bottom reaction flask containing a magnetic rotor, the molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate being 1:3, then add 150 mL of anhydrous acetonitrile as the reaction solvent, and stir under nitrogen for 15 h; after the reaction, remove the solvent anhydrous acetonitrile by rotary evaporation, and dissolve with 150 mL of ethyl acetate solution containing 150 μL of triethylamine, then extract once alternately with 0.5 mol / L potassium bisulfate solution and saturated sodium chloride solution, dry over anhydrous magnesium sulfate for 6 h, remove ethyl acetate by rotary evaporation, and collect the intermediate product.

[0071] The intermediate product and trimethyl orthoformate are placed together in a 500 mL dry reaction bottle with a molar ratio of the intermediate product to trimethyl orthoformate of 1:1. 150 mL of anhydrous acetonitrile is added as a reaction solvent, and a catalyst, p-toluenesulfonic acid monohydrate, is added. After stirring and reacting for 10 hours under nitrogen, triethylamine is added dropwise to terminate the reaction. The anhydrous acetonitrile is removed by rotary evaporation, and the mixture is dissolved with ethyl acetate, and then extracted once with saturated sodium bicarbonate and saturated sodium chloride. The organic phase is collected and the organic solvent, ethyl acetate, is removed by rotary evaporation to obtain FOE.

[0072] S2. Add 200 mg, 0.67 mmol of cisplatin to a 100 mL round-bottom flask, then add 12 mL of deionized water to dissolve the cisplatin, add 14 mL of 30 wt% H2O2 solution dropwise through a separatory funnel, and stir the reaction at 50 °C in the dark for 5 h. After the reaction is completed, remove most of the water and H2O2 solution by rotary evaporation, and then wash with deionized water and freeze-dry to obtain a yellow tetravalent cisplatin product.

[0073] 100 mg, 0.3 mmol of tetravalent cisplatin product and 120.8 mg, 0.6 mmol of norcantharidin were mixed, then dissolved in 5 mL of anhydrous N,N-dimethylformamide and added to a round-bottom reaction flask. 121.16 mg, 1.2 mmol of catalyst triethylamine were added, and the mixture was reacted at 60°C in the dark under nitrogen protection for 24 h. After the reaction, N,N-dimethylformamide was removed by vacuum distillation, and the mixture was precipitated with anhydrous ether under an ice bath. The precipitate was washed with acetone and finally dried to obtain a light yellow Pt(IV)-1 powder.

[0074] S3, 7.84 mg of Pt (IV) -1 and 0.33 mg of ICG were placed in 300 μL of FOE, and the solid was completely dissolved by ultrasound for 30 min to obtain an orthoester complex; 2% by mass of CMCS solution was added to the orthoester complex, and vortexed to emulsify the orthoester complex uniformly, and 5% by mass of GA solution was added while vortexing. -NH2 and n in GA -CHO The ratio is 1:2.4, V CMCS :V FOE The ratio of the organic composite hydrogel to the photothermal chemotherapy was 10:1, and an injectable organic composite hydrogel combined with photothermal chemotherapy was obtained.

[0075] Examples 1 to 3 of the present invention all produced injectable organic composite hydrogels with excellent performance for combined photothermal chemotherapy. Gel-1, Gel, Gel-2, Gel / FOE-1, Gel / FOE, Gel / FOE-2, and Gel / FOE / IP of Example 1 were used as examples for research. The specific research methods and results are shown below:

[0076] 1. Determination and analysis of injectable hydrogels and injectable organic composite hydrogels:

[0077] 1. Hydrogel Screening: Macroscopic Morphological Structure Observation: After the preparation of the injectable hydrogel and injectable organic composite hydrogel, a camera was used to photograph and record the macroscopic state. Microscopic Morphological Structure Observation: After the preparation of Gel / FOE-1, Gel / FOE, and Gel / FOE-2, they were removed and cut into thin slices with a blade. The slices were then observed under a microscope and photographed. Scanning Electron Microscope Sample Preparation: The injectable hydrogel and injectable organic composite hydrogel were rapidly frozen using liquid nitrogen and subjected to a brittle fracture treatment. They were freeze-dried and then affixed to the surface of a silicon wafer with conductive tape. A portion of the brittle fracture surface of the completely freeze-dried injectable hydrogel and injectable organic composite hydrogel was cut and adhered to the conductive tape. The silicon wafer with the hydrogel sample was then placed on the edge of the sample stage. The injectable hydrogel and injectable organic composite hydrogel were gold-sprayed and observed using an SEM. Single Frequency Scan: Gel-1, Gel, and Gel-2 were scanned using a Kinexus Lab+ rotational rheometer to examine changes in a single frequency. Determination of cross-linking degree: The amino group of CMCS reacts with the aldehyde group of GA to generate an imine bond and a molecule of water, which is used to determine the cross-linking degree of Gel-1, Gel and Gel-2.

