Injectable photo-thermal response type liposome hydrogel and preparation method thereof

Abstract

The invention belongs to the field of medicine, and relates to injectable photo-thermal response type liposome hydrogel and a preparation method thereof, the injectable photo-thermal response type liposome hydrogel comprises injectable hydrogel, IR820 and drug-containing thermosensitive liposome, and the IR820 and the drug-containing thermosensitive liposome are co-loaded on the injectable hydrogel; the drug-containing thermosensitive liposome comprises a thermosensitive liposome Lip and a pharmaceutical composition co-loaded on the thermosensitive liposome Lip, the pharmaceutical composition is prepared from bufalin Bu and apatinib Ap. The injectable photo-thermal response type liposome hydrogel for treating colon cancer and the preparation method provided by the invention have the advantages of high precision, strong persistence and good stability.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a hydrogel-type drug, particularly to an injectable photothermal responsive liposome hydrogel and its preparation method. Background Technology

[0002] Malignant tumors are among the most serious threats to human life and health, and cancer prevention and treatment have become a major issue urgently needing to be addressed in the global medical field. Globally, colorectal cancer ranks third in incidence and second in mortality among malignant tumors. Clinically, laparoscopic surgery combined with adjuvant chemotherapy (postoperative systemic intravenous chemotherapy and intraoperative intraperitoneal chemotherapy) is a common treatment for colorectal cancer patients. Studies show that approximately 30% to 50% of colorectal cancer patients experience tumor recurrence after treatment, with a recurrence rate exceeding 80% within the first three years post-surgery. Although intraoperative intraperitoneal chemotherapy can effectively reduce the postoperative recurrence rate of colorectal cancer, the drug dosage is difficult to control, and excessively high local concentrations and uneven distribution can easily cause acute gastrointestinal reactions such as nausea, vomiting, abdominal pain, and abdominal distension, as well as adverse reactions such as peritonitis, anastomotic leakage, intestinal obstruction, and malignant ascites, severely impacting patients' quality of life. Furthermore, chemotherapy drugs are prone to developing resistance. In addition, due to individual differences, some patients, such as the elderly, frail, or those with allergies, cannot tolerate intraoperative chemotherapy and can only undergo subsequent systemic intravenous chemotherapy. Therefore, researching novel local drug delivery systems for use after colon cancer resection and developing new medication regimens are of great significance for preventing postoperative recurrence of colon cancer, improving patients' quality of life, and prolonging their survival.

[0003] Compared with chemotherapy drugs, traditional Chinese medicine (TCM) has unique advantages in treating malignant tumors. Huachansu capsules, one of the advantageous TCM products from the "Qin Medicine" group, have the effects of detoxification, swelling reduction, and pain relief, and are mainly used for mid-to-late stage tumors and chronic hepatitis B. Clinical studies have shown that Huachansu capsules combined with oxaliplatin, leucovorin calcium, and 5-fluorouracil chemotherapy regimens have significant efficacy in treating postoperative patients with stage III colon cancer, improving their quality of life and reducing adverse reactions to chemotherapy. It is an effective TCM for preventing postoperative recurrence of colon cancer. Bufalin (hereinafter referred to as Bu) is one of the main active ingredients of Huachansu capsules. It can inhibit the growth and metastasis of colon cancer through pathways such as inhibiting proliferation, inducing apoptosis and autophagy, inhibiting invasion and metastasis, reversing multidrug resistance, inhibiting tumor angiogenesis, and regulating immune responses. However, bufalin has low solubility in water. Furthermore, bufalin belongs to the bufadienolide class of compounds, with a chemical parent structure similar to digitalis glycosides. Low doses have a cardiotonic effect, while high doses can cause severe cardiotoxicity. Studies by Zhang et al. showed that intraperitoneal injection of bufalin at concentrations of 1 mg / kg, 2 mg / kg, and 10 mg / kg all resulted in cardiac glycoside poisoning symptoms, such as palpitations, rapid breathing, and convulsions. In mice injected with 1 mg / kg bufalin, inverted T waves appeared on the electrocardiogram, and the mortality rates for mice injected with 2 mg / kg and 10 mg / kg bufalin reached 80% and 100%, respectively. Clearly, the poor water solubility and strong cardiotoxicity of bufalin limit its application in the treatment of colon cancer.

[0004] Molecular targeted therapy for tumors can treat specific genes or proteins on the surface of tumor cells, thus offering advantages such as precision and high specificity. Angiogenesis plays a crucial role in the development and progression of colorectal cancer, serving not only as a prerequisite for tumor growth but also as a significant factor promoting tumor metastasis. Tumor angiogenesis is regulated by various angiogenesis-promoting and inhibitory factors; disruption of this balance activates the "angiogenesis switch," prompting the tumor to generate new blood vessels. Vascular endothelial growth factor (VEGF) is currently the most effective specific promoter of angiogenesis. Binding to the VEGF receptor (VEGFR) activates downstream pathways such as PI3K / Akt, PTEN / Akt, Akt / mTOR, NF-κB, and MAPK / ERK, inducing the proliferation, differentiation, and migration of vascular endothelial cells, thereby promoting tumor angiogenesis. Therefore, anti-tumor angiogenesis therapy targeting VEGF is an important strategy for inhibiting the development and progression of colorectal cancer. Currently, the most widely used anti-angiogenic agents targeting VEGF include monoclonal antibodies and tyrosine kinase inhibitors. Apatinib (hereinafter referred to as Ap) is a small-molecule tyrosine kinase inhibitor with independent intellectual property rights in my country. It can selectively bind to and inhibit VEGFR-2, thereby inhibiting tumor angiogenesis. Clinical studies have shown that apatinib monotherapy has shown good therapeutic effects on colorectal cancer, and the combination of camrelizumab, 5-fluorouracil, and FOLFIRI chemotherapy regimen can further improve the therapeutic effect of colorectal cancer. However, apatinib has poor water solubility and low oral bioavailability (9.24% in rats and 15.4% in dogs), and the clinical oral dose is relatively high (850 mg / day), resulting in more side effects, mainly hypertension, hand-foot syndrome, and proteinuria.

[0005] Photothermal therapy (PTT) is an emerging cancer treatment method that utilizes photothermal conversion efficiency materials (PTA) to generate sufficient heat to kill tumor cells under near-infrared (NIR) light irradiation. Compared with traditional chemotherapy, PTT has advantages such as high selectivity, low invasiveness, reusability, and no systemic side effects, and can effectively inhibit tumor recurrence and metastasis. IR820 (neoindocyanine green) is an NIR dye widely used in the diagnosis and treatment of various tumors and has good water solubility. Currently, reports indicate that IR820-mediated PTT can effectively inhibit postoperative recurrence and metastasis of colon cancer. Furthermore, IR820-mediated PTT can achieve controlled drug release through photothermal response and can be used as an imaging agent to monitor drug release. However, free IR820 has poor tumor targeting ability in cancer treatment, thus affecting the efficacy of photothermal therapy. For example, Huang et al.'s study showed that after 24 hours of tail vein injection of IR820 (1.5 mg / kg) into the Hep 1-6 tumor-bearing nude mouse model, the IR820 was mainly distributed in the liver, kidneys, and lungs, while no fluorescent signal was detected in the tumor area. Liu et al.'s study also showed that after tail vein injection of free IR820 dye into the 4T1 tumor-bearing nude mouse model, no fluorescent signal was detected in the tumor area of ​​the mice, indicating that IR820 has low tumor targeting ability.

[0006] In conclusion, although bufotoxin, apatinib, and IR820 are all effective in treating colorectal cancer, the efficacy of each drug alone is currently insufficient, and there are no reports of the combined use of the three drugs to inhibit postoperative recurrence of colorectal cancer. Summary of the Invention

[0007] To address the aforementioned technical problems in the background art, this invention provides an injectable photothermal responsive liposome hydrogel for treating colon cancer with high precision, strong persistence, and good stability, as well as a method for its preparation.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] An injectable photothermal responsive liposome hydrogel, characterized in that: the injectable photothermal responsive liposome hydrogel comprises an injectable hydrogel and IR820 and drug-containing thermosensitive liposomes co-loaded on the injectable hydrogel; the drug-containing thermosensitive liposomes comprise thermosensitive liposomes Lip and a drug composition co-loaded on the thermosensitive liposomes Lip; the drug composition is bufotalin Bu and apatinib Ap.

[0010] The mass ratio of the above-mentioned pharmaceutical composition to the thermosensitive liposome Lip is 1:10-30; preferably, the mass ratio of the pharmaceutical composition to the thermosensitive liposome Lip is 1:20.

[0011] The mass ratio of bufotalin Bu to apatinib Ap in the above drug composition is 1:500.

[0012] The aforementioned thermosensitive liposomes (LIPs) include dipalmitoylphosphatidylcholine (DPPC), monostearate phosphatidylcholine (MSPC), and DSPE-PEG. 2000 The ratio of dipalmitoylphosphatidylcholine (DPPC) to monostearate phosphatidylcholine (MSPC) is 5–15:1, and the DSPE-PEG... 2000 The ratio of dipalmitoylphosphatidylcholine (DPPC) to monostearate phosphatidylcholine (MSPC) is 1.5:1; preferably, the ratio of dipalmitoylphosphatidylcholine (DPPC) to monostearate phosphatidylcholine (MSPC) is 12.5:1.

[0013] The final concentration of IR820 mentioned above is 0.1 mg·mL. -1 ~0.2 mg·mL -1 Preferably, the final concentration of IR820 is 0.2 mg·mL. -1 .

[0014] The aforementioned injectable hydrogel is a triblock thermosensitive polyester polyethylene glycol copolymer, and the final concentration of the triblock thermosensitive polyester polyethylene glycol copolymer is 17.5%.

[0015] A method for preparing an injectable photothermal responsive liposome hydrogel, characterized in that: the method for preparing the injectable photothermal responsive liposome hydrogel includes the following steps:

[0016] 1) Drug-containing thermosensitive liposomes Bu&Ap-Lip were prepared using a thin-film dispersion method;

[0017] 2) Based on the drug-containing thermosensitive liposome Bu&Ap-Lip prepared in step 1), prepare an injectable photothermal responsive liposome hydrogel Bu&Ap-Lip@IR820 Gel.

