A method for preparing and applying a multifunctional cryogel

Abstract

This invention relates to the field of biomedical technology and discloses a method for preparing and applying a multifunctional cryogel. The preparation process is as follows: chitosan quaternary ammonium salt particles and sodium alginate particles are dissolved in deionized water to obtain chitosan quaternary ammonium salt solution and sodium alginate solution, respectively. Then, the chitosan quaternary ammonium salt solution and sodium alginate solution are mixed at a volume ratio of 1:4-4:1 and CaCl2 (1M) is added, or anlotinib is added after adding CaCl2 (1M), or anlotinib and black phosphorus nanosheets are added after adding CaCl2 (1M). After the mixed solution transforms into a gel, it is freeze-dried for 8-16 hours using a freeze dryer to obtain a composite cryogel. When this multifunctional cryogel is applied, after implantation of the composite cryogel into the excision site, infrared irradiation is applied to effectively stop bleeding, kill bacteria, and inhibit local tumor recurrence.

Description

Technical Field

[0001] This invention patent relates to the field of biomedical technology, specifically to a method for preparing and applying a multifunctional cryogel. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for 90% of all liver cancer cases. Radical surgical resection is the mainstream treatment for HCC. Intraoperative bleeding, postoperative infection, and a high postoperative tumor recurrence rate pose significant challenges to postoperative resection of liver cancer. Traditional clinical strategies such as hemostatic gauze, antibiotics for infection control, and radiotherapy / chemotherapy have not achieved satisfactory results. Therefore, developing a comprehensive "one-stop" strategy to simultaneously address hemostasis, antibiotic resistance, and recurrence issues after HCC resection is both urgent and promising.

[0003] However, the abundant blood supply in the liver and hepatocellular carcinoma makes intraoperative bleeding almost inevitable, rapid, and incompressible. Various existing hemostatic materials, such as nanofibers, sponges, and hydrogels, have been designed for rapid hemostasis. Cryogels, obtained at temperatures below the normal freezing point of the bulk solvent, are designed for rapid hemostatic applications due to their large pores, high porosity, and soft tissue-like elastic structure. Low-immunogenic sodium alginate (SA), a natural polysaccharide, is widely used in the preparation of cryogel platforms due to its instantaneous physical cross-linking properties. Frozen SA has internally unconnected macropores that allow for rapid absorption of plasma, adsorption of erythrocytes, and platelets. Furthermore, studies have shown that quaternized chitosan (QC) possesses excellent hemostatic properties and inhibits Gram-positive / negative bacteria. Cationic QC can cross-link with carboxyl-rich SA through electrostatic interactions to form stable SA / QC cryogels (SQ), exhibiting hemostatic and antibacterial properties.

[0004] Therefore, various composite cryogels of chitosan quaternary ammonium salt and sodium alginate have emerged in the prior art, and the composite cryogels have good antibacterial, hemostatic, liquid absorption and biocompatibility capabilities.

[0005] For example, Chinese invention patent application CN114887108A discloses a method for preparing a sodium alginate / chitosan quaternary ammonium salt double-crosslinked medical dressing with a gradient pore structure, comprising the following steps: preparing a mixed solution of sodium alginate and chitosan quaternary ammonium salt; using chloride salt to prevent the sodium alginate and chitosan quaternary ammonium salt from forming a precipitate; adding calcium carbonate nucleating agents of different particle sizes; lyophilizing the solution to obtain a solid dressing; washing the solid sample with an ethanol-acid mixed solution to prepare gradient pores and generate a secondary crosslinking reaction; finally washing the sample with anhydrous ethanol and lyophilizing it to obtain the sodium alginate / chitosan quaternary ammonium salt double-crosslinked medical dressing with a gradient pore structure. The dressing prepared by this invention has excellent antibacterial, hemostatic, absorbent, and biocompatible properties, and has good application prospects in the field of biomedical engineering.

[0006] For example, Chinese invention patent application CN114933718A also discloses a method for preparing a chitosan quaternary ammonium salt / oxidized sodium alginate self-healing hydrogel. The method involves: 1) preparing N-2-HACC and preparing an aqueous solution of N-2-HACC; 2) preparing oxidized sodium alginate and preparing an OSA solution; 3) adding NaCl to the N-2-HACC aqueous solution, dissolving it completely, then adding the OSA solution, and rapidly stirring to mix thoroughly to obtain the N-2-HACC / OSA hydrogel. The hydrogel obtained by this method exhibits good biocompatibility and self-healing effects, and can be used for biological tissue repair. It effectively solves the problems of poor mechanical properties and inability to self-repair in existing chitosan quaternary ammonium salt hydrogels, showing promising application prospects.

[0007] However, although the composite cryogel of chitosan quaternary ammonium salt and sodium alginate disclosed in the prior art can effectively stop bleeding and repair tissues in wounds and has good mechanical properties, it cannot effectively inhibit local tumor recurrence when used in surgical incisions after liver surgery. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing and applying a multifunctional cryogel, in order to solve the problems mentioned in the background art.

[0009] The embodiments of this application adopt the following technical solutions:

[0010] A method for preparing a multifunctional cryogel includes the following steps;

[0011] Step 1: Dissolve chitosan quaternary ammonium salt particles in deionized water and stir evenly at room temperature to obtain a 2wt% chitosan quaternary ammonium salt solution.