[0078] 2. Fourier transform infrared spectroscopy and X-ray diffraction determination of hydrogel: Gel freeze-dried product, Gel / FOE freeze-dried product and CMCS powder were fully mixed with potassium bromide powder, ground into fine powder and pressed into transparent thin slices; for liquid samples GA and FOE, potassium bromide powder was pressed into thin slices and then evenly applied on the surface of each sample, dried with a mercury lamp and heated at 500 cm -1 ~4000cm -1 Fourier transform infrared spectrometer scanning was performed within a wavenumber range. Freeze-dried Gel, freeze-dried Gel / FOE, freeze-dried Gel / FOE / IP, and CMCS powder were placed in the grooves of a glass slide and the surface was scraped flat. Liquid samples GA and FOE / IP were placed directly in the grooves of the glass slide and placed in an X-ray diffractometer for structural characterization.

[0079] 3. Determination of rheological properties of hydrogels: The rheological properties of Gel and Gel / FOE were evaluated using a Kinexus Lab+ rheometer. First, under constant stress conditions, Gel and Gel / FOE were frequency scanned in the range of 0.1 Hz to 10 Hz to monitor the changes in the storage modulus G' and loss modulus G". Subsequently, Gel / FOE was amplitude scanned in the range of 0.1% to 1000% to determine the linear viscoelastic region at a fixed frequency of 1 Hz. Next, the self-healing ability of Gel / FOE was quantitatively investigated by performing dynamic oscillation rheology tests at a constant frequency of 1 Hz. Finally, the change in viscosity was analyzed to evaluate the shear thinning behavior of Gel / FOE at different shear rates, with a shear rate range of 0.1s -1~100s -1 .

[0080] 4. Photothermal temperature rise, photothermal stability, and fluorescence intensity determination: Different groups containing 250 μg / mL ICG were prepared, specifically H2O / ICG, FOE / ICG, Gel / ICG, and Gel / FOE / ICG. In the Gel / FOE / ICG, the volume of Gel was 1052 μL and the volume of FOE was 300 μL. They were placed in centrifuge tubes and irradiated with an 808 laser at 0.5 W / cm 2 Irradiate for 5 minutes. During the irradiation process, take thermal photos with an infrared thermal imager and record the temperature. Re-prepare the above different groups of solutions and use an 808nm laser at 0.5W / cm 2 The cells were irradiated for 5 min. After the temperature of each group dropped to room temperature, the fluorescence intensity of the H2O / ICG and FOE / ICG groups was measured using a microplate reader. The above operation was repeated four times, and the data were recorded to observe the photothermal stability of each group and the effect of FOE on the fluorescence intensity of ICG.

[0081] 5. Determination of in vitro degradation: After the preparation of Gel / FOE is completed, it is weighed and recorded as M0, and placed in PBS solutions of different pH values, specifically pH7.4, pH6.8, pH5.0, and pH5.0 / GSH. pH5.0 / GSH means that glutathione is present in an environment of pH5.0 and oscillated at 37°C and 100 rpm. Gel, Gel / FOE, and Gel / FOE / ICG+NIR groups are placed in 0.2 mol / L, pH6.8 PBS solutions. The operation of the Gel / FOE / ICG+NIR group is as follows: the Gel / FOE / ICG is exposed to light once a day; three parallel groups are set up for each group, and the excess PBS solution is removed at the preset time point. The Gel, Gel / FOE, and Gel / FOE / ICG+NIR are weighed and recorded as Mr. The formula is used to calculate the degradation rate of Gel / FOE / ICG. Calculate the remaining mass percentage.