[0018] The specific implementation method of step 1) above is as follows:

[0019] 1.1) Weigh out the thermosensitive liposomes (Lip) and place them in a globular flask. The thermosensitive liposomes (Lip) comprise dipalmitoylphosphatidylcholine (DPPC), monostearate phosphatidylcholine (MSPC), and DSPE-PEG. 2000 ;

[0020] 1.2) Subsequently, a pharmaceutical composition consisting of bufotalin Bu and apatinib Ap is added to the flask;

[0021] 1.3) Add anhydrous methanol, sonicate to dissolve completely, then transfer to a rotary evaporator and rotary evaporate under reduced pressure until a transparent and uniform lipid film is formed on the inner wall;

[0022] 1.4) Add preheated PBS solution, rotate the water and combine with sonication, then squeeze through a microporous membrane to obtain the drug-containing thermosensitive liposome Bu&Ap-Lip;

[0023] Preferably, the mass ratio of the drug composition to the thermosensitive liposomes (Lip) is 1:10-30; more preferably, the mass ratio of the drug composition to the thermosensitive liposomes (Lip) is 1:20; the mass ratio of bufotoxin (Bu) and apatinib (Ap) in the drug composition is 1:500; the ratio of dipalmitoylphosphatidylcholine (DPPC) and monostearate phosphatidylcholine (MSPC) in the thermosensitive liposomes (Lip) is 5-15:1, and the DSPE-PEG... 2000 The ratio of dipalmitoylphosphatidylcholine (DPPC) to monostearate phosphatidylcholine (MSPC) is 1.5:1; preferably, the ratio of dipalmitoylphosphatidylcholine (DPPC) to monostearate phosphatidylcholine (MSPC) is 12.5:1; preferably, the rotary evaporation temperature is 30℃~50℃, more preferably 40℃; the rotary evaporation speed is 100 rpm; preferably, the preheating temperature of the PBS solution is 60℃; preferably, the rotary hydration temperature is 45℃~65℃, more preferably 60℃; the rotary hydration time is 35 min~55 min, preferably 50 min; the rotary hydration speed is 120 rpm; preferably, the ultrasonic power is 150W, and the ultrasonic method is to work for 5 seconds, pause for 2 seconds, then work for 5 seconds again, and so on, for a total ultrasonic time of 3 min; preferably, the microporous filter membrane includes a 0.45 μm microporous filter membrane and a 0.22 μm microporous filter membrane.

[0024] The specific implementation method of step 2) above is as follows:

[0025] 2.1) Weigh out the triblock thermosensitive polyester polyethylene glycol copolymer PLGA-PEG-PLGA and place it in a test tube;

[0026] 2.2) Add the drug-containing thermosensitive liposome Bu&Ap-Lip obtained in step 1) to the test tube in step 2.1), and allow it to swell at room temperature (25°C);

[0027] 2.3) After swelling is complete, add IR820 and stir until homogeneous to obtain injectable photothermal responsive liposome hydrogel Bu&Ap-Lip@IR820 Gel;

[0028] Preferably, the final concentration of the triblock thermosensitive polyethylene glycol copolymer PLGA-PEG-PLGA is 17.5%; the molecular weight of PLGA in the triblock thermosensitive polyethylene glycol copolymer PLGA-PEG-PLGA is 1800, the molecular weight of PEG is 1500, and the mass ratio of PLGA, PEG, and PLGA is 75:25:75; the final concentration of IR820 is 0.1 mg·mL. -1 ~0.2 mg·mL -1 Preferably, the final concentration of IR820 is 0.2 mg·mL. -1 .

[0029] An injectable photothermal responsive liposome hydrogel, Bu&Ap-Lip@IR820 Gel, prepared by a method based on injectable photothermal responsive liposome hydrogel, preferably has the following phase transition temperature from sol to gel state: 34.33±0.58℃; phase transition time from sol to gel state: 33.33±1.53s; and phase transition temperature from gel to sol state: 43.67±1.53℃.

[0030] The advantages of this invention are:

[0031] This invention provides an injectable photothermal-responsive liposome hydrogel and its preparation method. The injectable photothermal-responsive liposome hydrogel includes an injectable hydrogel and IR820 and drug-loaded thermosensitive liposomes co-loaded on the injectable hydrogel. The drug-loaded thermosensitive liposomes include thermosensitive liposomes (Lip) and a drug composition co-loaded on the thermosensitive liposomes (Lip). The drug composition is bufotalin (Bu) and apatinib (Ap). This invention co-loads bufotalin and apatinib onto thermosensitive liposomes, and then co-loads the drug-loaded thermosensitive liposomes and IR820 onto a PLGA-PEG-PLGA injectable hydrogel to construct an injectable photothermal-responsive liposome hydrogel co-loaded with bufotalin / apatinib / IR820 for postoperative local treatment. This invention utilizes the multi-target anti-tumor properties of bufotoxin, the ability of apatinib to specifically target VEGFR-2 and inhibit tumor angiogenesis, and the excellent photothermal conversion efficiency of IR820 to prepare Bu&Ap-Lip@IR820 Gel. The PLGA-PEG-PLGA hydrogel is thermosensitive; at room temperature, it can be injected into the tumor resection site in a sol state and rapidly transforms into a gel state at physiological temperature, completely covering the irregular wound surface and slowly and continuously releasing the drug. Under near-infrared light irradiation, the photosensitizer IR820 can raise the temperature of the tumor site to above 45°C through photothermal conversion, exerting a photothermal therapeutic effect. Simultaneously, it can transform the gel-state hydrogel back into a sol state and control the simultaneous release of bufotoxin and apatinib by the thermosensitive liposomes, increasing the drug concentration at the lesion site and thus effectively inhibiting postoperative recurrence of colon cancer. This invention achieves a multimodal synergistic anti-tumor effect combining "active ingredients of traditional Chinese medicine + anti-angiogenesis + photothermal therapy," addressing the shortcomings of insufficient efficacy of the three drugs used alone, as well as the drawbacks of bufotoxin's poor water solubility and strong cardiotoxicity, apatinib's poor water solubility and low oral bioavailability, and IR820's weak tumor targeting ability. It provides a precise, continuous, stable, and controllable postoperative tumor treatment strategy, offering a reference for exploring and developing a local delivery platform for postoperative treatment of colon cancer. Attached Figure Description

[0032] Figure 1 It is the synergistic effect of Bu and Ap on HCT116 cells;

[0033] Figure 2 This is the result of the optimization of the preparation process of Bu&Ap-Lip;

[0034] Figure 3 These are the characterization results of the Bu&Ap-Lip prepared in this invention;

[0035] Figure 4 The photothermal conversion properties of hydrogels with different IR820 concentrations;

[0036] Figure 5 These are the characterization results of the basic physical properties of the Bu&Ap-Lip@IR820 Gel prepared in this invention;

[0037] Figure 6 These are the characterization results of the rheological properties, photothermal properties, and in vitro drug release of the Bu&Ap-Lip@IR820 Gel prepared in this invention;

[0038] Figure 7 These are the experimental results of the in vitro biosafety, cytotoxicity, and photothermal effect of the Bu&Ap-Lip@IR820 Gel prepared in this invention;

[0039] Figure 8 These are the experimental results of the Bu&Ap-Lip@IR820 Gel prepared in this invention in cell apoptosis experiments, cell migration experiments, cell invasion experiments, and HUVCE tubule formation experiments;

[0040] Figure 9 These are the experimental results of the Bu&Ap-Lip@IR820 Gel prepared in this invention in Western blot experiments;

[0041] Figure 10 These are the results of in vivo biocompatibility and retention experiments and in vivo photothermal effect experiments of the Bu&Ap-Lip@IR820 Gel prepared in this invention;

[0042] Figure 11 This is the result of the hydrogel's in vivo efficacy in inhibiting postoperative tumor recurrence;

[0043] Figure 12 The results show the effects of the Bu&Ap-Lip@IR820 Gel prepared in this invention on tumor tissue and its influence on protein expression.

[0044] Figure 13 This invention relates to the effects of Bu&Ap-Lip@IR820 Gel prepared on tumor cell apoptosis and on CD31 protein expression in tumor tissue;

[0045] Figure 14 HE staining results of major organs, including the heart, liver, spleen, lungs, and kidneys, treated with different drugs;

[0046] Figure 15 Results on the biosafety of hydrogels. Detailed Implementation

[0047] Example 1: Preparation of Bu&Ap-Lip

[0048] 1.1) Screening of the Bu & Ap synergy ratio

[0049] The MTT assay was used to detect the toxicity of Bu and Ap alone and in different ratios to HCT116 colon cancer cells. Cells were cultured at 1.0 × 10⁶ cells / year. 4 Cells were seeded per well in 96-well cell culture plates and incubated at 37°C for 12 h. Each group received drug intervention for 24 h. Afterward, the culture plates were removed, and 20 μL of MTT was added to each well. Incubation continued for 4 h, followed by the addition of 150 μL of dimethyl sulfoxide. The absorbance (A) at 490 nm was measured using a microplate reader, and cell viability was calculated. The IC50 values ​​of Bu and Ap were used as the basis for cell viability determination. 50 Based on the Chou-Talalay combination therapy theory, the synergy index (CI) was calculated using CompuSyn software to analyze the toxic effects of Bu and Ap combination therapy on HCT116 cells.

[0050] See Figure 1 Bu and Ap alone reduced the IC50 of HCT116 colon cancer cells by 24 hours. 50 The values ​​were 22.87 ± 0.68 ng·mL. -1 and 14.72±0.44μg·mL -1 ( Figure 1 A~1B, among which Figure 1 A represents the toxicity of Bu to HCT116 cells; Figure 1 B is the toxicity of Ap to HCT116 cells; B and Ap's IC50 50 Based on the value, the synergistic effect of Bu and Ap combined was screened using CompuSyn software; the results are shown in […]. Figure 1 C~1D (where, Figure 1 C is the synergy index of different proportions of Bu & Ap; Figure 1 D is the validation of the synergistic effect of Bu and Ap. When the mass ratio (ng / μg) is 2:1, the CI value is the smallest and the synergistic effect of the drugs is the strongest.

[0051] 1.2) Preparation process of Bu&Ap-Lip

[0052] Bu&Ap-Lip was prepared using a thin-film dispersion method. Dipalmitoylphosphatidylcholine (DPPC), monostearate phosphatidylcholine (MSPC), and DSPE-PEG were accurately weighed in a mass ratio of 25:2:3:0.003:1.5. 2000Bu and Ap were mixed and placed in a round-bottom flask, then 1 mL of anhydrous methanol solution was added. After thorough sonication to dissolve, the mixture was rotary evaporated under reduced pressure at 40°C and 100 rpm until a transparent and homogeneous lipid film formed on the inner wall. Subsequently, 1 mL of preheated PBS at 60°C was added, and the mixture was rotary hydrated at 60°C and 120 rpm for 50 min. The mixture was then sonicated and passed through a membrane to obtain Bu&Ap-Lip, which was sealed and stored at 4°C for later use. The preparation steps for blank liposomes (Lip) are the same as for Bu&Ap-Lip, except that Bu and Ap are not included.