[0012] Step 2: Dissolve sodium alginate granules in deionized water and stir evenly at room temperature to obtain a 2wt% sodium alginate solution.

[0013] Step 3: Mix the chitosan quaternary ammonium salt solution obtained in Step 1 with the sodium alginate solution obtained in Step 2 at a volume ratio of 1:4-4:1, and add CaCl2 (1M) to obtain the SQ precursor. Then add anlotinib at a mass percentage of 0.05-2% of the SQ precursor and mix evenly. After the solution transforms into a gel, freeze the gel overnight at a gradient of any temperature from 2℃ to 8℃, any temperature from -30℃ to -10℃, and any temperature from -100℃ to 80℃. Then freeze-dry the gel for 8-16 hours using a freeze dryer to obtain the chitosan quaternary ammonium salt, sodium alginate, and anlotinib composite freeze gel, denoted as SQA.

[0014] Preferably, the process further includes adding black phosphorus nanosheets to step 3. The specific process is as follows: the chitosan quaternary ammonium salt solution obtained in step 1 and the sodium alginate solution obtained in step 2 are mixed at a volume ratio of 1:4-4:1, and CaCl2 (1M) is added to obtain the SQ precursor. Then, anlotinib and black phosphorus nanosheets, accounting for 0.05-2% of the mass percentage of the SQ precursor, are added and mixed evenly. After the solution transforms into a gel, the gel is subjected to a gradient freezing overnight at any temperature between 2°C and 8°C, any temperature between -30°C and -10°C, and any temperature between -100°C and 80°C. Then, it is freeze-dried for 8-16 hours using a freeze dryer to obtain a chitosan quaternary ammonium salt, sodium alginate, anlotinib, and black phosphorus composite freeze gel, denoted as SQAP.

[0015] Preferably, the mixing volume ratio of the chitosan quaternary ammonium salt solution to the sodium alginate solution is 0 / 10, 3 / 7, 1 / 1 or 7 / 3. After the mixed solution is converted into a gel, the gel is subjected to gradient freezing overnight at 4°C, -20°C and -80°C.

[0016] Preferably, the mixing volume ratio of the chitosan quaternary ammonium salt solution to the sodium alginate solution is 1 / 1, the added anlotinib accounts for 1% of the mass percentage of the SQ precursor, and after the mixed solution is converted into a gel, the gel is sequentially subjected to gradient freezing overnight at 4°C, -20°C and -80°C.

[0017] Preferably, the mixing volume ratio of the chitosan quaternary ammonium salt solution to the sodium alginate solution is 1 / 1, the added anlotinib accounts for 1% of the mass percentage of the SQ precursor, the added black phosphorus nanosheets account for 0.5% of the mass percentage of the SQ precursor, and after the mixed solution is converted into a gel, the gel is sequentially subjected to gradient freezing overnight at 4°C, -20°C and -80°C.

[0018] Preferably, after implanting the composite cryogel SQA or composite cryogel SQAP into the excision site, infrared irradiation is performed.

[0019] Preferably, when performing infrared irradiation, near-infrared light irradiation with a power of 0.1-3.5W / cm2 and a wavelength of 808nm is used.

[0020] Preferably, when performing infrared irradiation, near-infrared light irradiation with a power of 0.5W / cm2 or 0.8W / cm2 and a wavelength of 808nm is used.

[0021] Beneficial effects:

[0022] 1. The chitosan quaternary ammonium salt, sodium alginate and anlotinib composite cryogel prepared by the method provided in this invention can effectively inhibit angiogenesis of liver tumor cells and inhibit the proliferation of liver tumor cells and promote apoptosis of liver tumor cells by adding anlotinib;

[0023] The chitosan quaternary ammonium salt, sodium alginate and anlotinib composite cryogel prepared by this invention has certain photothermal conversion properties. By applying infrared light, the temperature of the composite cryogel can be increased above room temperature, thereby increasing the release rate of anlotinib to 62.41±4.10%.

[0024] 2. The chitosan quaternary ammonium salt, sodium alginate, anlotinib (AL3818), and black phosphorus nanosheets (BPNSss) composite cryogel SQAP prepared by the method provided in this invention can be used as a porous carrier. The positively charged QC component can absorb negatively charged red blood cells, thereby effectively reducing bleeding during hepatocellular carcinoma resection surgery and achieving rapid hemostasis.

[0025] Furthermore, black phosphorus nanosheets (BPNSss) under near-infrared light assistance can convert light energy into heat energy to sensitize bacterial membranes. Positively charged QCs can absorb negatively charged bacteria, causing bacterial membranes to rupture, thus playing a joint role in antibacterial action.

[0026] Black phosphorus nanosheets (BPNSss) have a mild photothermal therapy (MPTT) effect on residual tumor cells in the surgical resection area. At the same time, the controlled release of anlotinib (AL3818) by black phosphorus nanosheets (BPNSss) inhibits abnormal angiogenesis, thereby having an anti-tumor recurrence effect.

[0027] In summary, the SQAP multifunctional cryogel prepared by this invention has the effects of rapid hemostasis, antibacterial properties, and inhibition of local tumor recurrence. Attached Figure Description

[0028] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0029] Figure 1 This is a schematic diagram illustrating the preparation of the SQ-n cryogel of this invention patent;

[0030] Figure 2-3 SEM scanning electron microscope and pore size statistics of cryogels SQ-0, SQ-30, SQ-50 and SQ-70 prepared for this invention patent;

[0031] Figure 4This is a schematic diagram showing the water absorption rate and blood absorption rate of the SQ-n patent of this invention;

[0032] Figure 5 The images show the hemostasis effects of the SQ-n patented in this invention on various groups of mouse liver resection models.

