A calcium-containing chitosan hydrogel and its preparation method and application

Abstract

The invention discloses a kind of chitosan hydrogel containing calcium and its preparation method, 27.75‑111 mg / mL calcium chloride solution and 50‑200 mg / mL and molecular weight are 4000‑8000 Da sodium polyaspartate solution is mixed in equal volumes, stirred evenly, 21‑84 mg / mL sodium bicarbonate solution is added dropwise, stirred at room temperature for 5‑15min, calcium carbonate nanoparticle suspension is obtained; 300-450 μL suspension is added to 800 μL of 15‑45 mg / mL, chitosan solution having a degree of deacetylation greater than 80%, stirred to clarification, pH is measured, if not within 6.7-7.4, sodium bicarbonate solution is added, pH is made within 6.7-7.4; then 37°C water bath 5‑15min, chitosan hydrogel containing calcium is obtained. The present invention can enter cells after being incubated with cells, destroy intracellular lipid droplets by lipophage, and significantly reduce intracellular lipid content.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a calcium-containing chitosan hydrogel and a preparation method and application thereof. Background Art

[0002] Chitosan is a kind of cellulose with health-care effects, which exists in the bodies of crustaceans or arthropods. Chitosan can be used as a dietary supplement for fat reduction. It has been reported that chitosan has a relatively good effect on controlling blood lipids, especially for lowering cholesterol. It can prevent the absorption of fat in food and accelerate the metabolism of cholesterol in human blood. However, the current reports on chitosan fat reduction are only related to the removal of extracellular lipids, and the lipid-lowering effect is not obvious in clinical trials. Its potential lipid-lowering function is still unclear and the mechanism remains to be elucidated. The main reason is that the fat-reducing effect of the existing chitosan fat-reducing formula is unclear. Therefore, in order to solve the above problems, this patent designs a new type of chitosan gel, changes the assembly state of chitosan molecules, and adds calcium carbonate that can promote the combination of chitosan and lipids and promote lipid degradation.

[0003] In addition, one of the most important metabolic characteristics of tumor cells is the dysregulation of lipid metabolism and the massive accumulation of lipids. Cancer requires a large amount of fatty acids during its development. Cancer cells use de novo lipogenesis and fatty acid uptake to drive the overexpression of lipases to mediate lipid accumulation, further supporting the rapid proliferation, survival, migration, metastasis and chemotherapy resistance of tumors. Lipid droplets are the main organelles for tumor cells to store excess lipids and reduce cellular lipid stress. They are mainly composed of neutral lipids surrounded by a phospholipid monolayer. Excessive accumulation of lipids in tumor cells will be temporarily stored in the form of lipid droplets, and the formation of lipid droplets is a key adaptive response to the harmful environment. Lipid droplets are involved in various aspects of tumorigenesis and tumor progression, so targeting lipid droplets and tumor lipid metabolism is a promising strategy for treating human cancer.

[0004] With the development of hydrogel technology, hydrogels have been widely used in the biomedical field as emerging drug carriers. The biocompatibility, biodegradability, drug loading capacity and controllable drug release of hydrogels make them very competitive in the field of tumor treatment. Chitosan hydrogel has good tissue compatibility and biodegradability.