[0053] 1.3) Single-factor experiments to optimize the preparation process of Bu&Ap-Lip

[0054] Using the encapsulation efficiency of Bu and Ap as evaluation indicators, single-factor experiments were conducted to investigate different membrane material ratios (DPPC:MSPC) (5:1, 7.5:1, 10:1, 12.5:1, 15:1, m / m) and DSPE-PEG. 2000 The effects of dosage (1, 2, 3, 4, 5 mg), drug-lipid ratio (1:10, 1:15, 1:20, 1:25, 1:30, m / m), rotary evaporation temperature (30, 35, 40, 45, 50℃), hydration temperature (45, 50, 55, 60, 65℃), and hydration time (35, 40, 45, 50, 55 min) on the encapsulation efficiency of Bu and Ap in Bu&Ap-Lip were investigated to optimize the preparation process of Bu&Ap-Lip.

[0055] The effect of different membrane material ratios on drug encapsulation efficiency was investigated through single-factor experiments. When the membrane material ratio reached 12.5:1, the encapsulation efficiencies of Bu and Ap were the highest, at 81.42±1.61% and 85.03±2.61%, respectively. Figure 2 A represents the effect of different membrane material ratios on drug encapsulation efficiency. This invention also investigated different DSPE-PEG ratios. 2000 The effect of dosage on drug encapsulation efficiency was shown by adding 3 mg of DSPE-PEG. 2000 The highest encapsulation efficiencies were observed for Bu and Ap, at 79.64±2.71% and 89.59±1.07%, respectively. Figure 2 B is a different DSPE-PEG 2000 The effect of dosage on drug encapsulation efficiency was investigated. Furthermore, the effect of different drug-to-lipid ratios on encapsulation efficiency was studied. Results showed that when the total drug dosage was 7.5 mg and the drug-to-lipid ratio was 1:20, the encapsulation efficiencies for Bu and Ap were the highest, at 80.96±1.71% and 85.63±1.38%, respectively. Figure 2 C represents the effect of different drug-lipid ratios on drug encapsulation efficiency. The highest encapsulation efficiency was observed for Bu and Ap at a rotary evaporation temperature of 40℃. Figure 2D represents the effect of different rotary evaporation temperatures on drug encapsulation efficiency, which were 79.10±2.25% and 79.97±1.83%, respectively. The highest encapsulation efficiencies for Bu and Ap were observed at a hydration temperature of 60℃, at 85.23±1.71% and 84.99±1.11%, respectively. Figure 2 E represents the effect of different hydration temperatures on drug encapsulation efficiency. When the hydration time was 50 min, the encapsulation efficiencies of Bu and Ap were the highest, reaching 82.13±1.09% and 81.48±1.46%, respectively. Figure 2 F represents the effect of different hydration times on drug encapsulation efficiency.

[0056] 1.4) Box-Behnken optimizes the fabrication process of Bu&Ap-Lip

[0057] Based on the results of the single-factor study, the encapsulation efficiency (Y) of Bu and Ap in liposomes was used as the response value, and the membrane material ratio (A) and DSPE-PEG were selected. 2000 Three variables—addition amount (B), drug-to-liposome ratio (C)—were used as experimental factors. Design-Expert 13.0 software was used to design the process at three levels, with the encapsulation efficiency of Bu and Ap in Bu&Ap-Lip serving as the evaluation index to further optimize the preparation process. Subsequently, three batches of drug-loaded liposomes were prepared using the optimal process, and the reliability of the process was verified by measuring the encapsulation efficiency of Bu and Ap in the liposomes.

[0058] Based on the results of single-factor experiments, this invention selected three factors that significantly affected the encapsulation efficiency of Bu and Ap in liposomes: membrane material ratio (A), DSPE-PEG2000 addition amount (B), and drug-lipid ratio (C) as independent variables. A three-factor, three-level Box-Behnken design was optimized using Design-Expert 13 software (Table 1). The regression equation is as follows: Y = 87.47 - 0.6029A - 0.0302B - 3.77C - 2.62AB - 0.9063AC - 3.75BC - 11.23A 2 -10.03B 2 -5.87C 2 The results of the analysis of variance are shown in Table 2. The model was highly significant (F = 42.49, p < 0.001), R0 2 =0.9820, the corrected coefficient of determination is 0.9589, and the lack-of-fit term is not significant (p=0.0592), verifying the effectiveness and reliability of the model. The influence of three factors on EE was described using a three-dimensional response surface, and the results are shown in [Figure number missing]. Figure 2 G(Bu&Ap-Lip medium membrane material ratio, DSPE-PEG) 2000The response surface and contour plots of the equation fitted to the overall score (Y) for the dosage and drug-to-lipid ratio are shown. Response surface analysis results indicate that the optimal conditions are a film material ratio of 12.45:1 and DSPE-PEG. 2000 The dosage was 3.06 mg, and the drug-to-lipid ratio was 1:21. Considering actual production needs, the parameters were optimized and adjusted to 12.5:1, 3 mg, and 1:20. The results are shown in Table 3. The actual encapsulation rates obtained from the three batches of validation experiments fit well with the theoretical predictions, proving that the regression model can accurately predict and optimize the preparation process of Bu&Ap-Lip, and the process is reliable in practice.

[0059] Table 1. Box-Behnken Experimental Design and Results

[0060]

[0061] Table 2. Results of ANOVA for the regression model

[0062]

[0063]

[0064] Table 3. Predicted and Actual Drug Encapsulation Rate Results

[0065]

[0066] Example 2: Characterization of Bu & Ap-Lip

[0067] 2.1) Particle size distribution and zeta potential of Bu & Ap-Lip

[0068] Bu&Ap-Lip liposomes were stained with 2% phosphotungstic acid solution for 1 min, and the microstructure of the liposomes was observed by transmission electron microscopy (TEM). The particle size, zeta potential, and PDI changes of the liposomes were measured by dynamic light scattering (DLS) using a Malvern particle size potentiometer after the liposomes were heated at 50 °C or without heating.

[0069] 2.2) Determination of encapsulation efficiency and drug loading of Bu and Ap in Bu&Ap-Lip

[0070] Take an appropriate amount of Bu & Ap-Lip and separate the drug-loaded liposomes and free drug using ultrafiltration centrifugation. Determine the content of Bu and Ap in the liposomes using HPLC, and calculate the encapsulation efficiency and drug loading of the two drugs in the liposomes.

[0071]

[0072] Among them: W t W e and W l These are the total mass of the liposomal drug, the mass of the drug encapsulated in the liposomal capsule, and the total mass of the lipids.

[0073] 2.3) Stability test

[0074] Two batches of liposomes were divided into three portions and stored at 4℃ and 25℃ in the dark for 14 days, respectively. The particle size and PDI of the liposomes were measured every two days to investigate the stability of the prepared drug-loaded liposome solution at different temperatures.

[0075] See Figure 3 Transmission electron microscopy revealed that Bu&Ap-Lip was round or elliptical, with a smooth and flat surface and good dispersion. Figure 3 A is a transmission electron microscope (TEM) image of Bu & Ap-Lip. The encapsulation efficiencies of Bu and Ap were 84.76±2.51% and 85.43±0.33%, respectively, and the drug loadings were 0.0086±0.00025% and 4.27±0.02%, respectively. The average particle size of Bu & Ap-Lip was 151.1±1.380 nm, the PDI was 0.152±0.005, and the Zeta potential was -17.9±0.058 mV. After heating at 50 °C, the average particle size of Bu & Ap-Lip changed to 228.7±0.954 nm, the PDI changed to 0.350±0.012, and the Zeta potential changed to -6.7±0.264 mV, indicating that the temperature increase caused Bu & Ap-Lip to rupture. Figure 3 B is the particle size distribution and zeta potential distribution of Bu&Ap-Lip before and after heating. Figure 3 C(Changes in particle size and PDI of Bu&Ap-Lip after two weeks of storage at different temperatures) shows that after 14 days of storage at 4℃ and 25℃, there were no significant changes in the particle size and PDI of Bu&Ap-Lip, proving its good stability.

[0076] Example 3: Preparation of Bu&Ap-Lip@IR820 Gel

[0077] 3.1) Preparation of Bu&Ap-Lip@IR820 Gel

[0078] Accurately weigh 0.875 g of PLGA-PEG-PLGA and place it in a test tube. Add 5 mL of Bu&Ap-Lip solution and allow it to swell completely at room temperature (25°C) for 48 h. After swelling, accurately add 1 mg of IR820 and stir well to obtain the Bu&Ap-Lip@IR820Gel precursor solution. Store at 4°C in the dark for later use. The preparation steps for the blank hydrogel (Gel) are the same as for Bu&Ap-Lip@IR820 Gel, except that it does not contain Bu&Ap-Lip and IR820.

[0079] 3.2) Single-factor experiments to optimize the preparation process of Bu&Ap-Lip@IR820 Gel

[0080] In this experiment, PLGA-PEG-PLGA was selected as the matrix material for the thermosensitive hydrogel, and the optimal concentration was screened by phase transition temperature and phase transition time. Bu&Ap-Lip@IR820 gels with concentrations of 10.0%, 12.5%, 15.0%, 17.5%, and 20.0% were prepared sequentially. The optimal gel matrix concentration was determined by measuring the sol-gel-sol phase transition temperature within the 25-55℃ range and the sol-gel phase transition time at 37℃ using the inversion method. Subsequently, IR820 with a concentration of 0.1 mg·mL⁻¹ was prepared sequentially. -1 0.15 mg·mL -1 0.2 mg·mL -1 The Bu&Ap-Lip@IR820 Gel, via an 808nm laser (2.5W·cm²), -2 The optimal IR820 concentration was determined by photothermal conversion experiments under irradiation (5-minute temperature rise record) and 5 "ON / OFF" cycle stability tests.

[0081] Table 4 shows that the sol-gel phase transition temperature of Bu&Ap-Lip@IR820 Gel prepared with a matrix material concentration of 17.5% was 33.67±0.58℃, the gelation time at 37℃ was 32.33±2.52s, and the gel-sol phase transition temperature was 44.67±0.58℃. Subsequently, when the temperature was further increased to 45℃, the hydrogel underwent a gel-sol phase transition and returned to the flowing sol state. Therefore, a matrix material concentration of 17.5% was chosen for subsequent experiments. Figure 4 As shown in A (photothermal conversion curves of formulations with different IR820 concentrations), with an IR820 concentration of 0.2 mg·mL⁻¹, -1 The prepared Bu&Ap-Lip@IR820Gel was subjected to 808nm (2.5W·cm) -2 The temperature can rapidly reach 50℃ after 2 minutes of laser irradiation; Figure 4 As shown in Figure B, Bu&Ap-Lip@IR820 Gel maintained a stable temperature rise to approximately 50°C after five cycles of irradiation without significant degradation. Therefore, an IR820 concentration of 0.2 mg·mL was selected. -1 Further experiments will be conducted.

[0082] Table 4. Sol-gel phase transition temperature, gelation time at 37℃, and gel-sol phase transition temperature of hydrogels.

