Cis-platinum-loaded manganese phosphate nanoparticle cross-linked A-type gelatin nanoparticle hydrogel as well as preparation method and application thereof

Abstract

The invention discloses hydrogel. The hydrogel is obtained by crosslinking manganese phosphate nano particles with gelatin nano particles. The invention also discloses a preparation method of the hydrogel and application of the hydrogel as a drug carrier. The invention further discloses the hydrogel loaded with the anti-tumor medicine and a preparation method of the hydrogel. The invention also discloses a hydrogel loaded with cis-platinum. The hydrogel loaded with the cis-platinum comprises the cis-platinum and the hydrogel. The invention also discloses a preparation method of the cisplatin-loaded hydrogel, which comprises the following steps: preparing according to the preparation method of the hydrogel: firstly adding cisplatin into a suspension, and then adjusting the pH value of the suspension to 6-7 by using an acid-forming agent to obtain the cisplatin-loaded hydrogel. The invention also discloses an anti-breast cancer drug. The hydrogel disclosed by the invention has injectability and can be used as a drug carrier; the cisplatin-loaded hydrogel disclosed by the invention has a good anti-tumor effect, and the effect is better when the cisplatin-loaded hydrogel is combined with an anti-TIGIT monoclonal antibody.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a cisplatin-loaded manganese phosphate nanoparticle cross-linked type A gelatin nanoparticle hydrogel, and a preparation method and application thereof. Background Art

[0002] Breast cancer is the most common cancer in women, with 2.26 million new cases of breast cancer worldwide in 2023. In my country, the incidence of breast cancer is also increasing year by year. According to WHO forecasts, there will be 420,000 new cases of breast cancer in China in 2023. Breast cancer has replaced lung cancer as the world's number one cancer. Breast cancer is mainly treated with chemotherapy. Unfortunately, up to 70% of patients with this type of breast cancer are resistant to treatment, so it is extremely important to develop new breast cancer therapies.

[0003] In recent years, literature reports have found that the metal ion manganese can activate the cGAS-STING pathway and promote the affinity between cGAS and STING. The cGAS-STING pathway is a key part of innate immunity. STING is a cellular DNA receptor anchored in the endoplasmic reticulum. When double-stranded DNA binds to cGAS, it can catalyze the production of cAMP. cAMP changes STING from a monomer to a dimer, further activating TBK1 and p-IRF3, and promoting the production of type I interferon. Type I interferon has multiple immune stimulatory functions. It can enhance the maturation, migration and activation of various immune cells such as DC, T cells and NK cells.

[0004] The anti-tumor effect of chemotherapy is not only through cytotoxicity, but also through the regulation of tumor immunity. Cisplatin can increase double-stranded DNA in the cytoplasm by killing tumor cells, further activating the cGAS-STING pathway to induce immunogenic cell death. Although chemotherapy can effectively treat certain types of cancer, due to its disadvantages, such as high-frequency injections, rapid metabolism, low drug utilization, and severe side effects, treatment is often accompanied by severe side effects and a decline in quality of life. Hydrogels have great advantages as drug delivery platforms, which can deliver anti-tumor drugs directly to the tumor site, reduce systemic toxicity, and have good degradability.

[0005] The synergistic effect of chemotherapy and immunotherapy enhances the anti-tumor effect of chemotherapy, and this combination therapy has attracted great attention. Therefore, in order to improve the treatment efficiency of cancer and reduce the toxic side effects during treatment, the use of combined therapy for tumor treatment has very important practical significance. Summary of the invention

[0006] Based on the technical problems existing in the background technology, the present invention proposes a cisplatin-loaded manganese phosphate nanoparticle cross-linked type A gelatin nanoparticle hydrogel and its preparation method and application. The hydrogel of the present invention is injectable and can be used as a drug carrier; loading anti-tumor drugs with hydrogel can enhance innate and adaptive immune responses; the cisplatin-loaded hydrogel of the present invention has a good anti-tumor effect; when the cisplatin-loaded hydrogel is used in combination with the checkpoint inhibitor aTIGIT, a better effect can be achieved.

