Polyamino acid composite hydrogel and application thereof in preparation of osteosarcoma treatment preparation

Abstract

The invention provides a polyamino acid composite hydrogel, which comprises a polyamino acid hydrogel and an immunomodulator loaded in the polyamino acid hydrogel, the immunomodulator is selected from at least one of iron-based nanoparticles, a Toll-like receptor stimulant and a CD47 inhibitor, and the polyamino acid hydrogel is selected from polymethionine hydrogel. According to the invention, the immunomodulator with immunoregulation capability is efficiently loaded in a specific type of polyamino acid hydrogel, and the hydrogel capable of being locally and sustainably released is formed at a tumor part, so that the toxicity to normal tissues can be effectively reduced, the local anti-tumor immunocompetence of a tumor focus is improved, and the tumor tumor treatment effect is improved. Therefore, the purpose of improving the immune regulation ability of the tumor patient on the premise of ensuring no relapse after operation is achieved.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a polyamino acid composite hydrogel and an application thereof in preparing an osteosarcoma treatment preparation. Background Art

[0002] Osteosarcoma is one of the most common malignant tumors in children. It often occurs around the knee joint and has a high tendency to local invasion and metastasis. Although surgery combined with chemotherapy has greatly improved the prognosis of osteosarcoma patients, the prognosis of metastatic or recurrent osteosarcoma remains unsatisfactory. Immunotherapy has been shown to be a promising strategy for the treatment of human malignancies and has shown advantages in effective anti-osteosarcoma effects, inhibition of metastasis and recurrence.

[0003] The human immune system has been in a continuous battle with osteosarcoma cells, including three stages: immune clearance, balance and escape. In addition, the immune system plays an important role in the execution and development of anti-tumor immunity. Therefore, using the body's immune function to more effectively inhibit and treat cancer has become the focus of immunotherapy. The immunosuppressive microenvironment caused by osteosarcoma surgery greatly increases the risk of osteosarcoma recurrence and metastasis. Therefore, the development of new strategies to regulate the tumor immune microenvironment needs to be addressed.

[0004] Currently, a variety of biomaterials, including nano- or microparticles, scaffolds, hydrogels, etc., have been developed to improve anti-tumor immune responses, with obvious advantages: biomaterials such as nanoparticles have adjustable sizes, and appropriate particle sizes can promote their selective aggregation in draining lymph nodes; they can also release tumor-associated antigens by inducing immunogenic cell death, or precisely target specific immune cell types through surface modification; biodegradable hydrogels and other materials are used as carriers for local tumor microenvironment treatment. Due to their advantages such as strong adhesion, plasticity, and controlled release, they can effectively fill tissue cavity defects and promote regeneration and repair according to the needs of osteosarcoma surgery.

[0005] Specifically, hydrogels have a three-dimensional network structure with unique porous structure, mechanical properties, biocompatibility and bioactivity, and have become valuable drug delivery carriers in cancer treatment and tissue engineering. The sensitivity of hydrogels to the tumor microenvironment cannot be ignored in local sustained treatment, and the composition of hydrogels should be simplified as much as possible to facilitate mass production.

[0006] Therefore, it is necessary to develop a tumor microenvironment-responsive composite hydrogel that continuously releases immunomodulators to enhance the anti-tumor immunity of osteosarcoma patients while ensuring that there is no recurrence after surgery. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a polyamino acid composite hydrogel and its application in the preparation of osteosarcoma treatment preparations. The polyamino acid composite hydrogel provided by the present invention can be used to inhibit the recurrence and metastasis of osteosarcoma.

[0008] The present invention provides a polyamino acid composite hydrogel, comprising a polyamino acid hydrogel and an immunomodulator loaded in the polyamino acid hydrogel, wherein the immunomodulator is selected from at least one of iron-based nanoparticles, Toll-like receptor agonists and CD47 inhibitors, and the polyamino acid hydrogel is selected from polymethionine hydrogel.

[0009] Preferably, the mass ratio of the polyamino acid hydrogel to the immunomodulator is in the range of 80:1 to 80:60.

[0010] Preferably, the polyamino acid is selected from polyethylene glycol-polymethionine.

[0011] Preferably, the iron-based nanoparticles are selected from carboxymethyl nano-iron oxide nanoparticles;

[0012] The Toll-like receptor agonist is selected from R848;

[0013] The CD47 inhibitor is selected from PBD-150.