[0083]

[0084] Example 4: Characterization of Bu&Ap-Lip@IR820 Gel

[0085] 4.1) Determination of the phase transition temperature of "sol-gel-sol"

[0086] The gel phase transition temperature of Bu&Ap-Lip@IR820 Gel was determined using the inversion method. 1 mL of the Bu&Ap-Lip@IR820 Gel precursor solution was placed at the bottom of a glass vial, which was then placed in a constant-temperature water bath. The temperature was increased by 1°C at intervals, and each temperature point was incubated for 25 min. The test temperature range was 25°C–50°C. The glass vial was repeatedly inverted, and the sample flow was observed as the temperature increased. The temperature at which the sample remained in a non-flowing state for 30 s after inversion was recorded as the sol-gel phase transition temperature of the hydrogel. Subsequently, the sample flow was observed again, and the temperature at which the sample returned to a flowing state after inversion was recorded as the gel-sol phase transition temperature.

[0087] 4.2) Determination of phase transition time

[0088] The gel phase transition time of Bu&Ap-Lip@IR820 Gel was determined using the inversion method. 1 mL of the Bu&Ap-Lip@IR820 Gel precursor solution was injected into the bottom of a glass vial, which was then placed in a 37°C water bath. The vial was inverted every 5 seconds, and the sample flow was observed. The time it took for the sample to remain in a non-flowing state for 30 seconds after inversion was recorded as the gel phase transition time.

[0089] The results showed that the sol-gel phase transition temperature of Bu&Ap-Lip@IR820 Gel, measured by the flipping method, was 34.33±0.58℃, the sol-gel phase transition time was 33.33±1.53s, and the gel-sol phase transition temperature was 43.67±1.53℃.

[0090] 4.3) Appearance and preliminary stability assessment

[0091] The gel and Bu&Ap-Lip@IR820 gel were placed at room temperature, 37℃, and 50℃ for 15 min, respectively, and the changes in their state were observed. In addition, 1 mL of Bu&Ap-Lip@IR820 gel was accurately pipetted into a glass bottle, and 3 mL each of purified water, physiological saline, pH 7.4 phosphate-buffered saline (PBS), complete culture medium, and fetal bovine serum were added. After vortexing and mixing, each sample was placed at room temperature in the dark for 7 days.

[0092] See Figure 5Both the gel and the Bu&Ap-Lip@IR820 gel exhibit good flowability at room temperature (25°C). After being placed at 37°C for 15 minutes, they rapidly transform into a non-flowing gel state. When the temperature is further increased to 50°C, they can return to a flowable sol state. Figure 5 A. Reversible sol-gel-sol transition characteristics of Bu&Ap-Lip@IR820 Gel and Bu&Ap-Lip@IR820 Gel. The entire transition process from 25℃ to 50℃ is reversible, indicating that Bu&Ap-Lip@IR820 Gel has good thermosensitive properties and that Bu&Ap-Lip and IR820 do not affect the crosslinking of the gel. Bu&Ap-Lip@IR820 Gel is uniformly dispersed and stable in different solutions, and no clumping or layering occurs after 7 days at room temperature. Figure 5 B, Preliminary stability study of Bu&Ap-Lip@IR820 Gel at room temperature (from a to e: purified water, physiological saline, PBS, complete culture medium, fetal bovine serum), demonstrates its good stability.

[0093] 4.4) Microstructure and injectability investigation

[0094] The microstructure of the cross-section of Bu&Ap-Lip@IR820 Gel was observed using scanning electron microscopy. Plastic culture dishes were placed in a 37°C water bath. Using a 1mL syringe, the letters "ABC" were written in the plastic culture dish along with the gel and the Bu&Ap-Lip@IR820 Gel precursor solution, or the solution was injected into pure water at 37°C to assess the injectability of the hydrogel.

[0095] Scanning electron microscopy results show that the Bu&Ap-Lip@IR820 Gel exhibits a loose and uniform porous structure with numerous interconnected pores. Figure 5 C, SEM images of Bu & Ap-Lip@IR820 Gel). Figure 5 D (Photos of Gel and Bu&Ap-Lip@IR820 Gel injected into 37°C water and the letters “ABC” written) show that Gel and Bu&Ap-Lip@IR820 Gel have good injectability, and the injectability is not affected by the addition of Bu&Ap-Lip and IR820.

[0096] 4.5) Rheological property evaluation

[0097] The rheological properties of the gel and the Bu&Ap-Lip@IR820 gel were tested using a Haake Mars III rheometer. The tests were conducted at a constant strain of 1% and a constant frequency of 1 Hz, with temperatures ranging from 25°C to 55°C and a heating rate of 2°C / min. -1Temperature scanning experiments were conducted; isothermal time scanning experiments were performed for 60 s under constant temperature of 37℃, constant frequency of 1 Hz, and constant strain of 1%; and frequency scanning experiments were conducted with frequencies ranging from large to small from 200 rad·s under constant temperature of 37℃ and constant strain of 1%. -1 ~0.1 rad·s -1 Frequency scanning experiment; strain scanning experiment with strain range from small to large of 0.01% to 100% under constant temperature of 37℃ and constant frequency of 1Hz; cyclic strain scanning experiment with a large strain (500%) applied in the first 120s and then a small strain (1%) applied under constant temperature of 37℃ and constant frequency of 1Hz, repeated five times.

[0098] See Figure 6 Temperature scanning experiments showed that both the storage modulus (G′) and loss modulus (G″) of Gel and Bu&Ap-Lip@IR820 Gel underwent a sol-gel transition at 33℃, and a reversible gel-sol transition occurred with increasing temperature. Figure 6 A- Figure 6 B, of which Figure 6 A is the temperature scan experiment of gel. Figure 6 B is a temperature scan experiment of Bu&Ap-Lip@IR820 Gel). At 37°C, Bu&Ap-Lip@IR820 Gel can complete gelation within 34 seconds. Figure 6 C, isothermal time-scan experiment of Bu & Ap-Lip@IR820 Gel). In the frequency scan experiment, G′ was always higher than G″ and there was no intersection. Figure 6 Frequency scanning experiments of Bu&Ap-Lip@IR820Gel (D) demonstrate its good injectability. A wide linear viscoelastic region was observed within the strain range of 0.01% to 100%, indicating that Bu&Ap-Lip@IR820 Gel can maintain a gel state over a broad strain range. Figure 6 E. Strain scanning experiments of Bu&Ap-Lip@IR820 Gel). The self-healing properties of Bu&Ap-Lip@IR820 Gel were investigated by cyclic strain scanning experiments. The hydrogel maintained a gel state at small strains (G'>G”), and transformed into fluid-like behavior at large strains (G”>G'). After multiple damage repairs, the G' value of the hydrogel did not decrease, which proves that Bu&Ap-Lip@IR820 Gel has good self-healing properties. Figure 6 F, Cyclic strain scanning experiment of Bu&Ap-Lip@IR820 Gel).

[0099] 4.6) Photothermal performance evaluation

[0100] After gelling 1 mL of pure water, Bu&Ap-Lip Gel, IR820 Gel, and Bu&Ap-Lip@IR820 Gel at 37 °C, the gel was then subjected to an 808 nm laser (2.5 W·cm⁻¹). -2 Irradiation was performed for 5 minutes, and images were acquired and the temperature of the hydrogel was recorded every 0.5 minutes using a FLUKE infrared thermal imager, and a temperature rise curve was plotted. The Bu&Ap-Lip@IR820 Gel was subjected to 5 laser "ON / OFF" cycles (1 minute irradiation / cooling to 37°C), and the temperature was recorded every 0.1 minutes, and a cycle curve was plotted to test its photothermal stability.

[0101] Real-time images of different hydrogels under laser irradiation, such as Figure 6 G(Gel, Bu&Ap-Lip Gel, IR820 Gel and Bu&Ap-Lip@IR820 Gel use 808nm(2.5W·cm -2 The photothermal images of laser irradiation at different times are shown, indicating that the temperature change area is limited to the irradiated point of the gel. The photothermal curves of different gels are shown below. Figure 6 As shown in H(photothermal curves of Gel, Bu&Ap-Lip Gel, IR820 Gel, and Bu&Ap-Lip@IR820 Gel over time), the gel without IR820 and Bu&Ap-Lip Gel showed no significant temperature change after 5 minutes of continuous NIR irradiation. Conversely, the IR820 Gel and Bu&Ap-Lip@IR820 Gel containing IR820 showed a rapid temperature increase under NIR irradiation, exceeding 55°C. This indicates that Bu&Ap-Lip@IR820 Gel possesses excellent photothermal conversion performance. Results of multi-cycle irradiation experiments are shown in […]. Figure 6 I(Bu&Ap-Lip@IR820 Gel uses 808nm(2.5W·cm -2 (Photothermal curves after 5 cycles of laser irradiation) The maximum temperature of Bu&Ap-Lip@IR820 Gel did not decrease under multiple cycles of irradiation, indicating that Bu&Ap-Lip@IR820 Gel has good photothermal stability.

[0102] 4.7) Drug release in vitro

[0103] The drug release characteristics of Bu&Ap-Lip@IR820 Gel under near-infrared light (808 nm) irradiation were investigated. Two groups were selected: Group I, Bu&Ap-Lip@IR820 Gel, and Group II, Bu&Ap-Lip@IR820 Gel + L. At room temperature, 1 mL of the Bu&Ap-Lip@IR820 Gel precursor solution was injected into a 5 mL amber glass bottle, which was then placed in a 37°C water bath. After gel formation, 1 mL of release medium (PBS containing 0.5% Tween 80) was injected, and the bottle was slowly shaken in a 37°C water bath. In Group II, the release was measured at 1 and 2 hours using an 808 nm laser (2.5 W·cm²). -2 Irradiate for 1 min, and collect all release media from two groups at pre-set times (0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24 h), while immediately adding 1 mL of isothermal and equal volume of fresh release media. Dilute and filter the collected release media, and determine the Ap content using HPLC.

[0104] In vitro release results showed that, without laser irradiation, the drug was released stably and slowly from the hydrogel without significant burst release. This is likely due to the retention effect of the liposome-hydrogel composite system on drug molecules. At 48 hours, 57.44% of apatinib was released from the hydrogel. In contrast, under two laser irradiations (1 hour and 2 hours), drug release significantly increased, with a cumulative release of 81.22% of the drug from the hydrogel within 48 hours. Figure 6 J, Bu & Ap-Lip@IR820Gel (cumulative drug release curves with and without laser irradiation). These results indicate that the temperature rise caused by laser irradiation can accelerate drug movement in the liposome-hydrogel system and promote the diffusion of drug molecules out of the gel.

[0105] 4.8) Investigation of swelling properties

[0106] After hydrogel is implanted in the body, the swelling stress it generates may have adverse effects on surrounding tissues. Therefore, the swelling properties of Bu&Ap-Lip@IR820 Gel were investigated using a gravimetric method. 1 mL of each of the gel, Bu&Ap-Lip Gel, and Bu&Ap-Lip@IR820 Gel was placed in a test tube, and 10 times the volume of water was added. After 24 hours, the water in the test tubes was discarded, and the contents were weighed and recorded.