[0033] Figure 6 This is a schematic diagram of the colony count and inhibition rate of LB plate after incubation of SQ-n with Escherichia coli and MRSA according to the present invention patent.

[0034] Figure 7 This is an experimental diagram of CCK8 cells after co-incubation of SQ-n and L929 cells according to the present invention.

[0035] Figure 8 These are scanning electron microscope images of the SQA and SQAP patents of this invention.

[0036] Figure 9 This is an EDS energy dispersive electron microscope image of the SQAP of this invention patent, where C is carbon, O is oxygen, N is nitrogen, F is fluorine, Na is sodium, Cl is chlorine, P is phosphorus, Ca is calcium, and Merge is a total image of C, O, N, F, Na, Cl, P and Ca.

[0037] Figure 10 For this invention patent, at 0.5 and 0.8 W / cm 2 Representative infrared thermal images of SQA and SQAP at 0, 30, 60, 120 and 300 s under near-infrared illumination;

[0038] Figure 11 For this invention patent, at 0.5 and 0.8 W / cm 2 Temperature profiles of SQA and SQAP in near-infrared light;

[0039] Figure 12 This invention patent is applicable at room temperature and at 0.5 and 0.8 W / cm². 2 The cumulative release of anlotinib by SQA and SQAP under near-infrared irradiation at 0.5 and 0.8 W / cm² was compared. 2 Response release curve of SQAP to AL3818 under near-infrared irradiation;

[0040] Figure 13 This is a CCK-8 experimental data graph of HUVECs from this invention patent;

[0041] Figure 14 This is a schematic diagram of the Transwell experiment of HUVECs in this invention patent;

[0042] Figure 15 These are representative images from the tube formation experiment of HUVECs in this invention patent;

[0043] Figure 16 This is an infrared thermographic image of the SQAP patent of this invention after it was applied to the tumor resection site under NIR irradiation;

[0044] Figure 17 This is a temperature curve under NIR irradiation after the SQAP patent of this invention is applied to the tumor resection site;

[0045] Figure 18 These are representative images of tumor sizes for each group in this invention patent;

[0046] Figure 19 These are the tumor volume and weight curves for each group in this invention patent;

[0047] Figure 20 This is a schematic diagram showing the tumor survival rates of each group in this invention patent;

[0048] Figure 21 These are representative images of HE staining and TUNEL staining from each group of this invention patent;

[0049] Figure 22 These are representative images of CD31 staining from each group in this invention patent;

[0050] Figure 23 These are survival rate images for each group in this invention patent. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0052] The technical solutions provided by the various embodiments of this application are described in detail below with reference to the accompanying drawings.

[0053] Example 1:

[0054] Please see Figure 2-8 A method for preparing and applying a multifunctional cryogel; comprising the following steps;

[0055] Step 1: Dissolve chitosan quaternary ammonium salt particles in deionized water and stir evenly at room temperature to obtain a 2wt% chitosan quaternary ammonium salt solution.

[0056] Step 2: Dissolve sodium alginate granules in deionized water and stir evenly at room temperature to obtain a 2wt% sodium alginate solution.

[0057] Step 3: The chitosan quaternary ammonium salt solution obtained in Step 1 and the sodium alginate solution obtained in Step 2 are mixed at a volume ratio of 0 / 10, 3 / 7, 1 / 1, or 7 / 3, and CaCl2 (1M) is added. After the mixed solution transforms into a gel, the gel is subjected to a gradient freezing overnight at 4℃, -20℃, and -80℃, and then freeze-dried for 8-16 hours using a freeze dryer to obtain a chitosan quaternary ammonium salt and sodium alginate composite freeze gel, denoted as SQ-n. The freeze gel prepared in Example 1 is scanned by SEM electron microscopy, and the results are as follows. Figure 2 and 3 As shown, from Figure 2 and 3 It can be seen that SQ-0 has a connected macroporous structure with smooth pore walls and high porosity (473.51±51.18μm). However, with the increase of QC content, the pore walls gradually become rougher and the pore size gradually decreases. SQ-50 has a pore size of 107.22±42.20μm, and SQ-70 has a pore size of 62.76±26.80μm. In step 3, S in SQ-n represents SA, Q represents QC, and n represents the QC content, and n can take the value of 0, 30, 50, or 70.

[0058] Test experiments on the gel prepared in Example 1:

[0059] Water absorption test:

[0060] After vacuum drying, the sample obtained through the above steps is cut into 3cm×3cm pieces, and its mass is recorded as M1. Then, it is immersed in a glass container containing 100mL of ultrapure water and left to stand at room temperature. At different time points, it is carefully removed, and the surface moisture is absorbed with filter paper. Its mass is recorded as M2. This process is repeated until equilibrium is reached, and its water absorption rate is measured.

[0061] Bloodsucking test:

[0062] After vacuum drying, the sample obtained through the above steps is cut into 3cm×3cm pieces, and its mass is recorded as W1. Then, it is immersed in a glass container containing 100mL of whole blood, left to stand at room temperature, and carefully removed after 1 minute. The surface whole blood is absorbed with filter paper, and its mass is recorded as W2. The blood absorption rate is then measured.

