A pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system and its preparation method and application

Abstract

The present invention discloses a pH / GSH program-responsive photothermal nanocomposite hydrogel co-drug-carrying system and a preparation method and application thereof, and belongs to the field of anti-tumor materials. The nanocomposite hydrogel co-drug-carrying system of the present invention is a pH-sensitive chitosan hydrogel co-carrying GSH stimulus-responsive drug-carrying nanoparticles encapsulated by hyaluronic acid and indocyanine green. The drug-carrying nanoparticles encapsulate tyrosine kinase inhibitors, and the chitosan hydrogel is obtained by cross-linking chitosan with polyethylene glycol-4-formylbenzoic acid. The nanocomposite hydrogel is easy to administer in situ, and the photothermal conversion of indocyanine green under near-infrared light irradiation is used to accurately thermally ablate the primary tumor; at the same time, the drug-carrying nanoparticles escape from the gel, respond to the release of the loaded tyrosine kinase inhibitor by GSH in the tumor cells, and inhibit tumor recurrence and metastasis. The nanocomposite hydrogel can achieve chemotherapy-photothermal synergistic therapy, enhance the therapeutic effect on tumors and reduce toxic side effects.

Description

Technical Field

[0001] The invention belongs to the field of anti-tumor materials, and specifically relates to a pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system and a preparation method and application thereof. Background Art

[0002] Cancer is the second leading cause of death after cardiovascular disease. Traditional cancer treatments mainly include surgical resection, chemotherapy, immunotherapy, and radiotherapy. Among them, chemotherapy is the most commonly used treatment option for clinical cancer patients, but it still has certain limitations. In the process of chemotherapy killing tumor cells, due to its non-specificity, it is widely distributed in the body after administration, and often causes toxicity to the body's normal and rapidly proliferating cells such as digestive tract cells, bone marrow hematopoietic cells, and immune cells, thereby causing a series of toxic side effects. In recent years, smart drug delivery systems for tumor treatment have been widely studied. These drug delivery systems can regulate the in vivo process of chemotherapy drugs, promote the distribution and accumulation of drugs in tumor tissues, improve the cellular uptake and release behavior of drugs, and achieve toxicity reduction and efficacy enhancement, and have good clinical development value.

[0003] Injectable hydrogels have attracted great attention in the field of anti-tumor drug delivery due to their low toxicity, minimal invasiveness, and adaptability to any irregular cavity. The route of administration of hydrogels is usually intratumoral / peritumoral injection. Compared with systemic administration, hydrogel delivery can directly deliver drugs to the tumor site at high concentrations, reach the therapeutic window concentration at a smaller dosage, and effectively reduce the toxic side effects of chemotherapy drugs. Although drug-loaded hydrogels have made certain research progress, they still have limitations in tumor treatment: (1) The encapsulation efficiency and homogeneity of hydrogels for hydrophobic drugs are limited, which can easily lead to local drug burst release; (2) Drug release is highly dependent on passive diffusion and lacks targeted selectivity. Most drugs themselves cannot reach deep tumor tissues and are potentially toxic to surrounding healthy tissues; (3) It is difficult to release combined therapeutic drugs synchronously according to the designed ratio, which reduces the synergistic therapeutic effect of drugs.

[0004] Combined with clinical needs, hydrogels with chemotherapy, photothermal therapy, and immunotherapy effects have been developed. Among them, non-invasive near-infrared (NIR)-responsive natural macromolecular hydrogel assembled drugs (NIR-NMHAD) have begun to become the focus of researchers. NIR can penetrate deeply into human tissues, cause little damage to paratumor tissues, and the irradiation time and space are controllable. The hydrogel transports the encapsulated drugs to the tumor tissue and releases the encapsulated drugs under the irradiation of NIR. The drugs enter the cells and inhibit the growth of tumor cells. Compared with heavy metal photothermal agents such as gold and lead, indocyanine green (ICG) is the only FDA-approved near-infrared imaging agent for clinical use. It can absorb near-red light and convert it into thermal energy, and can be used for photothermal therapy and photodynamic therapy. However, it is unstable in aqueous solution and easily decomposes, which brings great difficulties in long-term treatment. One of the methods to give full play to the functions of ICG while overcoming these shortcomings is to prepare near-infrared composite hydrogels. The good biocompatibility and drug encapsulation rate of nanohydrogels, coupled with the photothermal therapy with controllable time and space, near-infrared nanocomposite hydrogels have created good prospects for cancer treatment. The construction of a photothermal nanocomposite hydrogel co-drug delivery system by co-loading drug-loaded nanoparticles and photothermal conversion agents into hydrogels is expected to bring out the advantages of photothermal therapy and chemotherapy and overcome their respective shortcomings, enhance the tumor treatment effect and reduce toxic side effects, and has good clinical application prospects. Summary of the invention