[0082] 6. Verification of FOE escape from Gel / FOE: Six pieces of prepared Gel / FOE and Gel were soaked in 5 mL of 0.2 mol / L PBS solution, pH 6.8, and placed on a 37°C constant temperature shaker. At the preset time points of 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, the Gel / FOE and Gel were taken, cut into thin slices at the edge and center, and observed under a microscope and photographed.

[0083] 7. Experimental verification of whether FOE is degraded in Gel and whether it can escape from Gel: Gel / FOE was placed in a 4℃ refrigerator, FOE was extracted with dichloromethane at the preset time points of 3h, 6h, and 12h, and dichloromethane was removed by rotary evaporation. 1 The Gel / FOE was immersed in 0.2 mol / L PBS solution with a pH of 6.8. After 1 hour, part of the Gel / FOE buffer solution was lyophilized. 1 1H NMR was used for detection.

[0084] 8. Nanoparticle Size, Particle Size Stability, and Potential Change: The Gel / FOE / IP prepared in Example 1 was placed in a 0.2 mol / L PBS solution (pH 6.8) and placed in a 37°C incubator. After 12 hours, the supernatant was collected and centrifuged at 8000 rpm for 5 minutes to remove macromolecules from the Gel / FOE / IP solution, thereby obtaining nanoparticles. At room temperature, DLS was used to measure the particle size of the nanoparticles, the potential change at pH 7.4, 6.8, and 5.0, and the particle size stability at pH 7.4, 6.8, 5.0, and 5.0 / GSH.

[0085] 9. Drug release of cisplatin prodrug: Gel / IP and Gel / FOE / IP were sealed in 14000Da dialysis bags respectively, and then immersed in 5mL, 0.2mol / L PBS solutions of different ratios. The pH of the PBS solutions were 7.4, 6.8, 5.0, and 5.0 / GSH, respectively. Three parallels were set up for each group. At the preset time points of 1h, 3h, 6h, 12h, 24h, 48h, 72h, and 96h, the PBS solution was taken out and 5mL of new PBS solution was added. The obtained samples were measured by high performance liquid chromatography. The preparation method of Gel / IP is as follows: 7.3mg of Pt(IV)-1 and 0.33mg of ICG were mixed in a 2% by mass CMCS solution, vortexed, and a 5% by mass GA solution was added while vortexing. The n in CMCS was 0.5% by mass. -NH2 and n in GA -CHO The ratio is 1:1.6.

[0086] Result analysis:

[0087] 1. Screening of injectable hydrogels: Figure 1 As shown in Figure (b), at the same frequency, G' of the three groups of injectable hydrogels is always greater than G", indicating that the three groups of injectable hydrogels are already in the gel state. Figure 1As shown in Figure (c), the cross-linking degrees of Gel-1, Gel, and Gel-2 are 56.5%, 77.1%, and 61.4%, respectively. Compared with the Gel-1 and Gel-2 groups, the Gel group has higher mechanical strength. Figure 1 As shown in Figure (a), Gel-1 has a rough morphology and disordered pores. The Gel group has a uniform pore structure, presenting a regular, dense porous network. The Gel-2 group exhibits a stacked, sheet-like structure with covered pores. To minimize the impact of FOE on the injectable hydrogel structure, subsequent experiments selected a porous network with high mechanical strength and regular morphology.

[0088] 2. Screening of FOE amount in Gel / FOE: Figure 2 Although the SEM image of the Gel / FOE-1 group still shows a porous structure, microscopic observation of the Gel / FOE-1 slices shows that the amount of FOE added is too small, which is not conducive to the subsequent drug loading. The Gel / FOE group has a smooth morphology and still maintains a regular porous network structure. The micrograph shows that FOE is evenly dispersed in the Gel, which is conducive to the uniform release of the drug after subsequent drug loading. The Gel / FOE-2 group contains a large amount of FOE. The SEM image shows that some FOE has precipitated on its surface, and the original network pore structure has been completely destroyed. Taking all factors into consideration, we selected the Gel / FOE group for subsequent experiments.