[0007] The invention provides a hydrogel, wherein the hydrogel is obtained by cross-linking gelatin nanoparticles with manganese phosphate nanoparticles.

[0008] Preferably, the hydrogel is an injectable hydrogel.

[0009] Preferably, crosslinking is carried out under acidic conditions or neutral conditions.

[0010] Preferably, cross-linking is achieved by electrostatic interaction under acidic or neutral conditions.

[0011] Preferably, the pH of the acidic or neutral condition is 6-7.

[0012] Preferably, the gelatin is Type A gelatin.

[0013] The above-mentioned type A gelatin is also called acid-process gelatin.

[0014] Preferably, the particle size of the manganese phosphate nanoparticles is 50-100 nm.

[0015] Preferably, the particle size of the gelatin nanoparticles is 100-200 nm.

[0016] Preferably, 1 mL of hydrogel contains 0.1-5 mg of manganese phosphate nanoparticles and 70-150 mg of gelatin nanoparticles; more preferably, 1 mL of hydrogel contains 0.5-1 mg of manganese phosphate nanoparticles and 100 mg of gelatin nanoparticles.

[0017] Preferably, the cross-linking is carried out in water.

[0018] Preferably, the concentration of manganese phosphate nanoparticles in water is 0.1-5 mg / mL; more preferably 0.5-1 mg / mL.

[0019] The present invention also provides a method for preparing the hydrogel, comprising the following steps: mixing manganese phosphate nanoparticles, gelatin nanoparticles and an alkaline aqueous solution to obtain a suspension; and then adjusting the pH of the suspension to 6-7 with an acidifier to obtain a hydrogel.

[0020] Preferably, the pH of the suspension is 10-12.

[0021] The alkaline aqueous solution can be prepared with a water-soluble inorganic base; the water-soluble inorganic base can be sodium hydroxide, potassium hydroxide, etc.

[0022] The invention prepares manganese phosphate nanoparticles and gelatin nanoparticles respectively; both the manganese phosphate nanoparticles and the gelatin nanoparticles are negatively charged in an alkaline aqueous solution, so that the two can be evenly dispersed; then an acidifier is added to slowly hydrolyze and acidify the alkaline environment to slowly turn it into a weak acid or neutral state, so that the gelatin nanoparticles are converted into positively charged particles, and are cross-linked through electrostatic action to form a hydrogel.

[0023] Compared with traditional hydrogels, the hydrogel prepared by the present invention has better uniformity and good injectability, can be used as a drug carrier, and is beneficial for clinical application.

[0024] Preferably, the acid generator is D-glucono-δ-lactone.

[0025] Preferably, the dosage ratio of the acid generating agent to the suspension is 1-5.5 mg:1 mL, and more preferably, the dosage ratio is 5.5 mg:1 mL.

[0026] Preferably, the concentration of manganese phosphate nanoparticles in the suspension is 0.1-5 mg / mL; more preferably 0.5-1 mg / mL.

[0027] Preferably, in the preparation process of manganese phosphate nanoparticles, oleylamine, oleic acid, a phosphorus source, toluene, a manganese source and water are mixed, subjected to a hydrothermal reaction, and solid-liquid separation to obtain manganese phosphate nanoparticles.

[0028] Preferably, during the preparation of the manganese phosphate nanoparticles, the hydrothermal reaction is carried out at 170-190° C. for 8-10 h.

[0029] Preferably, in the preparation process of manganese phosphate nanoparticles, the phosphorus source is triethyl phosphate.

[0030] Preferably, in the preparation of manganese phosphate nanoparticles, the manganese source is manganese (II) acetylacetonate.

[0031] Preferably, in the preparation process of manganese phosphate nanoparticles, the molar ratio of phosphorus element to manganese element is 2-2.5:1.