[0014] The present invention also provides a method for preparing the polyamino acid composite hydrogel, comprising the following steps:

[0015] The polyamino acid solution and the immunomodulator solution are mixed and stirred and then gelled to obtain the polyamino acid composite hydrogel.

[0016] Preferably, the method for preparing the polyamino acid solution comprises the following steps:

[0017] A) reacting polyethylene glycol diamine with methionine-N-ring carboxylic anhydride to obtain polyethylene glycol-polymethionine;

[0018] B) Dissolve polyethylene glycol-polymethionine in PBS buffer solution to obtain a polyamino acid solution.

[0019] Preferably, the methionine-N-intra-ring carboxylic anhydride is prepared from methionine and triphosgene.

[0020] Preferably, the mixing and stirring temperature is 0-20°C.

[0021] Preferably, the gelling temperature is 10-50° C. and the time is 1-10 min.

[0022] The present invention also provides an application of the polyamino acid composite hydrogel in preparing a preparation for treating osteosarcoma.

[0023] Compared with the prior art, the present invention provides a polyamino acid composite hydrogel, including a polyamino acid hydrogel and an immunomodulator loaded in the polyamino acid hydrogel, wherein the immunomodulator is selected from at least one of iron-based nanoparticles, Toll-like receptor agonists and CD47 inhibitors, and the polyamino acid hydrogel is selected from polymethionine hydrogel. The present invention efficiently loads an immunomodulator with immunomodulatory ability in a specific type of polyamino acid hydrogel to form a local hydrogel that can be released sustainably at the tumor site, which can effectively reduce toxicity to normal tissues and improve the local anti-tumor immunity of the tumor lesion, thereby achieving the purpose of improving the immunomodulatory ability of tumor patients while ensuring no recurrence after surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the cytotoxicity test of polyamino acid materials;

[0025] Figure 2 This is a diagram showing the gelation effect of iron-based nanoparticle composite hydrogel;

[0026] Figure 3 The experimental results of iron-based nanoparticle composite hydrogel inhibiting postoperative tumor recurrence;

[0027] Figure 4 The regulation of immune microenvironment by iron-based nanoparticle composite hydrogel in postoperative tumor recurrence model;

[0028] Figure 5 Results of tumor inhibition and metastasis experiments for orthotopic osteosarcoma;

[0029] Figure 6 This is the result of the regulation of the immune microenvironment by the composite hydrogel loaded with immunomodulators in orthotopic osteosarcoma. DETAILED DESCRIPTION

[0030] The present invention provides a polyamino acid composite hydrogel, comprising a polyamino acid hydrogel and an immunomodulator loaded in the polyamino acid hydrogel, wherein the immunomodulator is selected from at least one of iron-based nanoparticles, Toll-like receptor agonists and CD47 inhibitors, and the polyamino acid hydrogel is selected from polymethionine hydrogel.

[0031] In the present invention, the polyamino acid is selected from polyethylene glycol-polymethionine.

[0032] The iron-based nanoparticles are selected from carboxymethyl nano-iron oxide nanoparticles;

[0033] The Toll-like receptor agonist is selected from R848;

[0034] The CD47 inhibitor is selected from PBD-150.

[0035] In some specific embodiments of the present invention, the immunomodulator is selected from iron-based nanoparticles, or a composite immunomodulator formed by a Toll-like receptor agonist and a CD47 inhibitor.

[0036] Preferably, in the composite immunomodulator, the mass ratio of the Toll-like receptor agonist and the CD47 inhibitor is 1:1 to 1:10, and can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any value between 1:1 and 1:10. When the Toll-like receptor agonist and the CD47 inhibitor are used in combination and loaded in a polyamino acid hydrogel, the tumor immune microenvironment in vivo can be significantly improved.

[0037] Among them, the mass ratio of the polyamino acid hydrogel to the immunomodulator is 80:1 to 80:60, and can be 80:1, 80:5, 80:10, 80:15, 80:20, 80:25, 80:30, 80:35, 80:40, 80:45, 80:50, 80:55, 80:60, or any value between 80:1 and 80:60.