[0005] To solve the above problems, the present invention embeds drug-loaded nanoparticles co-loaded with tyrosine kinase inhibitors into a pH-sensitive chitosan hydrogel containing a photothermal agent, indocyanine green, to construct a novel nanocomposite hydrogel co-drug loading system. The nanocomposite hydrogel designed by the present invention can not only target tumor cells and enhance the intratumoral penetration of drugs, but also respond to the high concentration of glutathione (GSH) in the tumor microenvironment to accurately release drugs and prevent premature release of drugs in the systemic circulation. Combining hydrogel and photothermal therapy can effectively prolong the drug release time and enhance the stability of the drug. By combining chemotherapy and photothermal therapy, the advantages of the two are effectively utilized to significantly enhance the tumor treatment effect and reduce toxic side effects.

[0006] In view of the shortcomings of existing drug delivery system technology, the purpose of the present invention is to provide a pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system. The nanocomposite hydrogel can improve the loading rate and bioavailability of drugs, enhance the tumor treatment effect and reduce toxic side effects, and synergistic chemotherapy and photothermal therapy can achieve the effect of significantly inhibiting tumor growth, recurrence and metastasis. The present invention also provides a preparation method and application of the above-mentioned pH / GSH programmed responsive nanocomposite hydrogel co-drug delivery system.

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0008] A pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system is a pH-sensitive chitosan hydrogel with GSH stimulus-responsive drug-loaded nanoparticles and indocyanine green encapsulated by hyaluronic acid (HA) embedded therein; the drug-loaded nanoparticles encapsulate tyrosine kinase inhibitors, and the tyrosine kinase inhibitors include lapatinib; the pH-sensitive chitosan hydrogel is a chitosan hydrogel cross-linked with polyethylene glycol-4-paraformylbenzoic acid (PEG-CBA). The pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system is easy to be injected in situ, can be slowly degraded in vivo, and the escaped nanoparticles are used to deliver drugs to tumor cells in a targeted and synchronous manner; and the tumor site where the hydrogel is injected can generate a constant high temperature (40-60°C) by irradiation with near-infrared light (NIR: 700-1100nm), and the photothermal treatment is realized by utilizing the photothermal conversion characteristics of indocyanine green, thereby inhibiting the proliferation, invasion and migration of tumor cells, and realizing the synergistic anti-tumor effect of chemotherapy-photothermal treatment.

[0009] In the above-mentioned pH / GSH program-responsive photothermal nanocomposite hydrogel co-drug-carrying system, the hyaluronic acid-encapsulated GSH stimulus-responsive drug-carrying nanoparticles are prepared by a method comprising the following steps: cholesterol succinate monoester is grafted to the hyaluronic acid side chain modified with cystamine dihydrochloride through an amide bond to form a GSH-responsive polymer; a tyrosine kinase inhibitor is encapsulated into the nanoparticles formed by the GSH-responsive polymer by a nanoprecipitation method to obtain drug-carrying nanoparticles loaded with tyrosine kinase inhibitors, i.e., hyaluronic acid-encapsulated GSH stimulus-responsive drug-carrying nanoparticles. The molecular weight of the hyaluronic acid is preferably 5000 to 14000 Da.

[0010] In the above-mentioned pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system, the particle size of the drug-loaded nanoparticles is preferably 30-200 nm.

[0011] In the above-mentioned pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system, the viscosity of the chitosan used is preferably 100-500 mPa.s.

[0012] In the above pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system, the nanocomposite hydrogel is prepared as an injection, and the tyrosine kinase inhibitor drug concentration in the injection is at least 1 mg / mL, preferably 1-6 mg / mL.

[0013] The preparation method of the above-mentioned pH / GSH program-responsive photothermal nanocomposite hydrogel co-drug delivery system comprises the following steps: blending hyaluronic acid-encapsulated GSH stimulus-responsive drug-loaded nanoparticles and indocyanine green in an aqueous solution containing 1-3% acetic acid, and after complete dissolution, dissolving chitosan in the blended solution, adding a cross-linking agent polyethylene glycol-4-paraformylbenzoic acid (PEG-CBA) aqueous solution, and stirring to obtain a pH / GSH program-responsive photothermal nanocomposite hydrogel co-drug delivery system.