[0107] The weight of Bu&Ap-Lip Gel, Bu&Ap-Lip Gel, and Bu&Ap-Lip@IR820 Gel showed no significant change after immersion in water for 24 hours, demonstrating that Bu&Ap-Lip@IR820 Gel has no significant swelling effect, and the addition of Bu&Ap-Lip and IR820 does not affect the safe swelling performance of the hydrogel. Figure 5E-5F, of which Figure 5 E represents swelling images of Gel, Bu&Ap-Lip Gel, and Bu&Ap-Lip@IR820 Gel. Figure 5 F represents the weight changes of Gel, Bu&Ap-Lip Gel, and Bu&Ap-Lip@IR820 Gel before and after 24 hours. These results indicate the potential of the obtained hydrogel for in vivo implantation.

[0108] Example 5: In vitro antitumor effect of Bu&Ap-Lip@IR820 Gel

[0109] 5.1) In vitro biocompatibility

[0110] The MTT assay was used to detect the toxicity of Gel and Lip@IR820 Gel to NCM460 cells: After the hydrogel was cured at 37℃, it was extracted with culture medium for 24 h, filtered to remove bacteria, and diluted to different concentrations (0%, 25%, 50%, 75%, 100%). NCM460 cells were then incubated at 1.0 × 10⁻⁶ cells / mL. 4 Red blood cells were seeded at a density of 1 cell / well in 96-well plates. After incubation for 12 hours, different concentrations of hydrogel extract were added, and the viability was measured after 24 hours of incubation. Blood compatibility was evaluated using an in vitro hemolysis test: 5% red blood cell suspension was incubated with different concentrations of hydrogel extract at 37°C for 4 hours, centrifuged, and the absorbance at 545 nm was measured. The hemolysis rate was calculated using water (positive) and PBS (negative) controls.

[0111] NCM460 cells incubated with hydrogel extracts containing high concentrations (100%) of Gel and Lip@IR820 Gel showed a survival rate greater than 90%, demonstrating good cell compatibility. Figure 7 A. Survival rate of NCM460 cells treated with different concentrations of hydrogel extract. This demonstrates that the prepared hydrogel and nanoparticles are safe and non-toxic. Blood compatibility results. Figure 7 As shown in B (hemolysis rate of hydrogels), compared to the hemolysis rate of Water (100%), the hemolysis rates of Gel, Lip Gel, and Lip@IR820 are all less than 5.0%, indicating that they have no significant hemolytic effect on blood cells. Therefore, it is demonstrated that the prepared hydrogels and nanoparticles have good blood compatibility.

[0112] 5.2) In vitro photothermal effect

[0113] Based on HCT116 colon cancer cells, the in vitro photothermal effect of Lip@IR820 Gel on HCT116 cells was further evaluated using a live-dead cell staining assay. The experiment was divided into six groups: I. Control group; II. Laser irradiation group (L); III. Blank liposome hydrogel laser irradiation group (Lip Gel+L); IV. Free IR820 laser irradiation group (IR820+L); V. Blank liposome IR820 hydrogel group (Lip@IR820 Gel); VI. Blank liposome IR820 hydrogel laser irradiation group (Lip@IR820 Gel+L). HCT116 colon cancer cells were inoculated at 1.0 × 10⁶ cells per cell line. 5 The hydrogel was seeded at a density of cells / well into 24-well plates and incubated for 12 h. The hydrogel was injected into Transwell chambers, and after gelation at 37°C, the chambers were placed into 24-well plates. After 10 min, groups II, III, IV, and VI were treated with an 808 nm NIR laser (2.5 W·cm⁻¹). -2 The hydrogel area was irradiated for 5 min. Then, staining was performed using the Calcein / PI cell viability and cytotoxicity assay kit, and images were captured using a Nikon inverted fluorescence microscope with excitation wavelengths of 470 nm and 525 nm.

[0114] The in vitro photothermal effect of IR820 was investigated using live and dead staining experiments, and the results are as follows: Figure 7 As shown in image D (staining results of live and dead HCT116 cells after different drug treatments), the central and peripheral regions of the bottom of the cell culture plates in the blank, L, Lip Gel+L, IR820+L, and Lip@IR820 Gel groups all exhibited strong green fluorescence, indicating high cell viability. The IR820+L group showed no significant red fluorescence, indicating a weaker tumor-killing effect. This may be because IR820 rapidly diffuses across the membrane from the upper Transwell chamber to the lower chamber, resulting in a lower IR820 concentration in the laser-irradiated area and a weakened photothermal effect. The Lip@IR820 Gel+L group showed strong red fluorescence in the central region of the cell culture plate near the upper Transwell chamber, and green fluorescence in the peripheral region, indicating a stronger tumor cell-killing effect in the central region. These results suggest that Lip@IR820 Gel can exert a good photothermal effect under laser irradiation. The hydrogel can extend the local retention time of IR820 to a limited extent, maintaining a high concentration of IR820 in the local area and thus exerting a good photothermal effect.

[0115] 5.3) In vitro cytotoxicity

[0116] The MTT assay was used to determine the cytotoxic effects of different drugs on HCT116 cells. Cells were cultured at 1.0 × 10⁶ cells / year. 4The liposomes were seeded at a density of 100 cells / well in 96-well plates and incubated for 12 hours. 100 μL of drug-loaded culture medium was added to each of the following groups: I. Control group; II. Laser-irradiated group (L); III. Control group (Lip Gel); IV. Control group (Lip@IR820 Gel+L); V. Drug-loaded liposomes (Bu&Ap-Lip@IR820 Gel); VI. Drug-loaded liposomes (Bu&Ap-Lip@IR820 Gel+L). Groups II, IV, and VI used an 808 nm NIR laser (2.5 W·cm²). -2 Irradiate the hydrogel region for 5 min, and continue incubation for 24 h. Then add 20 μL of MTT solution. After incubation for 4 h, remove the culture medium and add 150 μL of dimethyl sulfoxide to each well. Measure the absorbance of each well at 490 nm using a microplate reader and calculate the cell viability.

[0117] The in vitro antitumor effect of Bu&Ap-Lip@IR820 Gel was investigated using the MTT assay, and the results are as follows: Figure 7 As shown in C (Effect of different drugs on HCT116 cell survival), the Lip@IR820 Gel+L group showed higher cell survival, exhibiting a weaker cytotoxic effect. This may be because the aqueous environment of the culture medium affects temperature propagation compared to the in vitro photothermal effect. Compared with the control group (100%), the tumor cell survival rates of both the Bu&Ap-Lip@IR820 Gel and Bu&Ap-Lip@IR820 Gel+L groups were significantly reduced (p<0.01), exhibiting a stronger cytotoxic effect. Compared with the Bu&Ap-Lip@IR820 Gel group, the tumor cell survival rate of the Bu&Ap-Lip@IR820 Gel+L group was further reduced (p<0.01), exhibiting an even stronger cytotoxic effect. This is because the local high heat energy caused by laser irradiation promotes the release of more Bu and Ap from Bu&Ap-Lip@IR820 Gel, thereby inducing more tumor cell death.

[0118] 5.4) Cell migration experiment

[0119] HCT116 cells were fed at a concentration of 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 1 cell / well in 6-well plates and incubated in a cell culture incubator. When the cell confluence reached 90%, the cell layer was rapidly and gently scratched with a 10 μL sterile pipette tip. The culture medium was discarded, and the cells were washed three times with PBS to remove floating cells and cell debris. The scratch width was observed and recorded under an inverted microscope, and this time point was recorded as 0 h. The experimental groups were the same as in 5.3. Groups II, IV, and VI used an 808 nm NIR laser (2.5 W·cm²). -2After irradiating the hydrogel area for 5 minutes and incubating for 24 hours, the scratch conditions of each group were photographed and recorded using an inverted microscope. The scratch area of ​​each group was statistically analyzed using ImageJ 1.52 software, and the differences in cell scratch healing among the groups were calculated and compared.

[0120] The effects of different drugs on the migration ability of tumor cells were evaluated using a scratch healing assay. The results are as follows: Figure 8 B (Anti-migration effects of different drugs on HCT116 cells (scale bar: 500 μm)) and Figure 8 F (semi-quantitative results of cell migration) shows that cell migration was not inhibited in the L, Lip Gel, and Lip@IR820 Gel+L groups. Compared with the control group, the relative cell migration rates of the Bu&Ap-Lip@IR820 Gel and Bu&Ap-Lip@IR820 Gel+L groups were significantly reduced (p<0.01), showing enhanced tumor cell migration inhibition. Compared with the Bu&Ap-Lip@IR820 Gel group, the relative cell migration rate of the Bu&Ap-Lip@IR820 Gel+L group was further reduced (p<0.01), showing a stronger tumor cell migration inhibition effect.

[0121] 5.5) Cell invasion assay

[0122] Dilute the matrix gel to 1 mg / mL on ice with serum-free medium. -1 Add 60 μL / well to the upper chamber of a Transwell and incubate at 37°C for 1 h to allow solidification. Then add 100 μL of serum-free medium for 30 min to hydrate. Adjust the density of HCT116 cells cultured for 24 h in serum-free medium to 1.5 × 10⁻⁶. 5 Cells per mL were seeded, with 100 μL of cell suspension seeded into the upper chamber of a Transwell apparatus. Experimental groupings were the same as in 5.3. 1 mL of hydrogel extraction buffer was added to the lower chamber, and the cells were cultured for another 24 h. The culture medium from the upper chamber was then removed, and cells and matrix gel were wiped off with sterile cotton swabs. Cells were stained with 0.1% crystal violet for 30 min, washed three times with PBS, and dried. Cells that had transmembrane were randomly observed under a microscope, and quantitative analysis was performed using ImageJ 1.52.

[0123] The effects of different drugs on the invasive ability of tumor cells were evaluated using Transwell assays, and the results are as follows: Figure 8 C (the anti-invasion effect of different drugs on HCT116 cells (scale bar: 100 μm)) and Figure 8G (semi-quantitative statistical results of cell invasion) is shown. Compared with the control group, the number of invasive cells in the Bu&Ap-Lip@IR820 Gel and Bu&Ap-Lip@IR820 Gel+L groups was significantly reduced (p<0.01), showing enhanced inhibitory effects on tumor cell invasion. Compared with the Bu&Ap-Lip@IR820 Gel group, the number of invasive cells in the Bu&Ap-Lip@IR820 Gel+L group was further reduced (p<0.01), showing even stronger inhibitory effects on tumor cell invasion.

[0124] 5.6) Apoptosis experiment

[0125] The pro-apoptotic properties of Bu&Ap-Lip@IR820 Gel were investigated by flow cytometry. HCT116 cells were cultured at 5.0 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 1 cell / well in 6-well plates and incubated for 12 hours. Experimental groups were the same as in 5.3. The apoptosis detection kit was followed according to the instructions, and apoptosis was detected using flow cytometry (EPICS XL, COULTER, USA) and analyzed using the EXPO32ADC Analysis system.