[0005] We use the lipid binding properties of chitosan to design a chitosan oligomer hydrogel formula cross-linked by calcium carbonate nanoparticles. Among them, chitosan can enter the cell and destroy lipid droplets in the cell. Calcium carbonate can accelerate the lysosomal escape of chitosan. The generated calcium ions help to accelerate the oxidation of fatty acids in mitochondria and promote the degradation of lipids into fatty acids. Therefore, this formula has a strong function of removing intracellular lipids. In the tumor microenvironment, the composite chitosan hydrogel first deprives the lipids in the tumor cells and reduces the fat content in the tumor. On this basis, a cell shell polysaccharide-lipid complex is formed to promote the entry of immune cells and reverse the immunosuppressive microenvironment by activating macrophages. Using a composite chitosan gel alone in a tumor or combining chitosan gel with immune checkpoint inhibitors and chemotherapeutic drugs can improve the effect of tumor immunotherapy and can be applied to tumor treatment. In short, the present invention designs a chitosan hydrogel cross-linked by calcium carbonate, and uses it to reduce intracellular lipids, reduce lipids in tissues, and a sensitizer for tumor immunotherapy. The hydrogel has a clear lipid degradation mechanism, that is, it reduces intracellular lipid droplets by inducing autophagy of lipid droplets. Compared with chitosan solution, the hydrogel has a significant effect on removing intracellular lipid droplets, which significantly improves the lipid removal effect of existing chitosan solution and related preparations. At the same time, it can also be used as an adjuvant to improve the antigen presentation effect of the vaccine and achieve efficient immunity by stimulating the regulation of antigen recognition receptors of macrophages. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention proposes a calcium-containing chitosan hydrogel and a preparation method and application thereof, and prepares chitosan hydrogel by pH adjustment and temperature adjustment. On the one hand, the chitosan hydrogel enters the cell, and after escaping the lysosome and decomposing into small and uniform nanoparticles, it combines with lipid droplets in the cell and causes lipid droplet decomposition, and causes lipophagy by combining lipid droplets with LC3. Due to overactive fatty acid oxidation, the degradation of lipid droplets induced by chitosan hydrogel enhances the immunogenicity of tumor cells. On the other hand, the chitosan hydrogel deprives the cell of lipids, forms a cell capsid polysaccharide-lipid complex, increases Mincle in tumor-infiltrating lymphocytes, and reverses the immunosuppressive microenvironment. In summary, the calcium-containing chitosan hydrogel of the present invention can achieve the purpose of removing intracellular lipids and sensitizing in tumor immunotherapy.

[0007] A method for preparing a calcium-containing chitosan hydrogel, the method comprising the following steps:

[0008] Step 1: Mix 27.75-111 mg / mL calcium chloride solution and 50-200 mg / mL sodium polyaspartate solution with a molecular weight of 4000-8000 Da in equal volumes, stir until uniform, add 21-84 mg / mL sodium bicarbonate solution dropwise to the mixed solution, stir at room temperature for 5-15 minutes, and obtain a calcium carbonate nanoparticle suspension;

[0009] Step 2: Add 300-450 μL of calcium carbonate nanoparticle suspension to 800 μL of 15-45 mg / mL chitosan solution with a deacetylation degree greater than 80%, stir until clear, test the pH value of the solution, if it is not in the range of 6.7-7.4, add sodium bicarbonate solution to make the pH value of the solution in the range of 6.7-7.4; then bathe in 37° C. water for 5-15 minutes to obtain calcium-containing chitosan hydrogel.

[0010] The pH value of the solution is adjusted to 6.7-7.4 in order to reduce the electrostatic repulsion and enhance the hydrogen bonding and hydrophobic interactions between -NH2 groups.

[0011] Furthermore, in step 1, the concentration of the calcium chloride solution is 111 mg / mL, the molecular weight of sodium polyaspartate is 8000 Da, the concentration of the sodium polyaspartate solution is 200 mg / mL, and the concentration of the sodium bicarbonate solution is 84 mg / mL; in step 2, the volume of the calcium carbonate nanoparticle suspension is 300 μL, and the concentration of the chitosan solution is 25 mg / mL.

[0012] Furthermore, the stirring is magnetic stirring.

[0013] A calcium-containing chitosan hydrogel prepared by the above method.

[0014] Application of the calcium-containing chitosan hydrogel prepared by the method in removing intracellular lipids.

[0015] Application of the calcium-containing chitosan hydrogel prepared by the method in tumor treatment sensitization.

[0016] The beneficial effects of the present invention are as follows:

[0017] (1) Compared with pure chitosan solution, gel chitosan forms small and uniform nanoparticles after entering cells, which bind to and destroy lipid droplets, and cause lipophagy through the binding of LC3 and lipid droplets. Under the synergistic effect of calcium ions, gel chitosan exhibits a stronger effect of removing intracellular lipids.

[0018] (2) After lipid droplets are destroyed, free fatty acids are transported to mitochondria and fatty acid oxidation is activated. Therefore, calcium-containing chitosan hydrogel induces and regulates the lipid metabolism of tumor cells and enhances the immunogenicity of tumor cells.

[0019] (3) Calcium-containing chitosan hydrogel-treated immune cells showed reduced fatty acid oxidation and immune activation. Calcium-containing chitosan hydrogel upregulated the lipid-sensing C-type lectin receptor Mincle on macrophages, which led to a significant increase in macrophage phagocytosis of tumor cells and efficient antigen presentation.