[0032] Preferably, in the preparation process of manganese phosphate nanoparticles, the volume ratio of oleylamine, oleic acid, toluene and water is 1.4-1.6:0.8-1.2:4-5:0.1.

[0033] Preferably, in the preparation process of manganese phosphate nanoparticles, the dosage ratio of oleic acid to manganese element is 1.6-2.4 mL:1 mmol.

[0034] Preferably, in the preparation process of gelatin nanoparticles, the gelatin aqueous solution is added dropwise into the organic solvent, cooled to room temperature, a cross-linking agent is added for solidification, and the solid and liquid are separated to obtain gelatin nanoparticles.

[0035] Preferably, during the preparation of gelatin nanoparticles, the temperature of the gelatin aqueous solution is 40-45°C.

[0036] Preferably, during the preparation of gelatin nanoparticles, the concentration of gelatin in the gelatin aqueous solution is 0.02-0.025 g / mL.

[0037] Preferably, during the preparation of gelatin nanoparticles, the pH of the gelatin aqueous solution is 2-2.5, and the pH of the gelatin aqueous solution can be adjusted with inorganic acids such as hydrochloric acid.

[0038] Preferably, in the preparation process of gelatin nanoparticles, the organic solvent is acetone.

[0039] Preferably, in the preparation process of gelatin nanoparticles, the cross-linking agent is glutaraldehyde.

[0040] Preferably, during the preparation of gelatin nanoparticles, a neutralizing agent is added to allow the remaining cross-linking agent to react completely before solid-liquid separation.

[0041] Preferably, in the preparation process of gelatin nanoparticles, the neutralizing agent is guanidine hydrochloride.

[0042] The manganese phosphate nanoparticles and gelatin nanoparticles can be freeze-dried and set aside.

[0043] The invention also proposes the use of the hydrogel as a drug carrier.

[0044] Preferably, the composition is used as a carrier of an anti-tumor drug; more preferably, the anti-tumor drug is an anti-cancer drug.

[0045] The present invention also provides a hydrogel loaded with anti-tumor drugs, comprising: the anti-tumor drugs and the above hydrogel.

[0046] The anti-tumor drugs are uniformly dispersed in the hydrogel.

[0047] Preferably, the anti-tumor drug includes at least one of cisplatin and carboplatin.

[0048] Preferably, the hydrogel loaded with anti-tumor drugs is an anti-tumor implant.

[0049] The above-mentioned anti-tumor implant refers to a preparation made of drugs and excipients for implantation into the body.

[0050] Antitumor implants can be injected directly peritumorally to inhibit tumor growth.

[0051] The present invention also proposes a method for preparing the above-mentioned hydrogel loaded with anti-tumor drugs, comprising the following steps: preparing a suspension according to the above-mentioned hydrogel preparation method, first adding the anti-tumor drug to the suspension, and then adjusting the pH of the suspension to 6-7 with an acidifier to obtain a hydrogel loaded with anti-tumor drugs.

[0052] The present invention also provides a cisplatin-loaded hydrogel, comprising cisplatin and the above hydrogel.

[0053] Preferably, the dosage ratio of cisplatin to hydrogel is 0.2-5 mg:1 mL; more preferably 0.2-0.8 mg / mL.

[0054] The cisplatin is uniformly dispersed in the hydrogel.

[0055] The present invention also provides a method for preparing the above-mentioned cisplatin-loaded hydrogel, comprising the following steps: preparing a suspension according to the above-mentioned hydrogel preparation method, first adding cisplatin to the suspension, and then adjusting the pH of the suspension to 6-7 with an acidifier to obtain a cisplatin-loaded hydrogel.

[0056] Preferably, the dosage ratio of cisplatin to suspension is 0.2-5 mg:1 mL; more preferably 0.2-0.8 mg / mL.