[0014] Furthermore, the preparation method of the pH / GSH program-responsive photothermal nanocomposite hydrogel co-drug delivery system comprises the following steps: synthesizing hyaluronic acid (HA-CYS) modified with cystamine dihydrochloride by amidation reaction; grafting cholesterol succinate monoester to the side chain of HA by amidation reaction to obtain a GSH-responsive polymer HA-SS-Chol, the structural formula of which is shown below; subsequently, encapsulating the tyrosine kinase inhibitor into nanoparticles by nanoprecipitation method with the tyrosine kinase inhibitor and the GSH-responsive polymer molecules to obtain drug-loaded nanoparticles loaded with the tyrosine kinase inhibitor, i.e., hyaluronic acid-encapsulated GSH stimulus-responsive drug-loaded nanoparticles; subsequently, blending the hyaluronic acid-encapsulated GSH stimulus-responsive drug-loaded nanoparticles and indocyanine green in an aqueous solution containing 1-3% acetic acid, and after complete dissolution, dissolving chitosan in the blended solution, adding a crosslinking agent aqueous solution, and stirring to obtain a pH / GSH program-responsive photothermal nanocomposite hydrogel co-drug delivery system.

[0015]

[0016] In a preferred embodiment, the preparation method comprises the following steps:

[0017] (1) Synthesis of hyaluronic acid modified with cystamine dihydrochloride (HA-CYS): Hyaluronic acid, N-hydroxysuccinimide, and carbodiimide are dissolved in water to react to activate the carboxyl group of hyaluronic acid, and then an aqueous solution of cystamine dihydrochloride is added to react. During the reaction, the pH is controlled to be 4.5-5.0 at room temperature. After the reaction is completed, the mixed solution is transferred to a dialysis bag and dialyzed in water to remove the catalyst and unreacted small molecules. The mixed solution is freeze-dried to obtain white sponge-like HA-CYS.

[0018] (2) Synthesis of GSH-responsive polymer (HA-SS-Chol): Dissolve hyaluronic acid modified with cystamine dihydrochloride, cholesterol succinate, N-hydroxysuccinimide, and carbodiimide in organic solvents respectively. Under nitrogen protection, add N-hydroxysuccinimide solution, carbodiimide solution, and hyaluronic acid solution modified with cystamine dihydrochloride dropwise to the cholesterol succinate solution to react. After the reaction is completed, the mixed solution is transferred to a dialysis bag and dialyzed in water to remove unreacted small molecules and organic reagents. The mixed solution is freeze-dried to obtain a white sponge-like HA-SS-Chol. The organic solvent is preferably a mixture of one or more of dimethyl sulfoxide, formamide, and 1,4-dioxane.

[0019] (3) Preparation of GSH stimuli-responsive drug-loaded nanoparticles encapsulated by hyaluronic acid: GSH responsive polymer and tyrosine kinase inhibitor are co-dissolved in an organic solvent, and the mixed solution is slowly dripped into water under ultrasonic conditions. Subsequently, the solution is transferred to a dialysis bag and dialyzed in water. Finally, the obtained dialysate is filtered with a filter membrane to remove the unencapsulated drug to obtain hyaluronic acid-encapsulated GSH stimuli-responsive drug-loaded nanoparticles. The organic solvent is preferably a mixture of one or more of dimethyl sulfoxide and formamide.

[0020] (4) Preparation of crosslinking agent polyethylene glycol-4-p-formylbenzoic acid (PEG-CBA): polyethylene glycol, 4-p-formylbenzoic acid, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in organic solvents respectively, and the mixed solution of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine was slowly dripped into the 4-p-formylbenzoic acid solution, and then the mixed solution was slowly dripped into the polyethylene glycol solution and stirred for reaction. After the reaction was completed, water was added to the system to terminate the reaction. The above-obtained solution was post-treated: the solution was vacuum filtered and rotary evaporated to remove unreacted small molecules and organic reagents. The product after rotary evaporation is dissolved in an organic solvent and then frozen at 2-5°C until a white solid precipitates. The solution is filtered under reduced pressure, the organic solvent is removed by rotary evaporation, and freezing is continued. The freezing-filtration-rotary evaporation process is repeated for more than 3 times until no or only a small amount of white solid precipitates. The solution is rotary evaporated to obtain a milky white solid, water is added and dissolved in a water bath. After the milky white solid is completely dissolved, it is filtered under reduced pressure, and the filtrate is filtered through a membrane to obtain a clear aqueous solution. Finally, the solution is freeze-dried to obtain a white solid PEG-CBA.