[0020] (4) When combined with aPD-L1 and gemcitabine, calcium-containing chitosan hydrogels significantly slowed tumor growth rate and improved the survival rate of tumor-bearing mice during intratumoral treatment, indicating that calcium-containing chitosan hydrogels have the ability to sensitize cancer cells to treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The co-localization characterization of calcium-containing chitosan hydrogel and its effect on cell activity; wherein, Figure (A) is a co-localization fluorescence confocal image of calcium-containing chitosan gel, the left image of Figure (A) is calcium carbonate nanoparticles, the middle image is chitosan, and the right image is a merged image of calcium carbonate nanoparticles and chitosan; Figure (B) is a freeze-dried SEM image of calcium-containing chitosan gel; Figure (C) is an analysis of the results of chitosan gel and calcium-containing chitosan gel on tumor cell proliferation; Figure (D) is a statistical graph of CCK-8 experimental results after co-culture of different concentrations of calcium-containing chitosan and immune cell RAW 264.7 cells (n=5). Experimental group description: 4T1 (untreated tumor cell group), 4T1+Chito (tumor cell group treated with chitosan gel), 4T1+ChitoCa (tumor cell group treated with chitosan gel containing calcium).

[0022] Figure 2 The effect of calcium-containing chitosan gel on lipid droplets in tumor cells; Figure 2 (A) is a representative fluorescence confocal image of lipid droplets in tumor cells. The left 1st image in (A) is a representative image of the BC group, the left 2nd image is a representative image of the COS group, the right 2nd image is a representative image of the Chito group, and the right 1st image is a representative image of the ChitoCa group; Figure (B) is a quantitative data analysis of lipid droplets in tumor cells. Among them, n=15 for the BC group, n=10 for the COS group, n=9 for the Chito group, and n=15 for the ChitoCa group. *P represents <0.05, **P represents <0.01, ***P represents <0.001, and ****P represents <0.0001. Experimental group description: BC (untreated tumor cell group), COS (tumor cell group treated with chitosan solution), Chito (tumor cell group treated with chitosan gel), ChitoCa (tumor cell group treated with calcium-containing chitosan gel). Glossary: ​​LD (lipid droplet).

[0023] Figure 3Figure 2 shows the effect of calcium-containing chitosan gel on tumor cell metabolism and the phenotypic changes of tumor cells after co-culture with calcium-containing chitosan gel; Figure (A) shows the results of tumor cell mitochondrial stress test and the quantitative results of basal respiration (n=3-4); Figure (B) shows the results of tumor cell glycolysis stress test and the quantitative results of glycolysis (n=2-4); Figure (C) shows the mRNA level of CPT1A in tumor cells (n=3); Figure (D) shows the quantitative results of tumor cell scratch test (n=3); Figure (E) shows the quantitative results of tumor cell apoptosis (n=3), among which Annexin V + PI + Represents late apoptotic cells, Annexin V + PI - represents early apoptotic cells; Figure (F) is the quantitative result of calreticulin expression on the surface of tumor cells (n=4); Figure (G) is the quantitative result of MHC-I expression on the surface of tumor cells (n=4). Figure (H) is the quantitative result of ROS expression in tumor cells (n=4). Experimental group description: BC (untreated tumor cell group), Chito (tumor cell group treated with chitosan gel), ChitoCa (tumor cell group treated with chitosan gel containing calcium). *P represents <0.05, **P represents <0.01, ***P represents <0.001, ****P represents <0.0001. Glossary: ​​OCR: oxygen consumption rate; FCCP: oxidative phosphorylation uncoupler; ECAR: extracellular acidification rate; 2-DG: 2-deoxyglucose; CPT1A: carnitine palmitoyltransferase 1A; CRT: calreticulin; MHC-I: class I major histocompatibility complex; MFI: mean fluorescence intensity; ROS: reactive oxygen species.

[0024] Figure 4Figure 2 shows the effect of calcium-containing chitosan gel on immune cell metabolism and the secretion of proinflammatory factors and changes in surface Mincle receptors after co-culture of immune cells with calcium-containing chitosan gel; Figure (A) shows the results of immune cell mitochondrial stress test and the quantitative results of basal respiration (n=3-4); Figure (B) shows the results of immune cell glycolysis stress test and the quantitative results of glycolysis (n=4); Figure (C) shows the level of tumor necrosis factor (TNF-α) secreted by immune cells after co-culture with calcium-containing chitosan gel (n=3); Figure (D) shows the level of chemokine ligand 10 (CXCL10) secreted by immune cells after co-culture with calcium-containing chitosan gel (n=3); Figure (E) shows the level of interleukin 18 (IL-18) secreted by immune cells after co-culture with calcium-containing chitosan gel (n=3); Figure (F) shows the mRNA level of Mincle receptor after co-culture of immune cells with calcium-containing chitosan gel (n=3). Experimental group description: BC (untreated tumor cell group), Chito (tumor cell group treated with chitosan gel), ChitoCa (tumor cell group treated with chitosan gel containing calcium). *P represents <0.05, **P represents <0.01, ***P represents <0.001, ****P represents <0.0001.