[0038] The present invention also provides a method for preparing the polyamino acid composite hydrogel, comprising the following steps:

[0039] The polyamino acid solution and the immunomodulator solution are mixed and stirred and then gelled to obtain the polyamino acid composite hydrogel.

[0040] Specifically, the present invention first prepares a polyamino acid solution, and the preparation method of the polyamino acid solution comprises the following steps:

[0041] A) reacting polyethylene glycol diamine with methionine-N-ring carboxylic anhydride to obtain polyethylene glycol-polymethionine;

[0042] B) Dissolve polyethylene glycol-polymethionine in PBS buffer solution to obtain a polyamino acid solution.

[0043] Among them, methionine-N-intra-ring carboxylic anhydride is prepared from methionine and triphosgene.

[0044] Specifically, under protective atmosphere conditions, methionine is reacted with triphosgene to obtain a reaction product. The protective atmosphere conditions are selected from nitrogen atmosphere, and the triphosgene is reacted with methionine in multiple times. The reaction temperature is 40 to 80°C, which can be 40, 45, 50, 55, 60, 65, 70, 75, 80, or any value between 40 and 80°C, and the reaction time is 1 to 8 hours, which can be 1, 2, 3, 4, 5, 6, 7, 8, or any value between 1 to 8 hours.

[0045] Then, the reaction product is post-treated to obtain methionine-N-intra-ring carboxylic anhydride. The present invention has no particular limitation on the post-treatment method, and preferably, the post-treatment can be performed according to the following method:

[0046] After removing part of the solvent from the reaction product, the product is precipitated into ice n-hexane, then dissolved in ethyl acetate and poured into a separatory funnel, and washed with ice water several times. The upper liquid is mixed with anhydrous magnesium sulfate and dried at -20°C. After drying, it is filtered using a sand core funnel to obtain a filtrate, and the filtrate is vacuum dried to remove ethyl acetate to obtain a solid. Anhydrous THF is added to the solid, heated to dissolve completely, and anhydrous n-hexane is added several times until the precipitate cannot be dissolved back, and then anhydrous THF is added to dissolve the precipitate back. Cool down and crystallize, pour out the supernatant after complete crystallization, and vacuum dry the remaining part to obtain methionine-N-ring carboxylic anhydride.

[0047] After obtaining methionine-N-intracyclic carboxylic anhydride, polyethylene glycol diamine is reacted with methionine-N-intracyclic carboxylic anhydride to obtain polyethylene glycol-polymethionine.

[0048] Specifically, polyethylene glycol diamine is dehydrated and dissolved in anhydrous DMF, and then methionine-N-intra-ring carboxylic anhydride is added, and the reaction is carried out at room temperature for 24 to 120 hours, which can be 24, 36, 48, 60, 84, 120, or any value between 24 and 120 hours to obtain a reaction product. In the present invention, room temperature is defined as 25±5°C.

[0049] Wherein, the molecular weight range of polyethylene glycol diamine is 500-10000, and can be 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or any value between 500 and 10000.

[0050] The obtained reaction product is post-processed. The present invention has no particular limitation on the post-processing method. Preferably, the post-processing comprises the following steps:

[0051] The reaction product was precipitated with icy ether and then separated into solid and liquid to obtain a solid. The solid was redissolved with DMF and then dialyzed to obtain a dialyzed solution, and the dialyzed solution was lyophilized to obtain polyethylene glycol-polymethionine.

[0052] Wherein, the specification of the dialysis bag is 500-10000 KDa, and can be 500, 1000, 2000, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or any value between 500 and 10000.

[0053] After obtaining polyethylene glycol-polymethionine, the polyethylene glycol-polymethionine is dissolved in a PBS buffer solution to obtain a polyamino acid solution. The concentration of the polyamino acid solution is 4-20%, and can be 4%, 6%, 8%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, or any value between 4 and 20%.

[0054] The immunomodulator is dispersed in a solvent to obtain an immunomodulator solution, wherein the solvent is preferably a PBS solution.

[0055] Then, the polyamino acid solution and the immunomodulator solution are mixed and stirred to obtain a mixed solution, wherein the temperature of the mixed stirring is 0 to 20°C, which can be 0, 2, 4, 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, or any value between 0 and 20°C, and the time is 5 to 120 hours, which can be 5, 12, 24, 48, 60, 72, 84, 120, or any value between 5 and 120 hours. In the mixed solution, the mass concentration of the polyamino acid is preferably 6% to 9%, which can be 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, or any value between 6% and 9%.