[0057] The present invention uses hydrogel as a drug carrier. After loading anti-tumor drugs, the hydrogel loaded with anti-tumor drugs can accurately locate the tumor area after injection and gradually swell in the tumor area, slowly releasing manganese ions and cisplatin, activating the cGAS-STING pathway and promoting the activation of antigen-presenting cells to induce innate and adaptive immune responses, thereby inhibiting tumor growth. In addition, the hydrogel prepared by the present invention has pH and enzyme responsive drug release properties, which is conducive to the release of drugs located at the lesion of the body.

[0058] In addition, the hydrogel prepared by the present invention has no obvious toxicity when implanted in mammals, has good biosafety, and can be used to prepare anti-tumor implants that are directly injected around the tumor to inhibit tumor growth. Through local injection treatment of tumors, many difficult problems in cancer treatment, such as easy to produce toxic side effects and poor localization, can be solved.

[0059] The present invention also provides an anti-breast cancer drug, which is the above-mentioned hydrogel loaded with cisplatin;

[0060] Alternatively, the anti-breast cancer drug comprises: the above-mentioned cisplatin-loaded hydrogel and anti-TIGIT monoclonal antibody.

[0061] The above-mentioned cisplatin-loaded hydrogel and anti-TIGIT monoclonal antibody are injected separately when used; the specific injection method can be: in the order of day 0 and day 6, inject the cisplatin-loaded hydrogel peritumorally once every 6 days; in the order of day 1, 5, 9, and 13, inject the anti-TIGIT monoclonal antibody intraperitoneally once every 4 days.

[0062] The cisplatin-loaded hydrogel of the present invention has a good anti-tumor effect, which can enhance the innate and adaptive immune responses and induce the immunogenic death of cancer cells; when used alone, it has been shown in cell experiments and animal experiments that it can inhibit the growth of breast cancer tumors; when used in combination with anti-TIGIT monoclonal antibodies, it can achieve better effects.

[0063] Beneficial effects:

[0064] 1. Compared with traditional hydrogels, the hydrogel prepared by the present invention has better uniformity and good injectability, can be used as a drug carrier, and is conducive to clinical application.

[0065] 2. The present invention uses hydrogel as a drug carrier. After loading anti-tumor drugs, the hydrogel loaded with anti-tumor drugs can accurately locate the tumor area after injection and gradually swell in the tumor area, slowly releasing manganese ions and cisplatin, activating the cGAS-STING pathway and promoting the activation of antigen-presenting cells to induce innate and adaptive immune responses, thereby inhibiting tumor growth. In addition, the hydrogel prepared by the present invention has pH and enzyme responsive drug release properties, which is conducive to the release of drugs located at the lesion of the body.

[0066] 3. The hydrogel prepared by the present invention has no obvious toxicity when implanted in mammals and has good biosafety, and can be used to prepare anti-tumor implants for direct peritumoral injection to inhibit tumor growth. Through local injection treatment of tumors, many problems in cancer treatment such as easy to produce toxic side effects and poor localization can be solved.

[0067] 4. The cisplatin-loaded hydrogel of the present invention has a good anti-tumor effect. It can enhance the innate and adaptive immune responses and induce the immunogenic death of cancer cells. When used alone, it has been shown in cell experiments and animal experiments that it can inhibit the growth of breast cancer tumors. When used in combination with anti-TIGIT monoclonal antibodies, better effects can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a transmission electron microscopy image of the manganese phosphate nanoparticles prepared in Example 1.

[0069] Figure 2The scanning electron microscope images of type A gelatin nanoparticles and cisplatin-loaded hydrogel prepared in Example 1, wherein A represents type A gelatin nanoparticles and B represents cisplatin-loaded hydrogel.

[0070] Figure 3 This is a photo of the injectable cisplatin-loaded hydrogel prepared in Example 1.

[0071] Figure 4 Fluid mechanics of cisplatin-loaded hydrogel and unloaded hydrogel.

[0072] Figure 5 Cumulative release curves of cisplatin and manganese ions in cisplatin-loaded hydrogels under different pH conditions.