[0089] 3. Infrared spectrum and X-ray diffraction pattern analysis: Figure 3 Figure (a) shows the scattering of GA, CMCS, Gel, FOE and Gel / FOE at 500 cm -1 ~4000cm -1 FI-TR diagram of GA at 1722 cm -1 Stretching vibration absorption peak V C=O , and CMCS's 3442cm -1 Stretching vibration absorption peak V -NH2 Disappeared in Gel, and 1624cm appeared in Gel -1 V C=N , indicating that after the three-dimensional network structure of Gel is formed, the aldehyde and amino groups are consumed, forming a Schiff base dynamic cross-linking structure. -1 The characteristic peak V of FOE appears at C-F , and still appears 1624cm -1 Stretching vibration absorption peak V C=N , indicating that FOE was successfully added without destroying the cross-linking structure of Gel.

[0090] XRD analyzes the sample structure through the X-ray crystal diffraction effect. Figure 3As shown in Figure (b), CMCS exhibits a broad peak at 20.8°, GA exhibits a broad diffraction peak at 23.71°, and after gel formation, a broad peak at 22.3° emerges. This indicates cross-linking between CMCS and GA, altering the crystal structures of both CMCS and GA, confirming the successful preparation of gel. Furthermore, the broad peak position for Gel / FOE / IP is identical to that for Gel / FOE and FOE / IP, confirming the correct structure of Gel / FOE / IP.

[0091] 4. Analysis of rheological properties of Gel / FOE: Figure 4 As shown, Figure 4 Figure (a) shows that the storage modulus G' of Gel and Gel / FOE is greater than the loss modulus G", indicating that Gel and Gel / FOE have always maintained a cross-linked structure and have good stability. However, the G' and G" of Gel / FOE are lower than the G' and G" of Gel, indicating that the addition of FOE has reduced the mechanical strength of Gel to a certain extent. Figure 4 Figure (b) shows that the Schiff base crosslinking structure of Gel / FOE is destroyed under shear. As the shear rate increases, the viscosity of Gel / FOE decreases rapidly and eventually remains constant, demonstrating that Gel / FOE has good injectability. The inset of Figure (b) also demonstrates the injectability of Gel / FOE on a macroscopic scale. Figure 4 Figure (c) determines the critical strain values of G' and G" of Gel / FOE, based on which high and low strain scans are performed in Figure (d). Figure 4 Figure (d) shows that under alternating high and low strain sweeps, Gel / FOE transitions between a gel and a sol state. At high strain, G' is less than G", indicating that Gel / FOE is in a sol state. At low strain, G' is greater than G", and Gel / FOE returns to a gel state. This sol-gel transition is reproducible, demonstrating the self-healing properties of Gel / FOE. This self-healing property is imparted by the dynamic imine bond formed between the amino and aldehyde groups. The inset in Figure (d) demonstrates the excellent self-healing properties of Gel / FOE on a macroscopic scale.

[0092] 5. Study on photothermal conversion performance and stability: Figure 5 As shown in (a) and (b), FOE / ICG at 0.5W / cm 2 After 5 minutes of laser irradiation, the temperature can reach 74.3℃, while the temperature of H2O / ICG is only 47.2℃ after 5 minutes of irradiation, indicating that FOE with low specific heat capacity is beneficial to improving the photothermal efficiency of ICG. The Gel / ICG and Gel / FOE / ICG groups finally heated up to 48.7℃ and 54℃ respectively, indicating that FOE loaded in Gel is beneficial to improving the overall photothermal heating. Figure 5 As shown in Figures (c) and (d), the FOE / ICG group maintained a high photothermal effect after multiple heating cycles. Under the same conditions, the photothermal capacity of H2O / ICG was significantly reduced, while the Gel / FOE / ICG group exhibited superior photothermal stability compared to the Gel / ICG group. Furthermore, the FOE / ICG group maintained high fluorescence intensity after multiple heating cycles, indicating that FOE enhances the structural stability of the ICG molecule, which will further facilitate the role of ICG in subsequent cancer treatments.

[0093] 6. In vitro degradation study: Gel / FOE degradability plays an important role in stable drug release. Figure 6 As shown, Gel / FOE degraded rapidly within 48 hours. At 48 hours, the remaining mass percentages at pH 7.4, pH 6.8, pH 5.0, and pH 5.0 / GSH were 39.3%, 35.0%, 15.7%, and 14.7%, respectively. The results indicate that a decrease in pH accelerates the cleavage of the dynamic imine bond in Gel / FOE, thereby accelerating the disintegration of Gel / FOE. At pH 6.8, Gel / FOE degraded faster than the Gel group. This is because the dynamic imine bond is acid-sensitive. In a slightly acidic environment, the imine bond cleavage and FOE escape accelerate the overall degradation rate. The degradation rate of the Gel / FOE / ICG+NIR group was the fastest, indicating that photothermal treatment is beneficial to the disintegration of Gel / FOE.