[0056] Next, gelation is carried out at 10 to 50° C. for 1 to 10 min to obtain a polyamino acid composite hydrogel. The gelation temperature can be 10, 15, 20, 25, 30, 35, 40, 45, 50, or any value between 10 and 50° C., and the gelation time can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 1 and 10 min.

[0057] The present invention also provides an application of the polyamino acid composite hydrogel in preparing a therapeutic preparation for osteosarcoma. The therapeutic preparation is used to inhibit the recurrence and metastasis of osteosarcoma.

[0058] The polyamino acid composite hydrogel provided by the present invention can inhibit the recurrence of osteosarcoma in vivo, and continuously release immunomodulators with different responsiveness under different microenvironments to perform immune regulation.

[0059] The polyamino acid composite hydrogel provided by the present invention continuously releases immunomodulators in the local microenvironment after osteosarcoma surgery, regulates tumor-associated macrophages, enhances anti-tumor immunity, and reduces the recurrence rate of osteosarcoma after surgery. The polyamino acid composite hydrogel provided by the present invention can prevent the recurrence of osteosarcoma, reduce the side effects of immune adjuvants, and enhance the anti-tumor immunity of tumor patients after surgery.

[0060] The present invention efficiently loads immunomodulators with immune regulation capabilities into a specific type of polyamino acid hydrogel to form a local hydrogel that can be sustainably released at the tumor site, which can effectively reduce toxicity to normal tissues and enhance the local anti-tumor immunity of the tumor lesion, thereby achieving the purpose of enhancing the immune regulation ability of tumor patients while ensuring no recurrence after surgery.

[0061] In order to further understand the present invention, the polyamino acid composite hydrogel provided by the present invention and its application in the preparation of bone and flesh treatment preparations are described below in conjunction with examples. The protection scope of the present invention is not limited by the following examples.

[0062] Example 1

[0063] 1. Synthesis of methionine-N-intracyclic carboxylic anhydride (L-methionine NCA)

[0064] Select an anhydrous and oxygen-free three-necked flask, weigh 20g of methionine (L-methionine), add it and evacuate it for 30min, add the reaction solvent anhydrous tetrahydrofuran (THF), and stir it in an oil bath at 60℃ under nitrogen protection. Add triphosgene 30g (10g+10g+10g) three times under nitrogen conditions, and react for 3h under nitrogen circulation. After the reaction, turn on the nitrogen to evaporate the reaction solution to 50mL, and slowly settle it in 300mL of ice n-hexane. Then dissolve it in ethyl acetate (250mL) and pour it into a separatory funnel, and wash it with ice water three times. Pour the upper liquid into a conical flask, add anhydrous magnesium sulfate, and dry it at -20℃ for 12h. After drying, filter it with a sand core funnel to obtain the filtrate, and put the filtrate into an ampoule for vacuum drying to remove ethyl acetate to obtain a solid. Add 15 mL of anhydrous THF, heat at 40°C to dissolve completely, add 1 mL of anhydrous n-hexane each time until the precipitate cannot be dissolved back, then add 3 mL of anhydrous THF to dissolve the precipitate back. Cool down and crystallize, pour out the supernatant after crystallization is complete, and vacuum dry the remainder to obtain 11 g of the product L-methionine NCA with a yield of 55%, and the structure is shown in Formula I.

[0065]

[0066] 2-Polyethylene glycol-polymethionine (PMet n -PEG-PMet n )

[0067] Select an anhydrous reaction bottle with a branch, add 4g of NH2-PEG-NH2 with a molecular weight of 4000 and 400mL of toluene to perform azeotropic dehydration at 125°C. Then drain the toluene and dissolve the solid with about 80mL of anhydrous N,N-dimethylformamide (DMF). Add 8.7g of L-methionine NCA and react at room temperature for 3 days, during which the carbon dioxide produced by the reaction in the bottle is removed. After the reaction is over, slowly settle into 600mL of ice ether and filter to obtain a solid. The solid is re-dissolved with DMF and dialyzed with a 3500KDa dialysis bag. The dialysis water is replaced every 12h for three days. The dialyzed solution is freeze-dried with a freeze dryer to obtain 6.6g of light yellow solid, with an average of 25 methionines polymerized on each polyethylene glycol, a yield of 52%, and the structure is shown in Formula II.