[0021] (5) Preparation of pH / GSH programmed responsive photothermal nanocomposite hydrogel drug delivery system: GSH stimulus responsive drug delivery nanoparticles encapsulated by hyaluronic acid and indocyanine green are mixed in an aqueous solution containing 1-3% acetic acid, chitosan is dissolved in the mixed solution, polyethylene glycol-4-paraformylbenzoic acid aqueous solution is added, and stirred to obtain a pH / GSH programmed responsive photothermal nanocomposite hydrogel drug delivery system.

[0022] The present invention also provides the use of the pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system in the preparation of tumor therapeutic drugs. The tumors include skin melanoma, choroidal melanoma, breast cancer, colon cancer, cervical cancer, lung cancer, sarcoma, and glioblastoma. The drug is administered by intratumoral injection or paratumoral injection.

[0023] A tumor treatment drug comprises the pH / GSH programmed responsive photothermal nanocomposite hydrogel drug delivery system.

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

[0025] 1. The pH / GSH programmed responsive nanocomposite hydrogel co-drug delivery system provided by the present invention is easy to be injected into the tumor or adjacent to the tumor, and is slowly degraded in the body, with a long-term sustained release of anti-tumor drugs, thereby achieving continuous treatment of tumors.

[0026] 2. The present invention utilizes nanoparticles to encapsulate drugs into hydrogels, significantly improving the encapsulation efficiency of hydrophobic drugs. After escaping from the gel, the nanoparticles can target tumor cells and synchronously release chemotherapeutic drugs in response to the acidic microenvironment within the tumor cells, exerting a synergistic anti-tumor effect, thereby improving the tumor treatment effect.

[0027] 3. The present invention utilizes indocyanine green as a photothermal agent and utilizes its photothermal conversion characteristics to convert light energy into thermal energy through near-infrared light irradiation to achieve the effect of photothermal therapy.

[0028] 4. The present invention confirms through animal experiments that the pH / GSH programmed responsive nanocomposite hydrogel co-drug delivery system has good anti-tumor ability. Compared with drug-loaded nanoparticles and hydrogel drug delivery systems encapsulating free drugs, the pH / GSH programmed responsive nanocomposite hydrogel co-drug delivery system can better inhibit tumor growth and significantly improve the survival rate of mice. Based on this, the present invention provides the application of the pH / GSH programmed responsive nanocomposite hydrogel co-drug delivery system in the treatment of solid tumors, providing a new idea for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a transmission electron micrograph of the drug-loaded nanoparticles prepared in Example 2.

[0030] Figure 2 This is the particle size distribution of the drug-loaded nanoparticles prepared in Example 2.

[0031] Figure 3 It is the nanocomposite hydrogel prepared in Example 3.

[0032] Figure 4 3 are scanning electron microscope images of the nanocomposite hydrogel in Example 3 at different magnifications.

[0033] Figure 5 This is the degradation curve of the nanocomposite hydrogel in Example 4 in vivo.

[0034] Figure 6 This is the curve of the change of mouse tumor volume over time in Example 5.

[0035] Figure 7 This is the curve of the change in mouse body weight over time in Example 5.

[0036] Figure 8 is the tumor recurrence rate of mice in each group after postoperative medication.

[0037] Fig. 9 is the weight of the recurrent tumor in each group of mice after postoperative medication.

[0038] Fig.10 This is the body weight change curve of mice in each group after postoperative medication. DETAILED DESCRIPTION

[0039] The following further describes the pH / GSH program-responsive nanocomposite hydrogel co-drug delivery system provided by the present invention, and its preparation method and application. It is necessary to point out that the embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. If no specific technology or conditions are specified in the embodiments, they can be carried out according to the technology or conditions described in the literature in the field, or according to the product instructions. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0040] Example 1: Synthesis of GSH-responsive polymer HA-SS-Chol:

[0041] (1) Synthesis of hyaluronic acid modified with cystamine dihydrochloride (HA-CYS): 400 mg hyaluronic acid (molecular weight less than 10 KDa), 92 mg N-hydroxysuccinimide (NHS), 154 mg carbodiimide, and 2.25 g cystamine dihydrochloride were dissolved in 15 mL, 5 mL, 10 mL, and 10 mL deionized water, respectively. The NHS aqueous solution was first added dropwise to the hyaluronic acid aqueous solution using a Pasteur pipette under magnetic stirring. After sufficient mixing, the carbodiimide aqueous solution was then added dropwise to the mixture of hyaluronic acid and N-hydroxysuccinimide. The pH was controlled to be 4.8-5.0 at room temperature. After stirring for 2 hours, the cystamine dihydrochloride aqueous solution was added dropwise and the stirring reaction was continued for 24 hours. The mixed solution was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da and dialyzed in deionized water at room temperature for 72 hours, with the deionized water replaced every 6-12 hours. The mixed solution was freeze-dried to obtain white sponge-like HA-CYS, and the product was stored in a -20°C refrigerator for future use.

[0042] (2) Synthesis of GSH-responsive polymer (HA-SS-Chol): 1 g HA-CYS was weighed and dissolved in 10 mL formamide, 0.68 g cholesterol succinate monoester (Chol*-COOH), 0.288 g carbodiimide, and 0.173 g N-hydroxysuccinimide were dissolved in 50 mL, 3 mL, and 2 mL DMSO, respectively. Under nitrogen protection, the N-hydroxysuccinimide solution was added dropwise to the Chol*-COOH solution, and the carbodiimide solution was added dropwise after sufficient stirring. After stirring for 0.5 h, the HA-CYS solution was added dropwise, and the reaction was stirred for 48 h. The mixed solution was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da and dialyzed in deionized water at room temperature for 72 h, with the deionized water replaced every 6-12 h. The mixed solution was freeze-dried to obtain a white sponge-like HA-SS-Chol, which was stored in a -20 °C refrigerator for later use.

[0043] Example 2: Construction and characterization of drug-loaded nanoparticles co-loaded with lapatinib (LAP)

[0044] Drug-loaded nanoparticles were prepared by nanoprecipitation. 50 mg HA-SS Chol and 20 mg LAP were weighed and dissolved in 5 mL DMSO. After mixing evenly, they were slowly dripped into 50 mL deionized water under ultrasonic conditions. The mixed solution was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da. It was dialyzed in deionized water at room temperature for 72 hours, and the deionized water was replaced every 6-12 hours. The obtained solution was filtered under reduced pressure twice and then filtered with a 0.45 μm water filter membrane to obtain a yellow transparent solution, that is, the drug-loaded nanoparticle (HA-SS-Chol~LAP NPs, referred to as LAP NPs, the same below) solution was obtained and stored at 4 ° C for use. The absorbance at 365 nm was measured by an ultraviolet spectrophotometer. After the LAP concentration was measured by the standard curve, the LAP content in the LAP NPs was calculated to be 14.7% according to the drug loading formula.

[0045] The shape and size distribution of the obtained drug-loaded nanoparticles were determined by transmission electron microscopy and dynamic light scattering. Figure 1 and Figure 2 As shown. Figure 1 It can be seen that the drug-loaded nanoparticles are spherical in structure. Figure 2 It can be seen that the average particle size of the drug-loaded nanoparticles is 55.7 nm.

[0046] Example 3: Preparation and structural characterization of nanocomposite hydrogel

[0047] Weigh 3.26g of polyethylene glycol and dissolve it in 25mL of tetrahydrofuran at 50℃ in a water bath, weigh 0.49g of 4-p-formylbenzoic acid and dissolve it in 10mL of tetrahydrofuran, weigh 0.84g of N,N'-dicyclohexylcarbodiimide and 0.084g of 4-dimethylaminopyridine and dissolve them in 12mL of tetrahydrofuran. First, slowly drip the mixed solution of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine into the 4-p-formylbenzoic acid solution, stir at room temperature for 0.5h, slowly drip the polyethylene glycol solution, stir at 35℃ in a water bath for 48h, and after the reaction is completed, add 1mL of deionized water to the system to terminate the reaction. The above-obtained solution was post-treated: the solution was filtered under reduced pressure, tetrahydrofuran was removed by rotary evaporation, the product after rotary evaporation was dissolved with 25 mL of dichloromethane and then placed in a 4°C refrigerator for 30 minutes. At this time, a white solid precipitated from the solution, the solution was filtered under reduced pressure, dichloromethane was removed by rotary evaporation, and freezing was continued. The freezing-filtration-rotary evaporation process was repeated more than 3 times until no or only a small amount of white solid precipitated, and a milky white solid was obtained after rotary evaporation of the solution. 120 mL of deionized water was added and dissolved in a water bath at 80°C. After the milky white solid was completely dissolved, it was filtered under reduced pressure, and the filtrate was filtered through a 0.22 μm water filter membrane to obtain a clear aqueous solution. Finally, the solution was freeze-dried to obtain a white solid polyethylene glycol-4-paraformylbenzoic acid (PEG-CBA).