[0094] 7. Verification of FOE escape process from Gel / FOE: Figure 7 As shown in the figure, at 0 hours, the FOE at the edges and center of the Gel / FOE were uniformly dispersed in the form of droplets. Over time, the number of FOE in the edge Gel / FOE gradually decreased compared to the Gel, and the droplets gradually became smaller, almost completely escaping at 24 hours. The size and number of FOE droplets in the center remained almost unchanged over the 24 hours. These results indicate that the FOE escape process is a gradual process from the edge of the Gel / FOE to the center, while the droplets themselves gradually escape from the outside to the inside.

[0095] 8. Experimental verification of whether FOE degrades in Gel and whether it can escape from Gel: Whether FOE degrades in Gel and whether it can escape from Gel is the key to tumor treatment. Figure 8 It can be seen that within 12 hours, the characteristic peak and degradation peak of FOE in the Gel did not change significantly, proving that FOE would not degrade in the Gel within 12 hours. Partially degraded FOE was detected in the PBS solution, proving that FOE could successfully escape from the Gel and could be degraded in a slightly acidic environment.

[0096] 9. Nanoparticle size, particle size stability, and potential changes: Figure 9 As shown in Figure (a), after FOE degradation, the two drugs can self-assemble and form nanoparticles. The DLS test shows that the particle size of the nanoparticles is about 175nm, and the SEM image shows a smaller particle size. Figure 9 As shown in Figure (b), the nanoparticles maintained stable particle size and morphology within 72 hours under pH 7.4, pH 6.8, and pH 5.0 environments. However, under pH 5.0 / GSH environment, tetravalent cisplatin was reduced to divalent cisplatin, and the cisplatin prodrug disintegrated into cisplatin and norcantharidin, and the nanoparticles were destroyed. Figure 9 As shown in Figure (c), the surface potential of the nanoparticles increases with decreasing pH. This is because as the pH decreases, the carboxyl groups on the surface of the nanoparticles are protonated, which increases the surface potential, which is beneficial to enhancing the uptake of nanoparticles by tumor cells.

[0097] 10. Drug release of cisplatin: Figure 10 As shown in the figure, under pH 7.4, pH 6.8, and pH 5.0, the cisplatin drug release from the Gel and Gel / FOE groups was less than 20% of the total drug amount, while under pH 5.0 / GSH, the cisplatin drug release reached 58.8% and 70.3% of the total drug amount, respectively. This is because under the stimulation of pH 5.0 / GSH, tetravalent cisplatin is reduced to divalent cisplatin, the nanoparticle structure is destroyed, and cisplatin is released. The presence of FOE accelerates the effective escape of the drug from the Gel.

[0098] 2. In vitro application experiments:

[0099] 1. Cytotoxicity Assessment: Toxicity test for HepG2 cells: Use cultured cells, discard the supernatant medium, rinse adherent cells with PBS buffer, digest with trypsin solution, centrifuge, and resuspend the cells to obtain a cell suspension. Aspirate 180 μL of the cell suspension into a sterilized 96-well plate to a density of 4,000 cells per well. Incubate overnight to allow adherence before proceeding with subsequent procedures. The experimental groups included Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR, negative control group, and positive control group. Each group was set up in parallel with 6 groups. The negative control group was culture medium, and the positive control group was culture medium + cells. Among them, the concentration gradient of cisplatin in Gel / IP, Gel / IP+NIR, Gel / FOE / IP, and Gel / FOE / IP+NIR was 4.1μmol / L, 8.2μmol / L, 16.4μmol / L, 32.8μmol / L, and 65.6μmol / L. The cells were plated according to the needs of the group. After the cells adhered, the drugs of each concentration gradient experimental group were added. The non-illumination group was incubated for 48h, and the 96-well plate was taken out of the illumination group at 24h. An 808nm laser was used at 0.5W / cm2 Illuminate for 5 minutes, then continue incubating for 24 hours. After incubation, discard the culture medium and hydrogel residues, add 180 μL of fresh culture medium and 20 μL of thiazolyl blue solution to each well, and incubate for 4 hours. Discard the supernatant culture medium, add 150 μL of dimethyl sulfoxide to each well, and incubate at 37°C on a shaker for 20 minutes. After incubation, measure the absorbance of each well at a UV wavelength of 570 nm using a microplate reader.