[0063] from Figure 4 It can be seen that all SQ-n samples rapidly absorb water within 2 minutes and reach equilibrium within 15 minutes. With increasing QC content, the water absorption rate decreased from 41.42±2.18% for SQ-0 to 34.13±2.73% for SQ-70. This may be related to the pore size and the inherently lower absorbency of QC compared to SA. To better simulate the internal environment, we further immersed SQ-n samples in blood for 1 minute to explore the blood absorption rate. Figure 4It was also found that SQ-n has a blood absorption rate of over 10% within 1 minute, effectively solving the problem of massive blood leakage during tumor resection.

[0064] Hemostasis experiment:

[0065] A mouse liver injury model (MouLi) was used to evaluate the hemostatic potential of the composite cryogel. A 10 mm bleeding wound was created on the liver, and the prepared sample SQ-n was immediately applied to the bleeding site. The control group served as a negative control (NC group), and medical gauze served as a positive control (PC group). Bleeding time was recorded until bleeding stopped. During this process, gauze was used to absorb the outflowing blood, and the total blood loss was calculated by weighing the gauze before and after absorption. Results are as follows: Figure 5 As shown;

[0066] Because the risk of bleeding during surgical resection of hepatocellular carcinoma is extremely high, good hemostatic properties are very important. For example... Figure 5 As shown, the MouLi model was used to evaluate the hemostatic effect of SQ-n. Significant bleeding occurred in the BC group, while the bleeding was minimal in the SQ-50 and SQ-70 groups. Figure 5 It can also be seen that, compared with other groups, the blood loss and hemostasis time of SQ-50 and SQ-70 were reduced to 0.38±0.02g and 113.00±15.72s, and 0.32±0.03g and 104.00±9.85s, respectively, with statistically significant differences. The results show that with the increase of QC components, SQ-n exhibits good hemostatic effect. The possible reasons for the excellent hemostatic performance of SQ-n are: (1) QC activates the body's extrinsic and intrinsic coagulation pathways; (2) the positively charged quaternary ammonium salt groups in QC adsorb and aggregate negatively charged red blood cells, rapidly forming blood clots; (3) the low-temperature gel rapidly absorbs water from the blood, thereby accelerating blood coagulation and promoting hemostasis;

[0067] Antibacterial test:

[0068] The antibacterial activity of sample SQ-n was detected using methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). In addition, there were BC group (blank control group) and PC group (antibiotic group). Specifically, SQ-n was first mixed with MRSA or E. coli suspension (1×10⁻⁶). 8 The bacterial culture was incubated with the control group (CFU / mL) for 5 hours. The culture was then inoculated onto Luria-Bertani (LB) culture dishes and incubated at 37°C for 16-24 hours. Images of different groups of LB culture dishes were collected, and the colony-forming units (CFU) on the culture dishes were counted. The inhibition rate (IR) was calculated based on the colony count using the following formula: IR(%) = (NC - NS) / NC × 100%, where NC and NS represent the average colony count of the control and the sample, respectively.

[0069] Postoperative infection after HCC surgical resection is also a concern. For example... Figure 6 As shown, the antibacterial activity of SQ-n was evaluated using E. coli and MRSA as representative bacteria. Compared with the BC group and SQ-0 group filled with bacteria, SQ-n (n = 30, 50, 70) significantly reduced the survival rate of E. coli and MRSA on the plates, from 87.09±0.03% and 37.52±0.08% for SQ-30, to 98.94±0.01% and 90.03±0.02% for SQ-50, and even 99.06±0.00% and 91.14±0.01% for SQ-70. This excellent antibacterial performance may be due to the positively charged amino and quaternary ammonium groups in SQ-n, which can disrupt the bacterial wall and electrostatically adhere to the bacteria, leading to the release of intracellular fluid (Liang, Li, Huang, Yu, & Guo, 2021). In addition, the hydrophobic alkyl groups of QC can interact with the hydrophilic groups of bacteria, increasing membrane permeability and thus leading to the destruction of bacterial structure;

[0070] L929 cell CCK8 assay:

[0071] The samples prepared through the above steps were sterilized under ultraviolet light and set aside for later use. L929 cells were digested to prepare a cell suspension, and then the cells were seeded into 96-well plates and incubated in an incubator. After 24 hours, the original culture medium was aspirated, and the sterilized sample was added to each well. On days 1, 2, and 3, CCK-8 reagent was added to each well, and the plates were incubated for another 3 hours for color development. The OD value at 450 nm was measured to assess the cytotoxicity of the samples. The experiment was divided into 5 groups: BC group (untreated group), SQ-0 group, SQ-30 group, SQ-50 group, and SQ-70 group.

[0072] Biocompatibility is the most essential and fundamental requirement for biomaterials directly applied to the human body. We further evaluated the effect of SQ-n on L929 cell proliferation using the CCK8 assay. After 1–2 days of co-culture, the proliferation of L929 cells in the SQ-n group was similar to that in the BC group, indicating that SQ-n had no significant effect on L929 cell proliferation. However, after 3 days of co-culture, the proliferation of L929 cells in the SQ-70 group was lower than that in other groups, indicating that excessive QC is detrimental to cell proliferation (P<0.05). Therefore, considering the comprehensive evaluation results of hemostasis, antibacterial properties, and biocompatibility, both SQ-50 and SQ-70 exhibit similar excellent hemostatic and antibacterial properties. However, considering the potential cytotoxicity of the higher QC in SQ-70, which is detrimental to cell proliferation, we decided to use SQ-50 as the subject of subsequent research.