[0048] Weigh 1 mg of indocyanine green and dissolve it in 1 mL of LAP NPs aqueous solution containing 2% acetic acid (lapatini concentration is 1 mg / mL). After the indocyanine green is completely dissolved, add 0.03 g of chitosan (viscosity is 100-200 mPa.s), add 50 μL of 0.5 g / mL PEG-CBA aqueous solution, and stir rapidly to form LAP NPs gel containing ICG.

[0049] Figure 3 This is the gelation effect of the nanocomposite hydrogel after adding PEG-CBA solution. Figure 4 The scanning electron microscope images of the nanocomposite hydrogel at different magnifications show that the nanocomposite hydrogel has a loose and porous morphology, and the drug-loaded nanoparticles are attached to the pore walls of the gel.

[0050] Example 4: In vivo degradation of nanocomposite hydrogels

[0051] 100 μL of nanocomposite hydrogel was placed subcutaneously in female Balb / c nude mice, and the gel was taken out at 7 days, 14 days, 21 days and 28 days, and the gel weight was weighed.

[0052] Figure 5 This is the gel weight change curve. After 28 days of subcutaneous implantation, 40.3% of the gel still remains, indicating that the gel can slowly degrade in the body to release drugs.

[0053] Example 5: Establishment and treatment of MUM-2B cell subcutaneous anti-tumor model

[0054] Establishment of MUM-2B cell subcutaneous tumor model in nude mice: 100 μL containing 2×10 6 The cell suspension of MUM-2B cells was injected subcutaneously into the right ventral back of healthy female Balb / c nude mice aged 5 to 6 weeks. 3At about 14:00 p.m., the tumor-bearing mice were randomly divided into the following 6 groups and marked with ear tags: buffer solution intratumor injection group (PBS), free drug hydrogel intratumor injection group (Free LAP gel), drug-loaded nanoparticle hydrogel intratumor injection group (LAP NPs gel), blank drug indocyanine green hydrogel intratumor injection + near-infrared light irradiation group (ICG gel+NIR), hydrogel containing indocyanine green and drug-loaded nanoparticles intratumor injection + near-infrared light irradiation group (LAP NPs gel+NIR), drug-loaded nanoparticles intravenous injection + indocyanine green hydrogel intratumor injection + near-infrared light irradiation group (LAP NPs intravenous injection + ICG gel+NIR), 6 mice in each group. Before administration, the mice were weighed, the size of the mouse tumor was measured and recorded. Each mouse was injected with 100 μL of the corresponding drug by intratumoral injection. The LAP concentration was 10 mg / kg, the ICG content was 5 mg / kg, and NIR irradiation was performed twice each time, with an interval of 5 min before and after, and the irradiation time was 1 min / time. After administration, the ICG gel+NIR group, LAP NPs gel+NIR group, and LAPNPs (intravenous injection)+ICG gel+NIR group were irradiated with NIR once every 3 days, for a total of 5 times. The day of administration was recorded as day 0. The body weight and tumor volume of the mice were measured and recorded every 3 days after administration. When the tumor volume reached 1000nm 3 The experiment was then stopped. The tumor volume calculation formula is: tumor volume V (mm 3 )=0.52×a×b 2 , where a represents the length of the tumor and b represents the width of the tumor.

[0055] Figure 6 The curve of mouse tumor changes over time shows that compared with free drugs and nanoparticles, hydrogel encapsulation of drugs can improve the tumor treatment effect. The tumor inhibition rate of the LAP NPs gel+NIR group reached 91.4%, while the tumor inhibition rate of the Free LAP gel group was only 30.4%, which is the same as the result of tumor volume change, indicating that drug delivery through nanocomposite hydrogels can enhance the anti-tumor effect.

[0056] Figure 7 Figure 2 is the body weight change curve of mice in each group. All treatment groups did not show obvious body weight fluctuation, indicating that the intratumoral injection method and dosage have good safety.