[0100] 2. Qualitative and quantitative cell uptake: Qualitative uptake: Take the cultured adherent cells, digest them, and prepare a cell suspension for later use. Place a sterile cover glass in a six-well plate and add 1.8 mL of cell suspension. The cell density is about 1×10 5 After overnight culture to allow cells to adhere, Gel / IP and Gel / FOE / IP were added, both containing 65.6 μmol / L cisplatin, and the cells were cultured for 4 h. After the culture period, the residual hydrogel and culture medium were removed, and the coverslips were rinsed with PBS buffer. The cells were fixed with 4 wt% paraformaldehyde solution for 10 min, and the residual fixative was removed with PBS buffer. The excess PBS buffer was discarded, and DAPI stain was added to stain the cell nuclei for 10 min. The excess stain was rinsed with PBS buffer, and the cells were observed and photographed under a laser confocal microscope.

[0101] Quantitative uptake: After the cells were digested, plated, and adhered, drugs Gel / IP and Gel / FOE / IP were added. The concentration of cisplatin in both was 65.6 μmol / L. Three parallel controls were used in each group. After 4 h of co-culture, the residual hydrogel and culture medium were removed. The cells were washed three times with PBS buffer solution, and the excess PBS buffer solution was discarded. 200 μL of triple-formazan lysis solution was added to each well, shaken evenly, and placed in a 4°C refrigerator for 30 min. The cells in the wells were collected and incubated at 1.2×10 4 The mixture was centrifuged at 370 rpm for 10 min, the supernatant was aspirated and digested by adding concentrated nitric acid, and the drug content was detected by spectrometer to estimate the drug intake.

[0102] 3. Gel / FOE Adhesion Testing: Different Gels / FOEs were placed on pig skin, pork, and pig liver, and subjected to stretching, twisting, and squeezing. The adhesion of the Gels / FOEs to glass, plastic, rubber, and metal was observed and photographed. The Gels / FOEs were subcutaneously injected into mice, and the specimens were dissected 10 minutes later for observation and photographic documentation.

[0103] 4. Hemolysis experiment: 500 μL of blood was collected from the mouse heart and placed in a centrifuge tube containing sodium heparin. The tube was quickly mixed, and then 5 mL of PBS buffer solution was added. The tube was centrifuged at 2500 rpm for 5 minutes. The supernatant was discarded and the washing steps were repeated until the upper layer was clear. The red blood cells were collected by centrifugation and 10 mL of normal saline was added and gently mixed with the red blood cells to prepare a red blood cell suspension for subsequent experiments. This experiment set up a positive control group, a negative control group, and experimental groups with different concentration gradients. Three parallel controls were set up for each group. The positive control group was a mixture of 500 μL of cell suspension and 500 μL of 0.1% Triton X-100, and the negative control group was a mixture of 500 μL of cell suspension and 500 μL of normal saline. 500 μL of cell suspension was mixed with each group of materials and incubated in a 37°C constant temperature incubator for 1 h. After incubation, each group of samples was centrifuged at 10,000 rpm for 5 min, photographed and recorded, and the supernatant was aspirated into a 96-well plate. The absorbance of each group was detected at an ultraviolet wavelength of 570 nm using a microplate reader, and the data was recorded.

[0104] .

[0105] Result analysis:

[0106] 1. Cytotoxicity evaluation: Figure 11 It can be seen that with the increase in drug concentration in each group, the cytotoxicity to HepG2 cells increased in a dose-dependent manner. Compared with the non-irradiated group, the irradiated group showed higher cytotoxicity, indicating that photothermal therapy has a certain killing effect on tumor cells. The Gel / IP group has a certain cytotoxicity due to the degradation of Gel, the shedding of GA, and the toxicity of drug release to cells. Gel / FOE / IP has higher cytotoxicity than Gel / IP. On the one hand, the oil-water incompatibility allows more drugs to be released from Gel / FOE. On the other hand, after the degradation of FOE, the two drugs self-assemble to form nanoparticles, which are more easily taken up by cells, resulting in stronger cytotoxicity. The results of Gel / FOE / IP+NIR show that the presence of FOE is conducive to further improving the combined effect of photothermal therapy and chemotherapy.