[0025] Figure 5 The phagocytic ability and antigen presentation efficiency of immune cells after stimulation with calcium-containing chitosan gel on tumor cells. Figure (A) is a representative fluorescence confocal image of immune cells phagocytosing tumor cells after stimulation with calcium-containing chitosan gel, in which the left 1st column is the cell nucleus, the left 2nd column is the immune cell cytoplasm, the right 2nd column is the tumor cell membrane, the right 1st column is a merged image, the first row is a representative image of the BC group, and the second row is a representative image of the ChitoCa group; Figure (B) is the quantitative result of immune cell phagocytosis of tumor cells (n=2); Figure (C) is a representative image of immune cell antigen presentation efficiency (n=5). Description of experimental groups: BC (untreated tumor cell group), Chito (tumor cell group treated with chitosan gel), TcChito (tumor cell-loaded chitosan gel), ChitoCa (tumor cell group treated with calcium-containing chitosan gel), TcChitoCa (tumor cell-loaded calcium-containing chitosan gel), Mincle - / - RAW (RAW 264.7 cells with Mincle knockout and 4T1 cells simply mixed group), Mincle - / -RAW+TcChitoCa (RAW 264.7 cells with Mincle knockout and 4T1 cells mixed in a calcium-containing chitosan environment), RAW (wild-type RAW 264.7 cells and 4T1 cells simply mixed), RAW+TcChitoCa (RAW 264.7 cells with Mincle knockout and 4T1 cells mixed in a calcium-containing chitosan environment). *P represents <0.05, **P represents <0.01, ***P represents <0.001, ****P represents <0.0001. Glossary: ​​BMDC (bone marrow-derived dendritic cells).

[0026] Figure 6 The effect of calcium-containing chitosan gel combined with aPD-L1 in treating tumors; Figure (A) is the experimental scheme of combined treatment; Figure (B) is the growth curve of tumors in each group of mice (n=4); Figure (C) is the survival curve of each group of mice (n=4). Experimental group description: PBS (PBS-treated mice group), TcChitoCa (tumor cell-loaded calcium-containing chitosan gel-treated mice group), aPD-L1 (aPD-L1-treated mice group), TcChitoCa+aPD-L1 (tumor cell-loaded calcium-containing chitosan gel-treated mice and aPD-L1 combined treatment mice group). *P represents <0.05.

[0027] Figure 7 The results of combined treatment of tumors with calcium-containing chitosan gel and gemcitabine (GEM); Figure (A) is the experimental scheme of combined treatment; Figure (B) is the growth curve of tumors in each group of mice (n=6); Figure (C) is the survival curve of each group of mice (n=6). Experimental group description: PBS (PBS-treated mice group), TcChitoCa (tumor cell-loaded calcium-containing chitosan gel-treated mice group), GEM (gemcitabine-treated mice group), TcChitoCa+GEM (tumor cell-loaded calcium-containing chitosan gel-treated mice group and gemcitabine-treated mice group). *P represents <0.05, **P represents <0.01. DETAILED DESCRIPTION

[0028] The present invention will be described in detail below based on the accompanying drawings and preferred embodiments, and the purpose and effects of the present invention will become more clear. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] 1. Material Characterization and In Vitro Safety Evaluation

[0030] Example 1

[0031] 100 μL of 111 mg / mL calcium chloride solution and 200 mg / mL sodium polyaspartate (molecular weight 8000Da) were mixed evenly, 100 μL of 84 mg / mL sodium bicarbonate solution was added dropwise, and stirred for 10 minutes to obtain a prefabricated calcium carbonate nanoparticle suspension. 300 μL of the prefabricated calcium carbonate nanoparticle suspension was added dropwise to 800 μL of 25 mg / mL chitosan solution (molecular weight 5000Da) with a deacetylation degree greater than 80%, stirred until clear, the pH value of the solution was in the range of 6.7 to 7.4, and incubated in a water bath at 37°C for 15 minutes to obtain a calcium-containing chitosan hydrogel.