[0073] Example 2:

[0074] Please see Figure 1-3A method for preparing and applying a multifunctional cryogel includes the following steps;

[0075] Step 1: Dissolve chitosan quaternary ammonium salt particles in deionized water and stir evenly at room temperature to obtain a 2wt% chitosan quaternary ammonium salt solution.

[0076] Step 2: Dissolve sodium alginate granules in deionized water and stir evenly at room temperature to obtain a 2wt% sodium alginate solution.

[0077] Step 3: Mix the chitosan quaternary ammonium salt solution obtained in Step 1 with the sodium alginate solution obtained in Step 2 at a volume ratio of 1 / 1, add 1% AL3818 and mix evenly, then add CaCl2 (1M). After the solution transforms into a gel, freeze the gel sequentially at 4℃, -20℃ and -80℃ overnight, and then freeze-dry it for 8-16 hours using a freeze dryer to obtain the chitosan quaternary ammonium salt, sodium alginate and anlotinib composite freeze gel, denoted as SQA.

[0078] Example 3:

[0079] Please see Figure 1-23 A method for preparing and applying a multifunctional cryogel includes the following steps;

[0080] Step 1: Dissolve chitosan quaternary ammonium salt particles in deionized water and stir evenly at room temperature to obtain a 2wt% chitosan quaternary ammonium salt solution.

[0081] Step 2: Dissolve sodium alginate granules in deionized water and stir evenly at room temperature to obtain a 2wt% sodium alginate solution.

[0082] Step 3: The chitosan quaternary ammonium salt solution obtained in Step 1 and the sodium alginate solution obtained in Step 2 are mixed at a volume ratio of 1:1. 1% AL3818 and 0.5% BPNSss are added and mixed thoroughly. Then, CaCl2 (1M) is added. After the solution transforms into a gel, the sample is sequentially placed in a gradient freeze-drying environment at 4℃, -20℃, and -80℃ overnight. Then, it is freeze-dried for 8-16 hours using a freeze dryer to obtain a chitosan quaternary ammonium salt / sodium alginate / anlotinib / black phosphorus composite cryogel, denoted as SQAP. The morphology of SQA and SQAP is observed by scanning electron microscopy of the cryogels prepared in Examples 2 and 3. Figure 8 and 9 As shown, with the addition of AL3818 and BPNSss, the surfaces of SQA and SQAP become rougher and the pore size decreases. The AL3818 and BPNSss components in SQAP were detected by EDS. Figure 6The characteristic elements nitrogen (N, 0.62%) and fluorine (F, 0.08%) of AL3818 and the characteristic element phosphorus (P, 0.05%) of BPNSss were uniformly distributed in SQAP, further confirming the successful loading of AL3818 and BPNSss into SQ-50 cryogel.

[0083] Test experiments on the gels prepared in Examples 2 and 3:

[0084] Photothermal conversion performance test:

[0085] First, the samples prepared in Examples 2 and 3 were cut into 1×1cm blocks; then, at power densities of 0.5 and 0.8 W / cm², respectively... 2 The samples were irradiated with an 808nm NIR laser for 4 minutes. Then, a photothermal imager was used to acquire photothermal images of the samples, and temperature curves of the samples were recorded to evaluate the photothermal conversion performance of the samples.

[0086] The introduction of BPNSss endows it with excellent photothermal conversion properties. Considering that mPTT has good anti-tumor effects while protecting surrounding normal tissues, SQA and SQAP were subjected to concentrations of 0.5 and 0.8 W / cm², respectively. 2 Its photothermal conversion performance was evaluated by 808 nm NIR irradiation. Temperature was monitored in real time, and representative photothermal images are shown below. Figure 10 As shown. Figure 11 As shown, at a power of 0.5W / cm 2 and 0.8W / cm 2 In the 808nm near-infrared range, the SQA temperatures were 27.73±0.49℃ and 28.70±1.20℃, respectively, both showing a slight increase. At 0.5W / cm²... 2 Under near-infrared irradiation, the temperature of SQAP gradually increased to 38.17±0.91℃, at 0.8W / cm². 2 Under near-infrared irradiation, the temperature of SQAP rapidly increased to 45.53±2.30℃. Considering that the optimal temperature for mPTT inactivation of tumor cells is 42-48℃, a near-infrared power of 0.8W / cm² was selected. 2 For use in subsequent experiments;

[0087] AL3818 release test:

[0088] The samples prepared in Examples 2 and 3 were cut into 1×1cm pieces; SQA and SQAP were soaked in 5mL PBS at 0.5 and 0.8 W / cm, respectively. 2Power exposure was performed at room temperature (RT) and 808 nm NIR. The system was maintained at a vibration rate of 50 rpm. At predetermined time intervals, 1.0 mL of the release solution was collected for analysis and added to 1.0 mL of fresh PBS. The release rate of AL3818 was measured at different time points (0-14 days). The experiment consisted of 6 groups: SQA+RT group, SQAP+RT group, SQA+0.5 W / cm² group, and SQA+0.5 W / cm² group. 2 Group, SQAP+0.5W / cm 2 SQA+0.8W / cm 2 Group 2 and SQAP+0.8W / cm 2 Irradiate with NIR for 10 minutes daily.