[0126] The results are as follows Figure 8 A (the effect of different drugs on the apoptosis rate of HCT116 cells) and Figure 8 E (Statistical results of quantitative analysis of apoptosis) shows that, compared with the control group, the apoptosis rates of Bu&Ap-Lip@IR820 Gel and Bu&Ap-Lip@IR820 Gel+L groups were significantly increased (p<0.01), demonstrating enhanced pro-apoptotic ability of HCT116 cells. Compared with the Bu&Ap-Lip@IR820 Gel group, the apoptosis rate of Bu&Ap-Lip@IR820 Gel+L group was further increased (p<0.01), demonstrating stronger pro-apoptotic ability.

[0127] 5.7) Tube Formation Experiment

[0128] After thawing Matrigel at 4°C, it was diluted with pre-chilled DMEM basal medium (DMEM: Matrigel = 1:1). 50 μL of the Matrigel dilution was added to each well of a 96-well plate, which was then incubated at 37°C for 1 hour to solidify. The HUVEC cell density was adjusted to 6 × 10⁶ cells / well. 5 50 μL of each sample was inoculated into a 96-well plate at a density of 1 / mL. Experimental groups were the same as in 2.9.3. After drug administration, the plate was incubated for 4 h. Fields of view were selected using an inverted microscope for observation and recording. Finally, quantitative analysis was performed using the ImageJ plugin Angiogenesis Analyzer.

[0129] The results are as follows Figure 8 D (Effects of different drugs on HUVEC angiogenesis (scale bar: 100 μm)) and Figure 8 The results (Hsemi-quantitative statistical results of HUVEC angiogenesis) are shown. Compared with the control group, the number of tubular branching points in the Bu&Ap-Lip@IR820 Gel and Bu&Ap-Lip@IR820Gel+L groups was significantly reduced (p<0.01), demonstrating enhanced tumor angiogenesis inhibition. Compared with the Bu&Ap-Lip@IR820 Gel group, the number of tubular branching points in the Bu&Ap-Lip@IR820 Gel+L group was further reduced (p<0.01), demonstrating even stronger tumor angiogenesis inhibition.

[0130] 5.8) Western blot

[0131] HCT116 cells treated with the drug were washed with PBS and proteins were extracted with 100 μL of pre-chilled protein lysis buffer (RIP). GAPDH was used as an internal control protein. After protein separation by 10% SDS-PAGE gel electrophoresis, the samples were transferred to PVDF membranes, blocked, and incubated overnight at 4°C with Ki-67, Cleaved Caspase-3, MMP-2, VEGF, and CD31. Secondary antibody was added, and the cells were incubated at room temperature on a shaker for 2 h. After incubation, developing solution was added at room temperature, and the cells were incubated in the dark for 3 min. The cells were then exposed and visualized using a high-sensitivity chemiluminescence imaging system. Image J 1.52 software was used to statistically analyze the grayscale values ​​of the protein bands and compare the differences in protein expression among the groups.

[0132] The results are as follows Figure 9 A (Effects of different drugs on Ki-67, Cleaved Caspase-3, MMP-2, VEGF, and CD31 proteins in HCT116 cells) and Figure 9B (Semi-quantitative statistical results of protein expression, note: Ⅰ. Control; Ⅱ. L; Ⅲ. Lip Gel; Ⅳ. Lip@IR820 Gel+L; Ⅴ. Bu&Ap-Lip@IR820 Gel; Ⅵ. Bu&Ap-Lip@IR820 Gel+L) shows that, compared with the control group, the expression levels of Ki-67, Cleaved Caspase-3, MMP-2, VEGF, and CD31 proteins in HCT116 cells of the L, Lip Gel, and Lip@IR820 Gel+L groups were not significantly different. However, the expression level of Cleaved Caspase-3 protein in HCT116 cells of the Bu&Ap-Lip@IR820 Gel and Bu&Ap-Lip@IR820 Gel+L groups was significantly increased (p<0.01), while the expression levels of Ki-67, MMP-2, VEGF, and CD31 proteins were significantly decreased (p<0.01). Compared with the Bu&Ap-Lip@IR820 Gel group, the expression level of Cleaved Caspase-3 protein in HCT116 cells was significantly increased (p<0.01), while the expression levels of Ki-67, MMP-2, VEGF, and CD31 proteins were significantly decreased (p<0.01). This is consistent with the results of the cytotoxicity experiment. The local hyperthermia caused by laser irradiation led to the release of more Bu and Ap proteins from Bu&Ap-Lip@IR820 Gel, which promoted the expression of Cleaved Caspase-3 in HCT116 cells and inhibited the expression of Ki-67, MMP-2, VEGF, and CD31, thereby exerting a more efficient anti-tumor effect.

[0133] Example 6: In vivo animal experiments

[0134] 6.1) Biocompatibility and retention in vivo

[0135] Based on the BALB / c nude mouse model, 100 μL of Bu&Ap-Lip@IR820 Gel precursor solution was implanted subcutaneously into the back of BALB / c nude mice. The general condition of the mice (including activity, swelling, redness, and other clinical signs) and mortality were observed. At predetermined time points (1 week, 2 weeks, 3 weeks, and 4 weeks), tissues around the injection site were carefully removed for histopathological examination using HE staining to assess the in vivo biocompatibility of Bu&Ap-Lip@IR820 Gel. The in vivo retention of Bu&Ap-Lip@IR820 Gel in nude mice was investigated using an IVIS in vivo imaging system. The hydrophobic fluorescent dye DiD was selected instead of Bu and Ap to prepare drug-loaded liposome hydrogels (DiD-Lip@IR820 Gel) for in vivo experiments. BALB / c nude mice were randomly divided into three groups: I. DiD (DiD: 1.5 mg / mL)-1 Group II. DiD-Lip@IR820 Gel (IR820: 0.2 mg·mL⁻¹) (100 μL) -1 In the III group (100 μL), and the DiD-Lip@IR820 Gel+L group (100 μL), different drugs were implanted subcutaneously into the back of BALB / c mice. In the III group, an 808 nm laser (2.5 W·cm²) was used on days 1 and 2 post-implantation. -2 Irradiation for 1 min. DiD fluorescence images were acquired at specified time points (1d, 7d, 14d, 21d, 28d) using the IVIS in vivo imaging system with excitation and emission wavelengths of 646nm and 663nm, respectively, and quantitative analysis of fluorescence intensity was performed using Clinx IVScope EQ Capture software.

[0136] The biocompatibility of Bu&Ap-Lip@IR820 Gel was investigated by subcutaneous injection into nude mice. Results showed that, during the observation period, the skin around the injection site appeared normal without swelling or redness, as observed visually. Histopathological examination results using HE staining are as follows: Figure 10 As shown in Figure A (HE staining results of subcutaneous tissue at different time points (scale bar: 100μm, 100×)), no obvious pathological changes were observed around the injection site at 1, 2, 3, and 4 weeks post-injection. No fibrous capsules or tissue hyperplasia were found caused by hydrogel implantation; only mild inflammatory cell infiltration was observed, which resolved over time. This indicates that the Bu&Ap-Lip@IR820 Gel exhibits mild inflammatory response and good biocompatibility. The drug release performance of the hydrogel in vivo was monitored using an IVIS system, and the results are as follows: Figure 10 As shown in B (fluorescence images of the backs of nude mice injected with different formulations recorded using an IVIS system with DiD as the fluorescent label), fluorescence signals were detectable in the DiD group on day 1, but disappeared on day 7, indicating that free DiD readily diffuses or degrades rapidly in vivo. Compared to day 1, the fluorescence intensity of the DiD-Lip@IR820 Gel group continued to decrease on days 7, 14, 21, and 28. Figure 10 (C, Semi-quantitative analysis of fluorescence intensity) but fluorescence signals were detected throughout the monitoring period, indicating that the liposome hydrogel can slowly release the loaded drug, maintaining a stable and long-term drug concentration in vivo for sustained treatment. Compared with the DiD-Lip@IR820 Gel group, the DiD-Lip@IR820 Gel+L group showed significantly reduced fluorescence intensity on days 7, 14, 21, and 28 after two NIR laser irradiations, indicating that the prepared hydrogel formulation can intelligently regulate the release of the loaded therapeutic agent by "turning the NIR laser on / off".

[0137] 6.2) In vivo photothermal effect

[0138] Based on a BALB / c nude mouse subcutaneous colon cancer resection model, this study investigated the temperature rise at the injection site of Bu&Ap-Lip@IR820 Gel under NIR laser irradiation. The experiment was divided into four groups: I. Saline group (100 μL); II. Lip Gel group (100 μL); III. Lip@IR820 Gel group (IR820: 0.2 mg / mL). -1 , 100μL); IV.Bu&Ap-Lip@IR820 Gel group (Bu: 0.003mg·mL -1 Ap: 1.5 mg / mL -1 100 μL). The sample was injected into the tumor resection site of BALB / c nude mice after surgery, and 808 nm (2.5 W·cm) was used in each group. -2 The sample was irradiated with an NIR laser for 5 minutes, and images were acquired using a FLUKE infrared thermal imager, with the temperature of the implantation site recorded every 0.5 minutes.

[0139] In vivo photothermal experiments such as Figure 10 D-10E (of which, Figure 10 D is a thermal image of different drugs after subcutaneous injection followed by continuous irradiation with an 808nm laser for 5 minutes. Figure 10 E represents the temperature change over time after subcutaneous injection of different drugs. As shown in the figure, the saline group and the Lip Gel group were at 808 nm (2.5 W·cm⁻¹). -2 Under NIR laser irradiation, the temperature at the injection site remained around 37℃ with no significant change. However, the Lip@IR820 Gel and Bu&Ap-Lip@IR820 Gel+L groups showed no significant change at 808nm (2.5W·cm⁻¹). -2 Under NIR laser irradiation, the temperature at the injection site rapidly reached approximately 52°C within 1 minute of irradiation, and then remained at approximately 52°C, which was sufficient to kill tumor cells and promote the release of the loaded drug. These results indicate that Bu&Ap-Lip@IR820 Gel possesses good and stable in vivo photothermal properties.