[0068]

[0069] 3 Safety of polymethionine hydrogel

[0070] The safety of polymethionine materials was tested using a fibroblast cell line (L929). L929 cells were seeded at 3000 cells / well on a 96-well plate containing DMEM medium. After 24 hours, different concentrations of PMet were used to n -PEG-PMet n Solution (concentrations: 0, 0.75, 1.5, 3, 6, 12 mg mL -1 ) were treated with the cells. The cells were placed in a humidified incubator at 37°C and 5% CO2 for 72 h, then the medium was removed and the cells were washed three times. CCK-8 reagent was then added and the cells were incubated at 37°C for 1.5 h. The absorbance was obtained on a microplate reader at 450 nm (Bio-Rad, USA) to measure cell viability. The results are shown in Figure 1 , Figure 1 The results of the cytotoxicity test of polyamino acid materials are shown in Figure 2. Figure 1 It can be seen that polyamino acid materials are not toxic to normal cells.

[0071] 4 Iron-based nanoparticle composite hydrogel

[0072] 4.1 Preparation of iron-based nanoparticles

[0073] Under the protection of inert gas, carboxymethyl dextran sodium salt, ferric chloride and ferrous chloride are dissolved in 9mL of oxygen-free deionized water. The above solution is cooled to 0°C, and a pre-cooled alkaline solution of ammonium hydroxide (28%, 0.75mL) is added dropwise at a rate of 0.5mL / min under rapid magnetic stirring. After removing the inert gas protection, under vigorous stirring, the solution temperature is quickly raised to 80°C and maintained for 10min. After the solution is cooled to room temperature, a 100KDa dialysis bag is used for overnight dialysis to remove unreacted reagents. The above solution is filtered through a 0.22μm filter and stored at 4°C in the dark to obtain iron-based nanoparticles.

[0074] 4.2 Preparation of iron-based nanoparticle composite hydrogel

[0075] The polyamino acid (160.0 mg) prepared in step 2 was dissolved in 1.0 mL of PBS buffer solution and stirred at low temperature. Then 1 mL of PBS solution containing 40 mg of iron-based nanoparticles was prepared. The two solutions were mixed and stirred at low temperature of 2°C for 5 h. As for the hydrogel formed after 3 min at 37°C in a constant temperature box, the loading rate of iron-based nanoparticles in the hydrogel was 100%. The results are shown in Figure 2 ,Depend on Figure 2 It can be seen that the polymer can effectively load iron-based nanoparticles and form hydrogel at 37°C.

[0076] 4.3 Inhibition of postoperative tumor recurrence by iron-based nanoparticle composite hydrogel in vivo

[0077] (1) Establishment of animal model

[0078] Mouse osteosarcoma cells K7M2 were injected into the back of nude mice and grew into subcutaneous tumors. The tumor tissue blocks were separated into equal volumes and transplanted into the back of BALB / c mice (6-8 weeks old, about 18g). If the tumor growth curve showed a stable upward trend 8-12 days after transplantation, it means that the mouse osteosarcoma model was successfully established.

[0079] (2) Experiment on the inhibition of postoperative tumor recurrence by iron-based nanoparticle composite hydrogel

[0080] The mice with successful transplantation were randomly divided into three groups (n=6). After anesthesia, the dorsal tumor was partially removed, leaving 120 mm 3 Tumor tissue blocks were used as a postoperative recurrent tumor model for osteosarcoma. In the postoperative recurrent tumor model for osteosarcoma, 100 μL of iron oxide nanoparticles composite polyamino acid hydrogel was locally injected on the 3rd, 6th, and 9th days after partial tumor resection, and tumor growth and survival were monitored by continuous measurement of tumor volume. The tumor monitoring results of the first batch of mice showed that iron oxide nanoparticles composite polyamino acid hydrogel can reduce the postoperative recurrence rate and prolong the survival of the osteosarcoma mouse model. The tumor volume (V) was calculated using formula (1), where L and W (mm) represent the length and width of the tumor, respectively.