[0032] Example 2

[0033] 27.75mg / mL calcium chloride solution and 50mg / mL sodium polyaspartate (molecular weight 8000Da) were mixed evenly, and 100μL of 21mg / mL sodium bicarbonate solution was added dropwise, and stirred for 10 minutes to obtain a prefabricated calcium carbonate nanoparticle suspension. 300μL of the prefabricated calcium carbonate nanoparticle suspension was added dropwise to 800μL of 25mg / mL chitosan solution (molecular weight 5000Da) with a deacetylation degree greater than 80%, and stirred. At this time, because the pH value of the solution must be lower than 6.7, 84mg / mL sodium bicarbonate solution was added until the pH value of the solution was in the range of 6.7 to 7.4, stirred until clear, and incubated in a water bath at 37°C for 15 minutes to obtain a calcium-containing chitosan hydrogel.

[0034] Example 3

[0035] 55.5mg / mL calcium chloride solution and 100mg / mL sodium polyaspartate (molecular weight 8000Da) were mixed evenly, and 100mL of 42mg / mL sodium bicarbonate solution was added dropwise, and stirred for 10 minutes to obtain a prefabricated calcium carbonate nanoparticle suspension. 300μL of the prefabricated calcium carbonate nanoparticle suspension was added to 800μL of 25mg / mL chitosan solution (molecular weight 5000Da) with a deacetylation degree greater than 80%, and stirred. At this time, because the pH value of the solution must be lower than 6.7, 84mg / mL sodium bicarbonate solution was added until the pH value of the solution was within the range of 6.7 to 7.4, and stirred until clarified, and incubated in a 37°C water bath for 15 minutes to obtain a calcium-containing chitosan gel.

[0036] Example 4

[0037] 150 μL of 111 mg / mL calcium chloride solution and 200 mg / mL sodium polyaspartate (molecular weight 8000Da) were mixed evenly, and 150 μL of 84 mg / mL sodium bicarbonate solution was added dropwise, and stirred for 10 minutes to obtain a prefabricated calcium carbonate nanoparticle suspension. 450 μL of the prefabricated calcium carbonate nanoparticle suspension was added dropwise to 800 μL of 35 mg / mL chitosan solution (molecular weight 5000Da) with a deacetylation degree greater than 80%, and stirred until clarified, at which time the pH value of the solution was in the range of 6.7 to 7.4, and incubated in a 37°C water bath for 15 minutes to obtain a calcium-containing chitosan gel.

[0038] Example 5

[0039] 150 μL of 111 mg / mL calcium chloride solution and 200 mg / mL sodium polyaspartate (molecular weight 8000Da) were mixed evenly, and 150 μL of 84 mg / mL sodium bicarbonate solution was added dropwise, and stirred for 10 minutes to obtain a prefabricated calcium carbonate nanoparticle suspension. 450 μL of the prefabricated calcium carbonate nanoparticle suspension was added dropwise to 800 μL of 45 mg / mL chitosan solution (molecular weight 5000Da) with a deacetylation degree greater than 80%, and stirred until clarified, at which time the pH value of the solution was in the range of 6.7 to 7.4, and incubated in a 37°C water bath for 15 minutes to obtain a calcium-containing chitosan gel.

[0040] Example 6

[0041] 100 μL of 111 mg / mL calcium chloride solution and 200 mg / mL sodium polyaspartate (molecular weight 4000Da) were mixed evenly, and 100 μL of 84 mg / mL sodium bicarbonate solution was added dropwise, and stirred for 10 minutes to obtain a prefabricated calcium carbonate nanoparticle suspension. 300 μL of the prefabricated calcium carbonate nanoparticle suspension was added dropwise to 800 μL of 25 mg / mL chitosan solution (molecular weight 5000Da) with a deacetylation degree greater than 80%, and stirred until clear, the pH value of the solution was in the range of 6.7 to 7.4, and incubated in a water bath at 37°C for 15 minutes to obtain a calcium-containing chitosan hydrogel.

[0042] Taking the calcium-containing chitosan gel obtained in Example 1 as an example, calcium carbonate nanoparticles (green) were stained with calcein, chitosan (red) was stained with AF-555, and the distribution of each component was observed by confocal laser scanning microscope (CLSM). The porous structure of the gel was observed by cryo-scanning electron microscope (Cryo-SEM). The effect of the gel material on tumor cell proliferation was detected by RTCA-DP real-time unlabeled cell analyzer. The toxicity of the gel material to immune cells was detected by CCK-8 experiment.