[0057] Example 6: Establishment and treatment of postoperative recurrent subcutaneous tumor model using MUM-2B cells

[0058] MUM-2B cell postoperative recurrent subcutaneous tumor model: 100 μL containing 2×10 6The cell suspension of MUM-2B cells was injected subcutaneously into the right ventral back of healthy female Balb / c nude mice aged 5 to 6 weeks to a volume of 100 mm. 3 At about 14:00 p.m., the tumor-bearing mice were randomly divided into the following 5 groups and marked with ear tags: buffer solution intratumor injection group (PBS), free drug hydrogel intratumor injection group (Free LAP gel), drug-loaded nanoparticle hydrogel intratumor injection group (LAP NPs gel), blank drug indocyanine green hydrogel intratumor injection + near-infrared light irradiation group (ICG gel+NIR), and hydrogel containing indocyanine green and drug-loaded nanoparticles intratumor injection + near-infrared light irradiation group (LAP NPs gel+NIR), 5 mice in each group. After the mice were anesthetized, 99% of the tumor tissue was removed with a scalpel, sutured, and 100 μL of the corresponding drug was injected into each mouse at the surgical site. The LAP concentration was 10 mg / kg, the ICG content was 5 mg / kg, and NIR irradiation was performed twice each time, with an irradiation time of 1 min / time. After administration, the ICG gel+NIR group and the LAP NPs gel+NIR group were irradiated with NIR once every 3 days, for a total of 5 times. The day of drug administration was recorded as day 0. The weight of mice and tumor recurrence were measured and recorded every 3 days after drug administration. When the tumor volume reached 1000 nm 3 The experiment was then stopped. The tumor volume calculation formula is: tumor volume V (mm 3 )=0.52×a×b 2 , where a represents the length of the tumor and b represents the width of the tumor.

[0059] Figure 8 The tumor recurrence rate of each group after postoperative administration of mice. After 15 days of subcutaneous establishment of the MuM-2B tumor model, the tumor was removed and sutured. NIR irradiation was performed every 3 days after surgery to observe the recurrence of the tumor and record the weight changes of the mice. It can be seen in the figure that the tumors of the PBS group, Free LAP gel group and ICG gel+NIR group recurred on the 9th, 12th and 15th days respectively, and the tumor recurrence rate of the PBS group on the 18th day reached 100%. The tumor recurred in the LAP NPs group on the 18th day and reached 80% on the 21st day. The tumor recurred in the LAP NPs gel+NIR group on the 21st day, and the recurrence rate did not increase after 21 days, maintaining at 20%.

[0060] Fig. 9is the weight of the recurrent tumor in each group after the mice were given the drug after surgery. After the mice were killed on the 28th day, the tumors were dissected and weighed for analysis. The recurrent tumors in the PBS group and the Free LAP gel group were larger, with average weights of 0.706g and 0.486g, respectively. In comparison, the average tumor weights in the LAP NPs gel group and the ICG NPs gel group were 0.184g and 0.169g, respectively. The average tumor weight in the LAP NPs gel+NIR group was the lowest, only 0.018g, significantly lower than that in the other treatment groups. The above results show that nanocomposite hydrogels can reduce the postoperative tumor recurrence rate to a certain extent, and can significantly inhibit the growth of recurrent tumors.

[0061] Fig.10 This is the weight change curve of each group of mice after surgery. It examines the changes in mouse weight during the treatment process after tumor resection. As shown in the figure, during the entire experiment, the weight of the mice did not change significantly, and the mice grew well.

[0062] The above embodiments are only used to help illustrate the present invention, and the implementation of the present invention is not limited by the above embodiments. Any modifications made by those skilled in the art without departing from the spirit and principle of the present invention without creative contribution are included in the protection scope of the present invention.

Claims

1. A pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system, characterized in that: The invention relates to a pH-sensitive chitosan hydrogel with GSH stimulation-responsive drug-loaded nanoparticles and indocyanine green encapsulated by hyaluronic acid embedded therein; the drug-loaded nanoparticles encapsulate tyrosine kinase inhibitors; and the pH-sensitive chitosan hydrogel is a chitosan hydrogel cross-linked with polyethylene glycol-4-paraformylbenzoic acid.

2. The pH / GSH programmed responsive photothermal nanocomposite hydrogel drug delivery system according to claim 1, characterized in that: The hyaluronic acid-encapsulated GSH stimuli-responsive drug-loaded nanoparticles are prepared by a method comprising the following steps: cholesterol succinate monoester is grafted to the hyaluronic acid side chain modified with cystamine dihydrochloride through an amide bond to form a GSH responsive polymer; and a tyrosine kinase inhibitor is encapsulated into the GSH responsive polymer by a nanoprecipitation method to obtain hyaluronic acid-encapsulated GSH stimuli-responsive drug-loaded nanoparticles.