[0081]

[0082] Results Figure 3 , Figure 3 The results of the experiment on iron-based nanoparticle composite hydrogel inhibiting postoperative tumor recurrence. Figure 3 It can be seen that on day 18, the tumor volume of mice in the PBS group was 1640 mm 3 The tumor volume of mice in the iron-based nanoparticle group was 312 mm 3 The tumor volume of mice in the iron-based nanoparticle composite polyamino acid hydrogel group was 210 mm 3 . Regarding the recurrence rate, all mice in the PBS group relapsed, 30% of mice in the iron-based nanoparticle group relapsed, and the recurrence rate of the iron-based nanoparticle composite polyamino acid hydrogel was only 10%. Compared with the PBS component, the iron oxide nanoparticle composite polyamino acid hydrogel can effectively reduce the postoperative recurrence rate of the osteosarcoma mouse model to 10%, and prolong the survival of the mice to 180 days, achieving the purpose of reducing the recurrence of osteosarcoma and reducing the toxicity of the body.

[0083] 4.4 Iron-based nanoparticle composite hydrogels regulate postoperative tumor immunity

[0084] (1) Establishment of animal model

[0085] Mouse osteosarcoma cells K7M2 were injected into the back of nude mice and grew into subcutaneous tumors. The tumor tissue blocks were separated into equal volumes and transplanted into the back of BALB / c mice (6-8 weeks old, about 18g). If the tumor growth curve showed a stable upward trend 8-12 days after transplantation, it means that the mouse osteosarcoma model was successfully established.

[0086] (2) Detecting the effect of iron-based nanoparticle composite hydrogel on the immune microenvironment in a postoperative tumor recurrence model

[0087] The mice with successful transplantation were randomly divided into three groups (n=6). After anesthesia, the dorsal tumor was partially removed, leaving 120 mm 3 Tumor tissue blocks were used as a model of postoperative recurrent tumors in osteosarcoma. In the postoperative recurrent tumor model of osteosarcoma, 100 μL of iron oxide nanoparticles composite polyamino acid hydrogel was injected locally on the 3rd, 6th, and 9th days after partial tumor resection, and the changes in the tumor immune microenvironment were analyzed by flow cytometry of tumor tissue. The tumor monitoring results of the first batch of mice showed that iron oxide nanoparticles composite polyamino acid hydrogel can reduce the postoperative recurrence rate and prolong the survival of the osteosarcoma mouse model. The results are shown in Figure 4 , Figure 4The iron-based nanoparticle composite hydrogel regulates the immune microenvironment in the postoperative tumor recurrence model. The proportion of macrophages in tumor tissue in the PBS group was 8%, the proportion of macrophages in tumor tissue in the iron-based nanoparticle group increased by 28% compared with the PBS group, and the proportion of macrophages in tumor tissue in the iron-based nanoparticle composite polyamino acid hydrogel group increased by 41% compared with the PBS group. Figure 4 It can be seen that iron oxide nanoparticles composite polyamino acid hydrogel can polarize macrophages to M1 macrophages, and the phagocytic effect of activated macrophages on K7M2 (osteosarcoma cells) is significantly enhanced, accelerating the activation of acquired immune response while killing tumor cells.

[0088] Example 2

[0089] Toll-like receptor agonist and CD47 inhibitor composite hydrogel

[0090] 1. Preparation of composite hydrogel containing Toll-like receptor agonist and CD47 inhibitor

[0091] The polyamino acid (80.0 mg) obtained in step 3 of Example 1 was dissolved in 1.0 mL of PBS buffer solution and stirred at low temperature, and then Toll-like receptor agonist R848 (3.0 mg) and CD47 inhibitor PBD-150 (6.0 mg) were added, and the solution was mixed and stirred at low temperature for 3 days. After 37°C for 3 minutes in a constant temperature box, the gel was formed, and the loading rate of R848 and PBD-150 in the hydrogel was 100%.

[0092] Inhibition of tumor growth and metastasis in vivo by composite hydrogels of Toll-like receptor agonists and CD47 inhibitors

[0093] (1) Establishment of animal model

[0094] Mouse osteosarcoma RAW264.7 cells were injected into the tibia of BALB / c mice (6-8 weeks old, about 18 g) to establish an orthotopic osteosarcoma model. When the tumor volume reached 200 mm 3 This indicates that the mouse osteosarcoma model was successfully established and treatment began.