[0043] The experimental results are as follows:

[0044] The CLSM results showed that the calcium carbonate nanoparticles were evenly dispersed in the chitosan gel under this system. Figure 1 As shown in (A) in .

[0045] Cryo-SEM results show that the pore size of the gel material is about 12 μm. Figure 1 As shown in (B), its pore size allows the infiltration of immune cells.

[0046] The results of RTCA-DP real-time label-free cell analyzer showed that chitosan gel and chitosan gel containing calcium had no effect on the proliferation of tumor cells 4T1. Figure 1 As shown in (C) in .

[0047] The results of CCK-8 experiments showed that calcium-containing chitosan gel had no toxicity to immune cell RAW 264.7 cells within the concentration range of 0.25-8 mg / mL (calculated based on the concentration of chitosan). Figure 1 As shown in (D) in .

[0048] These data indicate that calcium carbonate nanoparticles are uniformly distributed in chitosan hydrogels, their pore size satisfies the infiltration of immune cells, and they are non-toxic to tumor cells and immune cells.

[0049] 2. Evaluation of the effect of materials in removing intracellular lipids

[0050] After incubating tumor cells with chitosan solution (COS), chitosan gel (Chito), and chitosan gel containing calcium (ChitoCa) for 24 hours, the materials were removed to obtain cells (the concentration of chitosan in each group was the same). The lipid droplets and nuclei of tumor cells were stained with BODIPY and Hoechst, respectively, the lipid droplet content of each group of cells was observed by CLSM, and the number and size of intracellular lipid droplets were quantitatively analyzed by ImageJ.

[0051] The experimental results are as follows:

[0052] Through CLSM and quantitative results, it can be observed that chitosan solution can only slightly reduce the number and size of intracellular lipid droplets in tumor cells, while calcium-containing chitosan gel greatly promotes the reduction of the number and size of intracellular lipid droplets after treating tumor cells for 24 hours. In the absence of calcium carbonate nanoparticles, the size of lipid droplets increases, indicating that lipids tend to redeposit in the absence of calcium. Figure 2 As shown in (A) to (B) in the figure.

[0053] These data suggest that, at the same concentration, cross-linked chitosan hydrogels are superior to chitosan solutions in eliminating lipid droplets.

[0054] 3. Changes in tumor cell metabolism and phenotype after treatment with calcium-containing chitosan gel

[0055] After the tumor cells were treated with calcium-containing chitosan gel for 24 hours, the aerobic respiration and glycolysis levels of the tumor cells were detected by the mitochondrial stress test and glycolysis stress test of the hippocampal test, respectively. The mRNA level of carnitine palmitoyltransferase 1A (CPT1A) in the cells was detected by real-time quantitative PCR (q-PCR) after the tumor cells were treated with calcium-containing chitosan gel for 24 hours. The migration ability of tumor cells in the environment of calcium-containing chitosan gel was detected by scratch test. The apoptosis, surface calreticulin (CRT) expression, surface MHC-I expression and the generation of intracellular reactive oxygen species (ROS) of tumor cells treated with calcium-containing chitosan gel for 24 hours were detected by flow cytometer.

[0056] The experimental results are as follows:

[0057] The mitochondrial stress test of the hippocampus test demonstrated that 4T1 cells treated with calcium-containing chitosan gel showed a significant increase in mitochondrial respiratory capacity, indicating enhanced oxidative phosphorylation. Consistent with the experiment of lipid droplet degradation, the presence of calcium significantly promoted oxidative phosphorylation, indicating that calcium has a synergistic effect on fatty acid degradation. Figure 3 As shown in (A) in .

[0058] The hippocampal glycolysis stress test showed that there was no significant change in the glycolysis of 4T1 cells after treatment with chitosan gel and chitosan gel containing calcium. Figure 3 As shown in (B) in .

[0059] The mRNA level of CPT1A in 4T1 cells was detected by q-PCR. The results showed that the intracellular CPT1A content increased after 4T1 cells were treated with chitosan gel for 24 hours. CPT1A mediates the transfer of long-chain fatty acids from the mitochondrial outer membrane to the matrix for β-oxidation. Figure 3 As shown in (C) in .

[0060] The above data prove that under the influence of calcium-containing chitosan gel, free fatty acids in tumor cells are transported to mitochondria for β-oxidation.