[0140] 6.3) In vivo anti-tumor recurrence effect

[0141] 6.3.1) Animal grouping and administration

[0142] 100 μL of HCT116-Luc cells were added at a concentration of 2 × 10⁻⁶. 7 100 cells / mL were injected into the right axilla of BALB / c nude mice, which were then fed normally. Seven days after inoculation, when the tumor volume reached approximately 100 mm², 3Nude mice were anesthetized with isoflurane, and approximately 95% of the visible tumor tissue was removed under aseptic conditions. 5% of the tumor tissue was intentionally left around the operating table to simulate real postoperative tumor cell residue. The nude mice were randomly divided into 6 groups (n=12): I. Saline group (100 μL); II. Bu + Ap group (Bu: 0.003 mg / mL). -1 Ap: 1.5 mg / mL -1 III. Lip Gel group (100 μL); IV. Lip@IR820 Gel+L group (IR820: 0.2 mg·mL⁻¹) -1 Groups IV and VI were administered the following treatments: Group 1 (100 μL); Group 2 (100 μL): Ⅴ. Bu&Ap-Lip@IR820 Gel group; Group 3 (100 μL): Ⅴ. Bu&Ap-Lip@IR820Gel+L group; Group 4 (100 μL): Ⅴ. Bu&Ap-Lip@IR820Gel+L group. The drugs were injected directly into the residual tumor site. After gel formation, the wound was closed with 6.0 silk sutures and disinfected. The drugs were administered once daily. Groups IV and VI were irradiated with an 808 nm NIR laser (5 min each time, power density manually adjusted to reach 45°C) 1 and 2 days after administration. Viability, status, and any visible toxicity changes at the injection site were monitored throughout the experiment. The weight of each mouse was measured and recorded every 2 days, and a weight-time curve was plotted. Tumor recurrence was monitored weekly using IVIS in vivo bioluminescence imaging under isoflurane anesthesia. Recurrence rate and mortality were calculated. Local recurrence was defined as the detection of a noticeably raised subcutaneous nodule at the surgical site on the day of the experiment. After the experiment, all excised tumors were photographed and weighed, and the tumor recurrence inhibition rate was calculated. The anti-tumor recurrence experiment lasted 28 days. After the experiment, blood samples were collected from mice by enucleation of the eyeballs, and serum samples were obtained by centrifugation. Mice were humanely euthanized after anesthesia with isoflurane, and tumor tissue, as well as major organs such as the heart, liver, spleen, lungs, and kidneys, were subsequently dissected to obtain tumor tissue.

[0143] In vivo experimental design for Bu&Ap-Lip@IR820 Gel to inhibit tumor recurrence, as follows: Figure 11 As shown in step A (Hydrogel Inhibition Postoperative Recurrence Procedure), through... Figure 11 The surgery was performed as shown in section B (tumor resection and hydrogel injection procedure). IVIS results are as follows: Figure 11As shown in C (IVIS detection of postoperative tumor recurrence), tumor recurrence occurred earlier and progressed more rapidly in the saline, Bu+Ap, and Lip Gel groups, with strong fluorescence signals observed as early as day 7. In contrast, while fluorescence signals were detected in the Lip@IR820 Gel+L, Bu&Ap-Lip@IR820 Gel, and Bu&Ap-Lip@IR820 Gel+L groups, the fluorescence intensity weakened or even disappeared in the later stages of the experiment (days 14, 21, and 28), indicating that tumor development was inhibited or even eliminated. The tumor images after treatment are shown below. Figure 11 Image D (image of the tumor removed on day 28 after resection and drug administration), the tumor recurrence inhibition rate was calculated as follows: Figure 11 As shown by E (tumor recurrence inhibition rate), the tumor recurrence inhibition rates of the saline, Bu+Ap, Lip Gel, Lip@IR820 Gel+L, Bu&Ap-Lip@IR820 Gel, and Bu&Ap-Lip@IR820 Gel+L groups were 0.00%, 16.67%, 0.00%, 16.67%, 8.33%, and 66.67%, respectively. The results indicate that the Bu&Ap-Lip@IR820 Gel+L group exhibited a more efficient anti-tumor recurrence effect. The tumor resection weights for each treatment group after the experiment are shown below. Figure 11 F (tumor weight in mice after different treatments) shows that the average tumor tissue weights in the saline, Bu+Ap, Lip Gel, Lip@IR820 Gel+L, Bu&Ap-Lip@IR820 Gel, and Bu&Ap-Lip@IR820 Gel+L groups were 539.17 mg, 347.00 mg, 305.00 mg, 295.00 mg, 255.00 mg, and 87.50 mg, respectively. The results indicate that although tumor recurrence occurred in the Bu&Ap-Lip@IR820 Gel+L group, tumor growth was inhibited during the treatment period. In conclusion, the Bu&Ap-Lip@IR820 Gel+L group showed the best anti-tumor recurrence effect. This may be because the photothermal ablation and photothermal triggering of Bu and Ap release generated by Bu&Ap-Lip@IR820 Gel under NIR laser irradiation significantly enhanced its inhibitory effect on postoperative tumor recurrence and metastasis. Apart from reduced postoperative comfort, no significant weight loss was observed in any of the treatment groups throughout the entire treatment period. Figure 11 G, body weight of mice treated with different drugs.

[0144] 6.3.2) Tumor histopathological examination

[0145] Tumor tissue was collected, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and subjected to pathological examination of the tumor tissue.

[0146] HE staining results of tumor tissues in each treatment group are shown below. Figure 12 A (Hematoxylin and eosin (HE) staining of tumor tissue and immunohistochemical images of Ki-67, Cleaved Caspase-3, MMP-2, and VEGF proteins). Tumor tissues in the saline, Bu+Ap, and Lip Gel groups showed a large number of neatly arranged cells with clearly visible nuclei. The Lip@IR820 Gel+L group showed moderate tumor necrosis. The Bu&Ap-Lip@IR820 Gel group also showed moderate tumor necrosis. The Bu&Ap-Lip@IR820 Gel+L group showed severe tumor necrosis, with irregularly arranged and significantly reduced tumor cells. These results indicate that Bu&Ap-Lip@IR820 Gel exhibits a stronger killing effect on tumor tissue under NIR laser irradiation.

[0147] 6.3.3) Immunohistochemical determination of tumor tissue-related protein expression

[0148] Tumor tissues were collected, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, and subjected to antigen retrieval → blocking of endogenous peroxidase → serum blocking → primary antibody incubation → secondary antibody incubation → DAB staining → counterstaining of cell nuclei → dehydration, clearing, mounting, and observation of the expression of Ki-67, Cleaved Caspase-3, MMP-2 and VEGF under a microscope.

[0149] The immunohistochemical results of Ki-67, Cleaved Caspase-3, MMP-2 and VEGF protein expression in tumor tissues of each treatment group are shown in the figure. Figure 12A and 12B (cumulative optical density values ​​of immunohistochemistry (200×, scale bar: 50μm), Note: Ⅰ. Saline; Ⅱ. Bu+Ap; Ⅲ. Lip Gel; Ⅳ. Lip@IR820 Gel+L; Ⅴ. Bu&Ap-Lip@IR820 Gel; Ⅵ. Bu&Ap-Lip@IR820Gel+L). The results showed that, compared with the saline group, the expression levels of Ki-67, Cleaved Caspase-3, MMP-2, and VEGF proteins in tumor tissues of the Bu+Ap and Lip Gel groups were not significantly different. However, the expression of Cleaved Caspase-3 protein in tumor tissues of the Lip@IR820 Gel+L and Bu&Ap-Lip@IR820Gel groups was significantly increased (p<0.01), while the expression of Ki-67, MMP-2, and VEGF proteins was significantly decreased (p<0.01). This indicates that both Lip@IR820 Gel-mediated photothermolysis and Bu&Ap-Lip@IR820Gel can exert good anti-tumor recurrence effects. Compared with the Lip@IR820 Gel+L and Bu&Ap-Lip@IR820 Gel groups, the Bu&Ap-Lip@IR820 Gel+L group showed a significant increase in Cleaved Caspase-3 protein in tumor tissue (p<0.05) and a significant decrease in the expression of Ki-67, MMP-2, and VEGF proteins (p<0.05). This demonstrates that under laser irradiation, Bu&Ap-Lip@IR820 Gel not only exerts photothermal tumor-killing effects but also releases more drugs, thereby increasing the expression of Cleaved Caspase-3 protein and decreasing the expression of Ki-67, MMP-2, and VEGF proteins in tumor tissue, resulting in a more efficient anti-tumor recurrence effect.

[0150] 6.3.4) Detection of apoptosis in tumor tissue

[0151] Tumor tissues were collected, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, and processed according to the TUNEL cell apoptosis detection kit before being placed under a laser confocal microscope to observe the apoptosis of tumor cells.

[0152] Results of tumor tissue cell apoptosis in each treatment group are shown below. Figure 13 A~13B( Figure 13 A is an immunofluorescence image of TUNEL apoptosis and CD31 protein in tumor tissue. Figure 13B represents the TUNEL apoptosis positivity rate. Compared with the saline group, the number of apoptotic cells stained with strong green fluorescence was significantly increased in the Lip@IR820 Gel+L, Bu&Ap-Lip@IR820 Gel, and Bu&Ap-Lip@IR820 Gel+L groups (p<0.01), indicating that both Lip@IR820 Gel-mediated photothermolysis under NIR irradiation and Bu&Ap-Lip@IR820 Gel can promote tumor cell apoptosis. Compared with the Lip@IR820 Gel+L and Bu&Ap-Lip@IR820 Gel groups, the number of apoptotic cells stained with strong green fluorescence was significantly increased in the Bu&Ap-Lip@IR820 Gel+L group (p<0.05), indicating that while photothermolysis kills tumor cells, it can also promote the release of more drugs from Bu&Ap-Lip@IR820 Gel, greatly enhancing its apoptosis-inducing effect on tumor tissue cells.

[0153] 6.3.5) Immunofluorescence analysis of the expression of related proteins in tumor tissues

[0154] To assess angiogenesis in mouse tumor tissues, immunofluorescence was used to perform histological analysis of CD31 protein in collected tumors. Sections were sequentially dewaxed three times with environmentally friendly dewaxing solution for 10 minutes each time, followed by elution three times with anhydrous ethanol, washing with distilled water, antigen retrieval, serum blocking, incubation with primary antibody, incubation with secondary antibody, counterstaining with DAPI, quenching with fluorescence quencher, and mounting. Finally, the sections were examined under an upright fluorescence microscope and images were acquired.

[0155] The results of immunofluorescence of CD31 protein expression in tumor tissues of each treatment group are shown in the figure. Figure 13Compared with the saline group, the red fluorescence signals of the Bu+Ap and Lip Gel groups showed no significant changes in A and 13C (immunofluorescence integral fluorescence values), indicating that the expression level of CD31 protein in tumor tissue was not significantly changed. However, the red fluorescence of the Lip@IR820 Gel+L and Bu&Ap-Lip@IR820 Gel groups was weakened (p<0.01), indicating that the expression level of CD31 protein in tumor tissue was reduced. This proves that both Lip@IR820 Gel-mediated photothermal and Bu&Ap-Lip@IR820 Gel have certain inhibitory abilities on angiogenesis. Compared with the Lip@IR820 Gel+L and Bu&Ap-Lip@IR820 Gel groups, the red fluorescence in the tumor tissue of the Bu&Ap-Lip@IR820 Gel+L group was further weakened (p<0.01), indicating that the expression level of CD31 protein in the tumor tissue was reduced. This proves that under laser irradiation, Bu&Ap-Lip@IR820 Gel not only exerted a photothermal anti-tumor recurrence effect, but also released more drugs, thereby reducing the expression of CD31 protein in the tumor tissue and exerting a more efficient inhibitory effect on tumor angiogenesis.