[0095] (2) Tumor growth and metastasis inhibition experiments

[0096] The mice with successful modeling were randomly divided into three groups (n=5). After anesthesia, the composite hydrogel was injected into the tumor site. The animal status was dynamically observed and the changes in tumor volume were recorded. On the 10th day after administration, the small animal Micro CT imaging technology was used to dynamically observe the differences in the bone status of the right knee joint of mice with tibial orthotopic osteosarcoma in each group. The experiment was terminated on the 25th day after administration. The results are shown in Figure 5 , Figure 5 Results of the in situ osteosarcoma tumor inhibition and metastasis experiment. Figure 5 It can be seen that in the tumor inhibition experiment, compared with other groups, the tumors in the R848+PBD-150 composite polyamino acid hydrogel group almost disappeared, and the combined treatment of R848+PBD-150 composite polyamino acid hydrogel (GEL / PBD+R848) achieved significant therapeutic effects. Micro CT imaging observations showed that the tibia and femur bones in the tumor invasion area were severely destroyed in the tumor tissues of the Control group and the GEL group. In the tumor tissue of the GEL / PBD+R848 group, the bones of the tibia and the lower part of the femur were intact and continuous, and the bone surface was smooth, indicating that the combined treatment hydrogel has the ability to inhibit osteosarcoma metastasis.

[0097] In vivo regulation of tumor immune microenvironment by composite hydrogels of Toll-like receptor agonists and CD47 inhibitors

[0098] After 10 days of treatment, the tumor tissues of each group were minced, digested and prepared into single cell suspensions. Flow cytometry was used to determine the level of tumor-associated macrophage repolarization and the expression of NK cells and CD4 + T cells, CD8 + The levels of other tumor-related immune cells such as T cells, MDSC cells, and Treg cells. Figure 6 , Figure 6 The results of drug-loaded composite hydrogel regulating the immune microenvironment in orthotopic osteosarcoma. Figure 6 It can be seen that R848+PBD-150 composite polyamino acid hydrogel can polarize macrophages to M1 macrophages. The content of MDSC cells and Treg cells in the GEL / PBD+R848 group was significantly lower than that in other experimental groups, indicating that the combined treatment of PBD-150 and R848 effectively reversed the immunosuppressive microenvironment of the tumor.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polyamino acid composite hydrogel, characterized in that: It comprises a polyamino acid hydrogel and an immunomodulator loaded in the polyamino acid hydrogel, wherein the immunomodulator is selected from at least one of iron-based nanoparticles, Toll-like receptor agonists and CD47 inhibitors, and the polyamino acid hydrogel is selected from polymethionine hydrogel.

2. The polyamino acid composite hydrogel according to claim 1, characterized in that: The mass ratio of the polyamino acid hydrogel to the immunomodulator is in the range of 80:1 to 80:

60.

3. The polyamino acid composite hydrogel according to claim 1, characterized in that: The polyamino acid is selected from polyethylene glycol-polymethionine.

4. The polyamino acid composite hydrogel according to claim 1, characterized in that: The iron-based nanoparticles are selected from carboxymethyl nano-iron oxide nanoparticles; The Toll-like receptor agonist is selected from R848; The CD47 inhibitor is selected from PBD-150.

5. A method for preparing the polyamino acid composite hydrogel according to any one of claims 1 to 4, characterized in that: The following steps are involved: The polyamino acid solution and the immunomodulator solution are mixed and stirred and then gelled to obtain the polyamino acid composite hydrogel.

6. The preparation method according to claim 5, characterized in that: The preparation method of the polyamino acid solution comprises the following steps: A) reacting polyethylene glycol diamine with methionine-N-intra-ring carboxylic anhydride to obtain polyethylene glycol-polymethionine; B) Dissolve polyethylene glycol-polymethionine in PBS buffer solution to obtain a polyamino acid solution.

7. The preparation method according to claim 6, characterized in that: The methionine-N-intra-ring carboxylic anhydride is prepared from methionine and triphosgene.

8. The preparation method according to claim 5, characterized in that: The mixing and stirring temperature is 0-20°C.

9. The preparation method according to claim 5, characterized in that: The gelling temperature is 10-50° C. and the time is 1-10 minutes.

10. Use of the polyamino acid composite hydrogel according to any one of claims 1 to 4 in preparing a preparation for treating osteosarcoma.

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