[0061] The scratch test proved that the migration ability of 4T1 cells was greatly reduced in the environment of calcium-containing chitosan gel due to the enhanced lipid catabolism. Figure 3 As shown in (D) in .

[0062] The results of flow cytometry analysis showed that compared with untreated 4T1 cells, the number of apoptotic 4T1 cells increased after 24 hours of treatment with calcium-containing chitosan gel. Figure 3 As shown in (E) in .

[0063] The results of flow cytometry analysis showed that compared with untreated 4T1 cells, the CRT on the surface of 4T1 cells increased after 24 hours of treatment with calcium-containing chitosan gel. In order to escape immune recognition, lipid droplets can isolate CRT to reduce CRT exposure. This result proves that the treatment of calcium-containing chitosan gel reverses the escape of tumor cell immune recognition. Figure 3 As shown in (F) in .

[0064] The results of flow cytometry analysis showed that compared with untreated 4T1 cells, the number of MHC-I on the surface of 4T1 cells increased after 24 hours of treatment with calcium-containing chitosan gel. In many types of cancer, major histocompatibility class MHC-I molecules are also a common immune escape mechanism. This result also proves that the treatment of calcium-containing chitosan gel reverses the immune recognition escape of tumor cells. Figure 3 As shown in (G) in .

[0065] Flow cytometry results showed that 4T1 cells produced more ROS after 24 h of treatment with calcium-containing chitosan gel compared with untreated 4T1 cells, indicating that lipid catabolism was overactivated, leading to lipid stress. Figure 3 As shown in (H) in .

[0066] These data indicate that the immunogenicity of 4T1 cells was greatly enhanced after treatment with calcium-containing chitosan gel, and the shift in immunogenic phenotype was attributed to the deprivation of intracellular lipids by chitosan gel.

[0067] 4. Effects of Calcium-containing Chitosan Gel on Immune Cells

[0068] After the immune cells were treated with calcium-containing chitosan gel for 24 hours, the aerobic respiration and glycolysis levels of the immune cells were detected by the mitochondrial stress test and glycolysis stress test of the hippocampus test, respectively. The levels of proinflammatory factors TNF-α, CXCL10, and IL-18 secreted by immune cells after treatment with calcium-containing chitosan gel for 24 hours were detected by ELISA kits. The mRNA level of C-type lectin receptor Clec4e (Mincle) was detected by real-time quantitative PCR (q-PCR) after immune cells were treated with calcium-containing chitosan gel for 24 hours.

[0069] The experimental results are as follows:

[0070] The RAW 264.7 cells treated with calcium-containing chitosan gel showed a decrease in mitochondrial respiratory capacity, indicating a decrease in oxidative phosphorylation, as demonstrated by the hippocampal test mitochondrial stress test. Figure 4 As shown in (A) in .

[0071] The glycolysis stress test of hippocampus test showed that the glycolysis of 4T1 cells treated with chitosan gel and chitosan gel containing calcium was slightly increased. Figure 4 As shown in (B) in .

[0072] ELISA results show that immune cells treated with chitosan gel secrete a large number of pro-inflammatory factors. Figure 4 As shown in (C), (D) and (E).

[0073] The mRNA level of Mincle in 4T1 cells was detected by q-PCR. The results showed that the content of Mincle increased after 4T1 cells were treated with chitosan gel for 24 hours. Figure 4 As shown in (F) in .

[0074] These data show that after treatment with calcium-containing chitosan gel, oxidative phosphorylation of immune cells was reduced, aerobic glycolysis was slightly increased, and pro-inflammatory factors were induced, proving that immune cells were activated. At the same time, the C-type lectin receptor Mincl on the surface of immune cells was upregulated, and the formation of anti-tumor immune niches in the tumor microenvironment was promoted.

[0075] 5. Macrophage phagocytosis of tumor cells and antigen presentation

[0076] 4T1 cells (red) were labeled with PKH26, and RAW 264.7 cells (green) were labeled with Cell-Tracker Green CMFDA, and co-cultured with calcium-containing chitosan gel. The phagocytosis of tumor cells by macrophages was observed by CLSM. Quantitative data of phagocytosis were obtained by flow cytometry. Surface co-stimulatory factors and specific antigen cross-presentation of immune cells after stimulation with calcium-containing chitosan gel were analyzed by flow cytometry.

[0077] The experimental results are as follows:

[0078] Through CLSM, we can observe that in the environment of calcium-containing chitosan gel, macrophages can engulf tumor cells and digest them into small particles. However, when the two types of cells are simply mixed, the tumor cells are still intact. Figure 5 As shown in (A) in .