[0156] 6.3.6) HE staining to observe the effects of different preparations on major organs and tissues

[0157] Major organ tissues from each group of mice were collected from dissection, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed and photographed under a microscope to observe and photograph the pathological changes of the major organ tissues.

[0158] HE staining results of major organs and tissues in each treatment group are shown below. Figure 14 (HE staining of major organs including heart, liver, spleen, lung and kidney treated with different drugs (scale bar: 50 μm)) No obvious pathological changes were found in the heart, liver, spleen, lung and kidney tissues of each treatment group, indicating that the hydrogel preparation has no obvious toxicity to major organ tissues at this drug dose.

[0159] 6.3.7) Complete blood count

[0160] Mouse blood was collected, and hematological parameters were analyzed using an automated blood analyzer, including erythrocyte system parameters: red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC); leukocyte system parameters: white blood cells (WBC), neutrophils (NEU), and lymphocytes (LYM); and platelet system parameters: red blood cell distribution width (RDW), platelets (PLT), and mean platelet volume (MPV).

[0161] Blood routine test results as follows Figure 15As shown in Group A (Ⅰ. Saline; Ⅱ. Bu+Ap; Ⅲ. Lip Gel; Ⅳ. Lip@IR820 Gel+L; Ⅴ. Bu&Ap-Lip@IR820 Gel; Ⅵ. Bu&Ap-Lip@IR820 Gel+L), compared with the saline group, the hematological parameters RBC, HGB, HCT, MCV, and MCHC in whole blood of the Bu+Ap, Lip Gel, Lip@IR820 Gel+L, Bu&Ap-Lip@IR820 Gel, and Bu&Ap-Lip@IR820 Gel+L groups showed no significant changes, proving that the hydrogel did not cause anemia or polycythemia. The WBC, NEU, and LYM showed no significant changes, proving that the hydrogel did not cause infection, inflammation, allergy, or immune response. The PDW, PLT, and MPV showed no significant changes, proving that the hydrogel did not pose a bleeding tendency or risk of thrombosis.

[0162] 6.3.8) Serum biochemical index detection

[0163] Mouse blood was collected and allowed to coagulate naturally at room temperature. After 30 minutes, the blood was centrifuged at 3500 rpm for 15 minutes at 4°C, and the supernatant was collected. Relevant biochemical indicators were detected using enzyme-linked immunosorbent assay (ELISA) according to the kit instructions. Biochemical indicators included: cardiac-related indicators: creatine kinase (CK), creatine kinase-MB (CK-MB), cardiac troponin T (cTnT), and cardiac troponin I (cTnI); liver-related indicators: alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and kidney-related indicators: uric acid (UA) and creatinine (Cr).

[0164] Serum biochemical results as follows Figure 15 As shown in Figures B (Ⅰ. Saline; Ⅱ. Bu+Ap; Ⅲ. Lip Gel; Ⅳ. Lip@IR820 Gel+L; Ⅴ. Bu&Ap-Lip@IR820 Gel; Ⅵ. Bu&Ap-Lip@IR820 Gel+L), compared with the saline group, there were no significant differences in serum cardiac-related indicators (CK, CK-MB, cTnT, and cTnI), liver-related indicators (ALT and AST), and kidney-related indicators (UA and Cr) in the Bu+Ap, Lip Gel, Lip@IR820 Gel+L, Bu&Ap-Lip@IR820 Gel, and Bu&Ap-Lip@IR820 Gel+L groups. This demonstrates that the hydrogel did not cause significant myocardial, liver, or kidney damage, which is consistent with the HE staining results of the heart, liver, and kidneys. In summary, the HE staining, complete blood count, and serum biochemical indicators of the major organs demonstrate that the hydrogel has good biocompatibility at this dosage.

Claims

1. An injectable photothermal responsive liposome hydrogel, characterized in that: The injectable photothermal response liposome hydrogel comprises an injectable hydrogel and IR820 and drug-containing thermosensitive liposomes co-loaded on the injectable hydrogel; the drug-containing thermosensitive liposomes comprise thermosensitive liposomes Lip and a drug composition co-loaded on the thermosensitive liposomes Lip; the drug composition is bufalin Bu and apatinib Ap.

2. The injectable photothermal responsive liposome hydrogel according to claim 1, wherein: The mass ratio of the drug composition to the thermosensitive liposomes Lip is 1:10-30; preferably, the mass ratio of the drug composition to the thermosensitive liposomes Lip is 1:

20.

3. The injectable photothermal responsive liposome hydrogel according to claim 2, wherein: The mass ratio of bufalin Bu to apatinib Ap in the drug composition is 1:

500.

4. The injectable photothermal responsive liposome hydrogel of claim 3, wherein the hydrogel is prepared by mixing the photothermal responsive liposome and the biocompatible polymer in a ratio of 1: 1 to 1: 10 (w / w). The thermosensitive liposome Lip comprises dipalmitoyl phosphatidylcholine DPPC, monostearoyl phosphatidylcholine MSPC and DSPE-PEG 2000 ; the ratio of dipalmitoyl phosphatidylcholine DPPC and monostearoyl phosphatidylcholine MSPC is 5-15:1, the ratio of DSPE-PEG 2000 to monostearoyl phosphatidylcholine MSPC is 1.5:1; preferably, the ratio of dipalmitoyl phosphatidylcholine DPPC and monostearoyl phosphatidylcholine MSPC is 12.5:

1.

5. The injectable photothermal responsive liposome hydrogel according to claim 1 or 2 or 3 or 4, wherein: The final concentration of IR820 is 0.1 mg mL -1 ~ 0.2 mg mL -1 ; preferably, the final concentration of IR820 is 0.2 mg mL -1 .

6. The injectable photothermal responsive liposome hydrogel of claim 5, wherein: The injectable hydrogel is a triblock thermosensitive polyester polyethylene glycol copolymer, and the final concentration of the triblock thermosensitive polyester polyethylene glycol copolymer is 17.5%.

7. A method for preparing an injectable photothermal responsive liposome hydrogel, characterized in that: The preparation method of the injectable photothermal response liposome hydrogel comprises the following steps: 1) preparing drug-containing thermosensitive liposomes Bu&Ap-Lip by using a thin film dispersion method; 2) preparing an injectable photothermal response liposome hydrogel Bu&Ap-Lip@IR820 Gel based on the drug-containing thermosensitive liposomes Bu&Ap-Lip prepared in step 1).

8. The method for preparing the injectable photothermal responsive liposome hydrogel according to claim 7, characterized in that: The specific implementation of step 1) is as follows: 1.1) Weigh out the heat-sensitive liposomes Lip comprising dipalmitoyl phosphatidylcholine DPPC, monostearoyl phosphatidylcholine MSPC, and DSPE-PEG 2000 and place in a jar. 2000 ; 1.2) then adding a drug composition, which is bufalin Bu and apatinib Ap, in an eggplant-shaped bottle; 1.3) adding anhydrous methanol, transferring to a rotary evaporator after ultrasonic dissolution, and performing rotary evaporation under reduced pressure until a transparent and uniform lipid film is formed on the inner wall; 1.4) adding a preheated PBS solution, rotating and hydrating and then extruding and filtering through a microporous filter to obtain drug-containing thermosensitive liposomes Bu&Ap-Lip; Preferably, the mass ratio of the pharmaceutical composition to the thermosensitive liposome Lip is 1:10-30; preferably, the mass ratio of the pharmaceutical composition to the thermosensitive liposome Lip is 1:20; the mass ratio of bufalin Bu and apatinib Ap in the pharmaceutical composition is 1:500; the ratio of dipalmitoyl phosphatidylcholine DPPC to monostearoyl phosphatidylcholine MSPC in the thermosensitive liposome Lip is 5-15:1, and the DSPE-PEG 2000 The ratio of dipalmitoyl phosphatidylcholine DPPC to monostearoyl phosphatidylcholine MSPC is 1.5:1; preferably, the ratio of dipalmitoyl phosphatidylcholine DPPC to monostearoyl phosphatidylcholine MSPC is 12.5:1; Preferably, the temperature of the rotary evaporation is 30-50°C, preferably 40°C; and the rotation speed of the rotary evaporation is 100 rpm; Preferably, the preheating temperature of the PBS solution is 60°C; Preferably, the temperature of the rotary hydration is 45-65°C, preferably 60°C; the time of the rotary hydration is 35-55 min, preferably 50 min; and the rotation speed of the rotary hydration is 120 rpm; Preferably, the ultrasonic power is 150 W, the ultrasonic mode is working for 5 s, stopping for 2 s, and then working for 5 s, and the ultrasonic time is 3 min; Preferably, the microporous filter comprises a microporous filter with a pore size of 0.45 μm and a microporous filter with a pore size of 0.22 μm.

9. The method for preparing the injectable photothermal responsive liposome hydrogel according to claim 7 or 8, characterized in that: The specific implementation of step 2) is as follows: 2.1) weighing a triblock thermosensitive polyester polyethylene glycol copolymer PLGA-PEG-PLGA and placing it in a test tube; 2.2) adding the drug-containing thermosensitive liposomes Bu&Ap-Lip obtained in step 1) to the test tube in step 2.1) and swelling at room temperature 25°C; 2.3) after the swelling is completed, adding IR820 and stirring uniformly to obtain an injectable photothermal response liposome hydrogel Bu&Ap-Lip@IR820 Gel; Preferably, the final concentration of the triblock thermosensitive polyester polyethylene glycol copolymer PLGA-PEG-PLGA is 17.5%; the molecular weight of PLGA in the triblock thermosensitive polyester polyethylene glycol copolymer PLGA-PEG-PLGA is 1800, the molecular weight of PEG is 1500, and the mass ratio of the PLGA, PEG and PLGA is 75:25:75; the final concentration of the IR820 is 0.1 mg·mL -1 ~0.2 mg·mL -1 ; preferably, the final concentration of the IR820 is 0.2 mg·mL -1 .

10. An injectable photothermal responsive liposome hydrogel Bu&Ap-Lip@IR820 Gel prepared by the method of claim 7 or 8 or 9, preferably, the phase transition temperature of the injectable photothermal responsive liposome hydrogel Bu&Ap-Lip@IR820 Gel from sol state to gel state is 34.33±0.58℃; the phase transition time of the injectable photothermal responsive liposome hydrogel Bu&Ap-Lip@IR820 Gel from sol state to gel state is 33.33±1.53s; the phase transition temperature of the injectable photothermal responsive liposome hydrogel Bu&Ap-Lip@IR820 Gel from gel state to sol state is 43.67±1.53℃.

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