[0107] 2. Qualitative and quantitative cell uptake: Observe the drug uptake using a laser confocal microscope. Figure 12 As shown in Figure (a), obvious red fluorescence appeared in the tumor cells, indicating that ICG was internalized by the cells. At the same time, the fluorescence intensity of Gel / IP was significantly weaker than that of Gel / FOE / IP. In order to further verify the ability of tumor cells to absorb various drug preparations, we also used cell lysis to detect the platinum content in the cell content extract. Figure 12 As shown in Figure (b), within 4 hours, the drug uptake by HepG2 cells in Gel / IP was 7.0 μg / 1×10 6The drug uptake in Gel / FOE / IP was 10.6 μg / 1×10 6 The qualitative and quantitative cell uptake results showed that the FOE in the Gel / FOE encapsulated the two drugs. The oil-water incompatibility accelerated the escape of the FOE as it escaped from the Gel / FOE. Furthermore, after the degradation of the FOE, the two drugs self-assembled into nanoparticles, which were more easily taken up by cells.

[0108] 3. Gel / FOE adhesion test: Figure 13 In the tissue adhesion experiment of Gel / FOE, it was found that it can firmly adhere to the surface of animal epidermis, liver and muscle tissue, and after the above tissues were flattened, squeezed, twisted and stretched, Gel / FOE can still firmly adhere to the surface of biological tissue. The adhesion of Gel / FOE is mainly formed by the interaction between covalent bonds and non-covalent bonds. CMCS is a natural polysaccharide derivative with many active groups. It can interact with the surface of proteins in biological tissues such as amino groups, carboxyl groups and hydroxyl groups through hydrogen bonds and imine bonds, and thus has good biological adhesion. In order to verify that Gel / FOE has in vivo injectability and in vivo adhesion, we injected Gel / FOE into the subcutaneous tissue of mice. The results showed that Gel / FOE can be injected into the mouse body, fixed to the subcutaneous tissue of the mouse, and has an elastic state and regular properties. Therefore, Gel / FOE has good biological tissue adhesion properties and in vivo injectability.

[0109] 4. Hemolysis test: passed Figure 14 It can be seen that in the positive control group treated with TritonX-100, due to the rupture of the red blood cell membrane, hemoglobin was released, the supernatant in the centrifuge tube was red, and no red blood cell sediment was produced at the bottom of the centrifuge tube, and the hemolysis rate was 100%. There was no significant difference between the Gel / FOE / Pt(IV)-1 at different concentrations and the negative control group. The supernatant was clear, and red blood cell sediment was produced at the bottom of the centrifuge tube. The hemolysis rate of all experimental groups was less than 5%, proving that no obvious hemolysis occurred and it had high blood compatibility. Therefore, it had good biosafety, which provided feasibility guarantee for the subsequent animal experiments. Gel / FOE / Pt(IV)-1 was prepared according to the following steps: 7.3 mg of Pt(IV)-1 was dispersed in 300 μL of FOE, and then a 2% by mass fraction CMCS solution was added and vortexed. While vortexing, a 5% by mass fraction GA solution was added. The n in CMCS was 1. -NH2 and n in GA -CHO The ratio is 1:1.6, V CMCS :V FOE The ratio of the molar ratio of FOE to Pt(IV)-1 was 10:3, and Gel / FOE / Pt(IV)-1 was obtained.

[0110] 3. In vivo application experiments:

[0111] When the tumor volume reaches 200 mm 3 At the same time, 42 model mice were randomly divided into 7 groups. They were FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR and blank control group. The blank control group was normal saline group. The tumor was injected orally on the 1st, 4th and 7th day. The irradiation group used 808 laser at 0.5W / cm2 every day for the first 7 days. 2 The mice were illuminated for 5 minutes. Except for the blank control group, all groups received 6 mg / kg of cisplatin. The mice were observed for 14 days, and their body weight, tumor volume, and survival were recorded daily. On the 14th day, the mice were sacrificed, and major organ tissues, including the heart, liver, spleen, lung, kidney, and tumor, were dissected and removed. The dissected tumors were cleaned with saline, wiped clean, weighed, and photographed.