[0089] To verify the controllable release of AL3818 via photothermal conversion of BPNSss, SQAP soaked in PBS was irradiated with near-infrared radiation for 10 minutes at 8-hour intervals, and the release of AL3818 was measured over 24 hours. The experiment was divided into two groups: SQAP + 0.5 W / cm². 2 Group and SQAP+0.8W / cm 2 .

[0090] Drug release behavior test:

[0091] At RT, SQA and SQAP showed similar cumulative AL3818 release at 14 days, at 57.59±4.71% and 60.12±4.57%, respectively. Figure 12 To evaluate the facilitating effects of near-infrared radiation and BPNSss on the release of AL3818 by SQA and SQAP, the release of AL3818 at different near-infrared powers was investigated. At 0.5 W / cm²... 2 Under near-infrared irradiation, the cumulative release of AL3818 by SQA was 62.41±4.10%, while the cumulative release of SQAP was 73.65±5.04%. Figure 12 This indicates that the photothermal conversion of BPNSss promotes the release of AL3818. Furthermore, at 0.8 W / cm²... 2 Under near-infrared irradiation, SQAP achieved a cumulative release of 85.37 ± 5.26% of AL3818 at 14 days, demonstrating sufficient drug release in both the short and long term. Figure 12 As shown in the two drug release curves, the drug release rate of SQAP increases significantly in a short time under a single near-infrared irradiation, but decreases after NIR irradiation. Furthermore, at 0.8 W / cm²... 2 Under near-infrared irradiation, the drug release effect is better than that at 0.5 W / cm². 2 The effect is more pronounced under near-infrared irradiation. This may be due to the fact that at 0.8 W / cm²... 2Under near-infrared irradiation, higher temperatures lead to more vigorous material movement, thus promoting the release of AL3818. This photothermal response-controlled drug release enables "on-demand" drug delivery and reduces adverse drug events and potential drug resistance.

[0092] CCK8 Experiment:

[0093] The samples prepared in Examples 2 and 3 were sterilized under ultraviolet light and set aside for later use. HUVECs cells were digested to prepare cell suspensions, and then the cells were seeded into 96-well plates and incubated in an incubator. After 24 hours, the original culture medium was aspirated, and the sterilized samples were added to each well. On days 1, 2, and 3, CCK-8 reagent was added to each well, and the plates were incubated for another 3 hours for color development. The OD value at 450 nm was measured to assess the cytotoxicity of the samples. The experiment was divided into four groups: BC group (untreated group), SQA group, SQAP group, and SQAP+NIR group.

[0094] Transwell experiments:

[0095] 5×10 4 HUVECs at a concentration of / mL were placed in the upper chamber, and the samples were placed in the lower chamber. After co-incubation for 36 hours, the migrating cells remaining on the filter membrane surface were removed with cotton swabs. The migrating cells on the lower surface of the filter membrane were stained with 0.5% crystal violet solution, fixed with 4% paraformaldehyde, photographed under a light microscope, and the number of migrating cells in each group was counted.

[0096] Tube forming experiment:

[0097] 200 μL of Matrigel (BDBiosciences, Franklin Lakes, NJ, USA) was added to a pre-cooled 24-well plate and polymerized at 37 °C for 30 min. Then, HUVECs (2 × 10⁻⁶) were added. 4 The suspension was seeded onto the Matrigel surface. After 6 hours, the cells were stained with Calciurin-AM and photographed under a fluorescence microscope. Statistical analysis was performed on the branching points and capillary lengths of the capillary-like structures.

[0098] To meet the needs of continuous proliferation, tumors obtain sufficient nutrients through angiogenesis (Viallard & Larrivée, 2017). Therefore, anti-angiogenesis is an important clinical strategy for preventing tumor recurrence. The in vitro anti-angiogenic effect of HUVECs was evaluated using this study. Figure 13 To illustrate the relative proliferation of HUVECs, on days 1, 2, and 3, the SQAP+NIR group showed lower cell proliferation than the other groups. Subsequently, the migration ability of HUVECs was evaluated using a Tanswell assay. Figure 14As shown, compared with the BC group (182.33±20.98), the SQA and SQAP groups showed less HUVEC migration (115.00±11.79 and 102.67±17.47, respectively), with the SQAP+NIR group showing the most significant difference (16.33±14.98), indicating that SQAP can effectively inhibit HUVEC migration under near-infrared irradiation. To evaluate the anti-angiogenic activity of SQA and SQAP, tube formation experiments were performed on HUVECs. Figure 15 As shown, compared with other groups, the tubular structures in the SQAP+NIR group were less mature and complete, and the branching points and capillary lengths were significantly reduced, demonstrating that the combination of SQAP and NIR has a good inhibitory effect on angiogenesis.

[0099] CCK8 Experiment:

[0100] The samples prepared in Examples 2 and 3 were sterilized under ultraviolet light and set aside for later use. H22 cells were digested to prepare a cell suspension, and then the cells were seeded into 96-well plates and incubated in an incubator. After 24 hours, the original culture medium was aspirated, and the sterilized sample was added to each well. On days 1, 2, and 3, CCK-8 reagent was added to each well, and the plates were incubated for another 3 hours for color development. The OD value at 450 nm was measured to evaluate the cytotoxicity of the samples. The experiment was divided into four groups: BC group (untreated group), SQA group, SQAP group, and SQAP+NIR group.