[0079] Flow cytometry results showed that in the presence of calcium-containing chitosan, the phagocytic ability of wild-type RAW 264.7 cells to tumors increased by more than 10 times, while the phagocytic ability of Mincle-knocked RAW 264.7 cells to tumors did not change much. This indicates that the activation of Mincle plays a key role in the phagocytosis of tumors. Figure 5 As shown in (B) in .

[0080] Flow cytometry results showed that after immune cells were stimulated by calcium-containing chitosan gel loaded with tumor cells B16-OVA, the surface co-stimulatory factor CD86 and the specific antigen cross-presentation marker SIINFEKL-H2Kb were greatly upregulated.

[0081] These data indicate that, under the stimulation of calcium-containing chitosan gel, immune cells phagocytose tumor cells and complete efficient antigen presentation.

[0082] 6. Calcium-containing chitosan gel combined with aPD-L1 therapy

[0083] Mice were inoculated with 10 6 4T1 tumor cells were injected intratumorally, followed by intratumoral injection of tumor cell-loaded calcium-containing chitosan gel on days 3, 6, 9, and 12, and intraperitoneal injection of 200 μg aPD-L1 on days 3, 6, 9, 12, 15, and 18. The tumor growth curve and survival curve of the mice were recorded during the following experiments.

[0084] The experimental results are as follows:

[0085] The results showed that the combined treatment of calcium-containing chitosan gel and aPD-L1 could significantly delay tumor growth and prolong the survival time of tumor-bearing mice.

[0086] These data suggest that chitosan reduces intracellular lipids and sensitizes cells to the antitumor effects of aPD-L1.

[0087] 7. Calcium-containing chitosan gel combined with aPD-L1 therapy

[0088] Mice were inoculated with 10 6 4T1 tumor cells were injected intratumorally, and then on days 3, 6, 9 and 12, tumor cell-loaded chitosan gel containing calcium was injected intratumorally, and 75 μg / g GEM was injected intraperitoneally on days 3, 6, 9 and 12. The tumor growth curve and survival curve of the mice were recorded during the following experiments.

[0089] The experimental results are as follows:

[0090] The results showed that the combined treatment of calcium-containing chitosan gel and GEM could significantly delay the growth of tumors and prolong the survival time of tumor-bearing mice with a survival rate of 33%.

[0091] These data suggest that reducing the levels of lipid droplets in tumor cells increases the immunosuppressive effect of gemcitabine on tumors.

[0092] Those skilled in the art can understand that the above are only preferred examples of the invention and are not intended to limit the invention. Although the invention is described in detail with reference to the above examples, those skilled in the art can still modify the technical solutions recorded in the above examples or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, etc. made within the spirit and principle of the invention shall be included in the protection scope of the invention.

Claims

1. A method for preparing a calcium-containing chitosan hydrogel, characterized in that: The method comprises the following steps: Step 1: Mix equal volumes of 27.75-111 mg / mL calcium chloride solution and 50-200 mg / mL sodium polyaspartate solution with a molecular weight of 4000-8000 Da, stir until uniform, add 21-84 mg / mL sodium bicarbonate solution dropwise to the mixed solution, stir at room temperature for 5-15 minutes, and obtain a calcium carbonate nanoparticle suspension; Step 2: Add 300-450 μL of calcium carbonate nanoparticle suspension to 800 μL of 15-45 mg / mL chitosan solution with a deacetylation degree greater than 80%, stir until clear, test the pH value of the solution, if it is not in the range of 6.7-7.4, add sodium bicarbonate solution to make the pH value of the solution in the range of 6.7-7.4; then bathe at 37 °C for 5-15 minutes to obtain calcium-containing chitosan hydrogel.

2. The method for preparing calcium-containing chitosan hydrogel according to claim 1, characterized in that: In step 1, the concentration of calcium chloride solution is 111 mg / mL, the molecular weight of sodium polyaspartate is 8000 Da, the concentration of sodium polyaspartate solution is 200 mg / mL, and the concentration of sodium bicarbonate solution is 84 mg / mL; in step 2, the volume of calcium carbonate nanoparticle suspension is 300 μL, and the concentration of chitosan solution is 25 mg / mL.

3. The method for preparing calcium-containing chitosan hydrogel according to claim 1, characterized in that: The stirring is magnetic stirring.

4. A calcium-containing chitosan hydrogel prepared by the method according to claim 1.

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