3. The pH / GSH programmed responsive nanocomposite hydrogel co-drug delivery system according to claim 1, characterized in that: The tyrosine kinase inhibitors include lapatinib.

4. The pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system according to claim 1, characterized in that: The particle size of the hyaluronic acid-encapsulated GSH stimulation-responsive drug-loaded nanoparticles is 30-200 nm.

5. The method for preparing the pH / GSH programmed light-heat-responsive nanocomposite hydrogel co-drug delivery system according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing hyaluronic acid-encapsulated GSH stimuli-responsive drug-carrying nanoparticles and indocyanine green in an aqueous solution containing 1-3% acetic acid, dissolving chitosan in the mixed solution, adding a crosslinking agent polyethylene glycol-4-p-formylbenzoic acid aqueous solution, and stirring to obtain a pH / GSH programmed light-heat-responsive nanocomposite hydrogel drug-carrying system.

6. The method for preparing the pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system according to claim 5, characterized in that: The following steps are involved: The invention discloses a method for synthesizing hyaluronic acid modified with cystamine dihydrochloride by amidation reaction; grafting cholesterol succinate monoester to the side chain of hyaluronic acid by amidation reaction to obtain a GSH responsive polymer; encapsulating a tyrosine kinase inhibitor into nanoparticles formed by GSH responsive polymer molecules by nanoprecipitation method to obtain hyaluronic acid encapsulated GSH stimuli responsive drug-loaded nanoparticles; then, mixing the hyaluronic acid encapsulated GSH stimuli responsive drug-loaded nanoparticles and indocyanine green in an aqueous solution containing 1-3% acetic acid, dissolving chitosan in the mixed solution, adding a crosslinking agent aqueous solution, and stirring to obtain a pH / GSH program responsive photothermal nanocomposite hydrogel co-drug loading system.

7. The method for preparing the pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system according to claim 5, characterized in that: The following steps are involved: (1) Synthesis of hyaluronic acid modified with cystamine dihydrochloride: hyaluronic acid, N-hydroxysuccinimide and carbodiimide are dissolved in water to react to activate the carboxyl group of hyaluronic acid, and then a cystamine dihydrochloride aqueous solution is added to react to obtain hyaluronic acid modified with cystamine dihydrochloride; (2) Synthesis of GSH-responsive polymer: hyaluronic acid modified with cystamine dihydrochloride, cholesterol succinate, N-hydroxysuccinimide, and carbodiimide are dissolved in organic solvents respectively. Under nitrogen protection, the N-hydroxysuccinimide solution, carbodiimide solution, and hyaluronic acid solution modified with cystamine dihydrochloride are sequentially dropped into the cholesterol succinate solution to react, thereby obtaining a white sponge-like GSH-responsive polymer. (3) Preparation of hyaluronic acid-encapsulated GSH stimuli-responsive drug-loaded nanoparticles: GSH responsive polymer and tyrosine kinase inhibitor are co-dissolved in an organic solvent, and the mixed solution is dripped into water under ultrasonic conditions to obtain hyaluronic acid-encapsulated GSH stimuli-responsive drug-loaded nanoparticles; (4) Synthesis of polyethylene glycol-4-p-formylbenzoic acid: polyethylene glycol, 4-p-formylbenzoic acid, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are dissolved in organic solvents respectively, a mixed solution of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is dropped into a 4-p-formylbenzoic acid solution, and then the mixed solution is dropped into the polyethylene glycol solution and stirred for reaction to obtain polyethylene glycol-4-p-formylbenzoic acid; (6) Preparation of pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system: GSH stimulus responsive drug-loaded nanoparticles encapsulated by hyaluronic acid and indocyanine green are mixed in an aqueous solution containing 1-3% acetic acid, chitosan is dissolved in the mixed solution, polyethylene glycol-4-p-formylbenzoic acid aqueous solution is added, and stirred to obtain a pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system.

8. Use of the pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system according to any one of claims 1 to 4 in the preparation of tumor therapeutic drugs.

9. The use according to claim 8, characterized in that: The tumors include skin melanoma, choroidal melanoma, breast cancer, colon cancer, cervical cancer, lung cancer, sarcoma, and glioblastoma.

10. A tumor treatment drug, characterized in that: A pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system comprising the pH / GSH programmed responsive photothermal nanocomposite hydrogel co-drug delivery system according to any one of claims 1 to 4.

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