[0101] Transwell experiments:

[0102] 5×10 4 H22 at a concentration of / mL was placed in the upper chamber, and the sample was placed in the lower chamber. After co-incubation for 36 hours, the migrating cells remaining on the filter membrane surface were removed with cotton swabs. The migrating cells on the lower surface of the filter membrane were stained with 0.5% crystal violet solution, fixed with 4% paraformaldehyde, photographed under a light microscope, and the number of migrating cells in each group was counted.

[0103] Considering the excellent photothermal conversion properties of the SQAP cryoprotectant and the photothermally responsive controlled-release molecular target AL3818, their synergistic antitumor effects with mPTT and molecularly targeted therapy were evaluated in vitro using H22 cells. The CCK8 assay was used to detect the proliferation capacity of H22 cells. Figure 15 In the study, the proliferation levels of H22 cells in the SQA and SQAP groups gradually decreased over time, while the SQAP+NIR group showed the best inhibitory effect on tumor proliferation. Furthermore, the migration ability of H22 cells was evaluated using a scratch assay. Figure 13 In the study, the number of infiltrating cells in the SQAP+NIR group was significantly lower than that in other groups. Overall, the synergistic anti-tumor effect of mPTT and molecular targeted therapy can effectively inhibit the proliferation and invasion of H22 cells, which is beneficial for inhibiting tumor recurrence after surgical resection of liver cancer.

[0104] Animal model of recurrent xenogeneic hepatocellular carcinoma:

[0105] To establish an animal model of xenograft liver cancer recurrence, Balb / C mice were injected with 5×10⁻⁶ ions of xenograft hepatocellular carcinoma into the anterior right dorsal side. 6 H22 cell concentration. When the tumor size reaches 150 mm. 3 At that time, all visible tumors were surgically removed, and this day was defined as day 0. Immediately after surgery, the mice were randomly divided into 4 groups: (1) no treatment after surgery (BC group); (2) oral administration of physiological saline containing AL3818 (5.0 mg / kg) every 3 days starting from day 2 after surgery, for a total of 5 times (AL3818 group); (3) implantation of SQAP gel at the resection site (SQAP group); (4) implantation of SQAP gel at the resection site, and near-infrared irradiation (0.8 W / cm²) every 3 days starting from day 2 after surgery. 2 Five trials were conducted (SQAP+NIR group), each lasting 1 minute, for a total of 5 trials. Mouse body weight and tumor volume were monitored and analyzed after resection.

[0106] In vivo photothermal properties:

[0107] Based on the results of in vitro photothermal performance testing, 0.8 W / cm² was selected. 2 In vivo evaluation was performed. Mice in the BC, AL3818, and SQAP+NIR groups received NIR irradiation during the first NIR treatment after surgery. Temperature change data were recorded, and temperature-time curves were plotted and analyzed.

[0108] Histological analysis:

[0109] After euthanasia of mice on day 21, tumor tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Subsequently, immunofluorescence staining (labeled with TUNEL, CD31, and HIF-1α) was performed according to the corresponding standard procedures.

[0110] Animal survival experiments:

[0111] Based on the xenogeneic hepatocellular carcinoma recurrence model established above, mice after surgical resection (recorded on day 0) were randomly divided into four identical groups: BC group, AL3818 group, SQAP group, and SQAP+NIR group. After treatment using the same method as described above, the mice were observed for 40 days, and the overall survival rate was assessed.

[0112] Based on the ideal results of the above in vitro validation experiments, an in vivo mouse model of xenogeneic hepatocellular carcinoma was established to further verify the in vivo efficacy of SQAP. Figure 16 and Figure 17 In the middle, at 0.8W / cm 2Under near-infrared irradiation, the local temperature of the control group and the AL3818 group did not change significantly, while the local temperature of the implanted SQAP rapidly increased to 45.50±1.05℃, which provided a guarantee for the implementation of mPTT treatment.

[0113] Due to tumor recurrence leading to mouse death, by day 21, 3 mice remained alive in the BC group, 4 mice each in the AL3818 and SQAP groups, and all mice in the SQAP+NIR group survived. Figure 18 Tumor volume dynamic curve () Figure 19 The results showed that, compared with other groups, all 5 mice in the SQAP+NIR group survived, and the recurrent tumor volume was the smallest (55.76±22.36 mm). 3 ). Figure 20 The results also showed that, over time, the recurrent tumor tissue in the BC group had the largest body weight, followed by the AL3818 group, the SQAP group, and the SQAP+NIR group.

[0114] On day 21, the obtained tumor tissue was subjected to histological evaluation. Figure 21 As shown, HE results revealed diffuse distribution of tumor cells in the BC group, exhibiting significant tumor atypia and pathological mitotic figures, with a low proportion of TUNEL-positive cells. In contrast, the proportion of TUNEL-positive cells was significantly upregulated in the SQAP+NIR group. These results indicate that NIR-assisted SQAP can inhibit tumor proliferation, promote tumor necrosis and apoptosis, thereby suppressing local recurrence after tumor resection.