[0112] Result analysis: On the 14th day, the tumor volume and tumor mass of the blank control group reached 3069.94 mm 3 However, compared with the control group, the tumor growth of FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, and Gel / FOE / IP+NIR was slower, with the tumor volume and tumor mass increasing slowly to 1000.71mm and 1000.71mm, respectively. 3 and 1.53g, 114.33mm 3 and 0.2g, 1846.21mm 3 and 3g, 798.33mm 3 and 1.1g, 1046.54mm 3 and 2.45g, 123.58mm 3The growth of mouse tumors was inhibited to varying degrees. Compared with the non-illumination group, the illumination group had smaller tumor volume and mass, showing a better tumor inhibition effect. The mortality rates of FOE / IP and FOE / IP+NIR on day 14 were 33.3% and 66.7%, respectively, due to the acute toxicity caused by the diffusion of the orthoester complex. The tumor cure rates of Gel / IP+NIR and Gel / FOE / IP+NIR on day 14 were 16.7% and 50%, respectively, and the mouse survival rate was 100%, demonstrating good biosafety and therapeutic ability. The therapeutic effect of the Gel / FOE / IP+NIR group was the most outstanding. This is because Gel / FOE achieves drug enrichment at the tumor site. During the degradation of Gel / FOE, the escape of FOE accelerates the escape of the drug, improving the photothermal performance while promoting the diffusion and penetration of the drug at the tumor site. After degradation in response to the microenvironment, the two drugs assist in self-assembly to form nanoparticles, which greatly improves the enrichment and retention of the drug at the tumor site, reduces systemic toxic side effects, and improves the therapeutic efficacy.

[0113] Obviously, those skilled in the art may make various modifications and variations to the invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the invention is intended to include such modifications and variations.

Claims

1. A method for preparing an injectable organic composite hydrogel combined with photothermal chemotherapy, characterized in that: The steps include: Under an inert atmosphere, 3-amino-1,2-propanediol is mixed with ethyl trifluoroacetate, and an amidation reaction is carried out in a liquid phase. The solvent is separated to obtain an intermediate product. The intermediate product is mixed with trimethyl orthoformate, and an acetalization reaction is carried out in a liquid phase under a catalytic effect. The solvent is separated to obtain an oily orthoester. Cisplatin prodrug and photothermal agent indocyanine green are co-dispersed in an oily orthoester to obtain an orthoester complex, and then a carboxymethyl chitosan solution is added. The orthoester complex is vortexed to emulsify the complex uniformly, and a glutaraldehyde solution is added while vortexing to obtain an injectable organic composite hydrogel for combined photothermal chemotherapy. The cisplatin prodrug is prepared according to the following steps: mixing divalent cisplatin in water, then adding H2O2 solution dropwise, stirring and performing oxidation reaction in the dark, separating the solvent, and freeze-drying to obtain a tetravalent cisplatin product; using anhydrous N,N-dimethylformamide as a reaction solvent and triethylamine as a catalyst, mixing the tetravalent cisplatin product, norcantharidin and triethylamine, reacting in the dark under nitrogen protection, performing esterification reaction in a liquid phase, and after the reaction, separating the solvent by rotary evaporation, settling, washing, collecting the precipitate, and drying to obtain the cisplatin prodrug; The mass fraction of carboxymethyl chitosan solution is 2%, the mass fraction of glutaraldehyde solution is 5%, and the n -NH2 With n in glutaraldehyde solution -CHO The ratio is 1:0.8~2.4; The volume ratio of carboxymethyl chitosan solution to oily orthoester is 10:1~5.

2. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The conditions for the amidation reaction are: stirring at room temperature for 10 h to 15 h.

3. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:1-3.

4. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The acetalization reaction conditions are: stirring at room temperature for 10 h to 15 h.

5. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The molar ratio of the intermediate product to trimethyl orthoformate is 1:1-3.

6. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The molar ratio of cisplatin prodrug to photothermal agent indocyanine green is 25~29:

1.

7. An injectable organic composite hydrogel for combined photothermal chemotherapy prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 7 in the preparation of a drug carrier.

9. Use of the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 7 in the preparation of anti-liver cancer drugs.

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