[0115] Figure 22 Immunofluorescence staining of CD31 and HIF-1α is shown. HIF-1α reflects the level of hypoxia in tumor tissue and is a core regulator of angiogenesis under hypoxic conditions; its upregulation can stimulate tumor angiogenesis (Liang, Li, Huang, & Guo, 2021). The expression levels of CD31 and HIF-1α were lowest in the SQAP+NIR group. Figure 22 This may be because NIR-assisted SQAP, in addition to releasing AL3818 to inhibit tumor angiogenesis, also reduces tumor oxygen demand by inhibiting tumor proliferation, thereby reducing the production of HIF-1α and blocking its further pro-angiogenic effects.

[0116] Figure 23As shown, survival analysis after 40 days of observation in each group revealed that the SQAP+NIR group had the longest overall survival and the best prognosis. Therefore, this study rationally designed and developed a NIR-assisted SQAP with hemostatic, antibacterial, anti-angiogenic, and anti-tumor functions as a "one-stop" strategy to meet clinical needs. Specifically, this multifunctional SQAP effectively reduces intraoperative bleeding and postoperative bacterial infection risk through QC, and inhibits local tumor recurrence through BPNSss-based photothermal conversion and AL3818 photothermal response release, in conjunction with mPTT and molecular targeted therapy.

[0117] This invention patent ultimately designed and synthesized a multifunctional cryogel based on SA, QC, BPNSss, and AL3818 for postoperative hemostasis, anti-infection, and in situ anti-tumor recurrence. Figure 1 As shown, we prepared a series of SQ-n cryogels to select the most suitable ratio. Due to the excellent physicochemical properties, hemostatic and antibacterial properties of SQ-50, we chose to load SQ-50 with AL3818 and BPNSs to construct SQAP. SQAP was transplanted into the subcutaneous xenograft tumor resection site in mice to evaluate its antitumor effect. As a porous carrier, SQAP can rapidly remove blood; the positively charged QC component can absorb negatively charged red blood cells, achieving rapid hemostasis. Near-infrared assisted BPNSss converts light energy into heat energy to sensitize the bacterial membrane, and the QC adsorbs negatively charged bacteria, causing the bacterial membrane to rupture, thus jointly exerting an antibacterial effect. Furthermore, BPNSss exhibits a mPTT effect on residual tumor cells in the resection area. Simultaneously, the controlled release of targeted AL3818 by BPNSss inhibits abnormal angiogenesis, thus having an anti-tumor recurrence effect. In summary, the SQAP multifunctional cryogel provides a "one-stop" synergistic strategy for postoperative resection of liver cancer.

[0118] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing a multifunctional cryogel, comprising the following steps; Step 1: Dissolve chitosan quaternary ammonium salt particles in deionized water and stir evenly at room temperature to obtain a 2wt% chitosan quaternary ammonium salt solution. Step 2: Dissolve sodium alginate granules in deionized water and stir evenly at room temperature to obtain a 2wt% sodium alginate solution. Step 3: The chitosan quaternary ammonium salt solution obtained in Step 1 and the sodium alginate solution obtained in Step 2 are mixed at a volume ratio of 1:4-4:1, and 1 mol / L CaCl2 is added to obtain the SQ precursor. Then, 0.05-2% anlotinib and 0.05-1.5% black phosphorus nanosheets are added to the SQ precursor and mixed evenly. After the solution transforms into a gel, the gel is subjected to a gradient freezing overnight at any temperature between 2℃ and 8℃, any temperature between -30℃ and -10℃, and any temperature between -100℃ and -80℃. Then, it is freeze-dried for 8-16 hours using a freeze dryer to obtain the chitosan quaternary ammonium salt, sodium alginate, anlotinib, and black phosphorus composite freeze gel, denoted as SQAP.

2. The method for preparing a multifunctional cryogel according to claim 1, characterized in that, The volume ratio of the chitosan quaternary ammonium salt solution to the sodium alginate solution is 3 / 7, 1 / 1, or 7 / 3. After the solution transforms into a gel, the gel is sequentially subjected to gradient freezing overnight at 4°C, -20°C, and -80°C.

3. The method for preparing a multifunctional cryogel according to claim 1, characterized in that, The mixing volume ratio of the chitosan quaternary ammonium salt solution to the sodium alginate solution is 1 / 1. The added anlotinib accounts for 1% of the mass percentage of the SQ precursor, and the added black phosphorus nanosheets account for 0.5% of the mass percentage of the SQ precursor. After the solution is converted into a gel, the gel is subjected to a gradient freezing overnight at 4°C, -20°C, and -80°C.

4. An application, wherein the multifunctional cryogel prepared by the method for preparing a multifunctional cryogel according to any one of claims 1-3 is used in the preparation of materials for postoperative hemostasis, anti-infection, and in situ anti-tumor recurrence, characterized in that: This includes implanting the composite cryogel SQAP into the excision site and then applying infrared irradiation.

5. The application of the multifunctional cryogel material according to claim 4, characterized in that: When applying infrared irradiation, a power of 0.1-3.5W / cm² is used. 2 Near-infrared light irradiation with a wavelength of 808nm.

6. The application of the multifunctional cryogel material according to claim 5, characterized in that: When applying infrared irradiation, a power of 0.5W / cm² is used. 2 Or 0.8W / cm 2 Near-infrared light irradiation with a wavelength of 808nm.

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