[0073] Figure 6 Biosafety of cisplatin-loaded hydrogels.

[0074] Figure 7 This is the MTT cytotoxicity experiment diagram of the hydrogel extract loaded with cisplatin.

[0075] Figure 8 After incubating RAW264.7 cells with the cisplatin-loaded hydrogel and the hydrogel extract prepared in Example 2 for 24 h, Western Blot was used to detect the activation of the cGAS-STING pathway in RAW264.7 cells.

[0076] Fig. 9 The data of flow cytometry detection of maturation of bone marrow-derived dendritic cells (BMDC) after incubation of BMDC with hydrogel extract loaded with cisplatin for 24 hours.

[0077] Fig.10 Tumor volume inhibition curve of cisplatin-loaded hydrogel in the treatment of mouse breast cancer model. DETAILED DESCRIPTION

[0078] The technical solution of the present invention is described in detail below through specific embodiments.

[0079] Example 1

[0080] A method for preparing a cisplatin-loaded hydrogel comprises the following steps:

[0081] 3 mL of oleylamine, 2 mL of oleic acid, 0.4 mL (2.3 mmol) of triethyl phosphate, 12 mL of toluene, 0.2 mL of deionized water, and 0.253 g (1 mmol) of manganese (II) acetylacetonate were added to a reaction kettle and mixed well, and the mixture was heated to 180°C for 9 h. After cooling, the mixture was centrifuged, and the solid was washed with anhydrous ethanol and dried to obtain manganese phosphate nanoparticles.

[0082] 1.8 g of type A gelatin powder was added to 75 mL of deionized water, heated to 42°C and stirred until dissolved, the pH was adjusted to 2.3 with hydrochloric acid, and the solution was slowly dripped into 210 mL of acetone at a rate of 1 second / drop until the solution became turbid, stirred and cooled to room temperature, and then 495 μl of a 25% glutaraldehyde aqueous solution was added, solidified overnight, 50 mL of a 1 mg / mL guanidine hydrochloride aqueous solution was added, and the solution was neutralized with the remaining glutaraldehyde, stirred at room temperature for 1 h, centrifuged, the solid was washed, and dried to obtain type A gelatin nanoparticles;

[0083] Manganese phosphate nanoparticles and an alkaline aqueous solution (sodium hydroxide aqueous solution) are used to prepare a manganese phosphate nanoparticle solution with a pH of 11 and a concentration of 1 mg / mL; type A gelatin nanoparticles and an alkaline aqueous solution (sodium hydroxide aqueous solution) are used to prepare a type A gelatin nanoparticle solution with a pH of 11 and a concentration of 200 mg / mL;

[0084] Take a manganese phosphate nanoparticle solution and a type A gelatin nanoparticle solution and mix them in a volume ratio of 1:1 to obtain a suspension; add cisplatin to the suspension (the dosage ratio of cisplatin to suspension is 0.8 mg:1 mL) and mix well, then add D-glucono-δ-lactone to make its final concentration 5.5 mg / mL, stir at room temperature for 10-15 minutes, at which time the pH drops to 6-7, and a cisplatin-loaded hydrogel is obtained.

[0085] The manganese phosphate nanoparticles, type A gelatin nanoparticles, and cisplatin-loaded hydrogels prepared in Example 1 were tested. The results are as follows: Figure 1-4 shown.

[0086] Figure 1 This is a transmission electron microscopy image of the manganese phosphate nanoparticles prepared in Example 1.

[0087] Figure 2 The scanning electron microscope images of type A gelatin nanoparticles and cisplatin-loaded hydrogel prepared in Example 1, wherein A represents type A gelatin nanoparticles and B represents cisplatin-loaded hydrogel.

[0088] Figure 3 This is a photo of the injectable cisplatin-loaded hydrogel prepared in Example 1.

[0089] Depend on Figure 1-3 It can be seen that the particle size of manganese phosphate nanoparticles is about 50 nm, the particle size of type A gelatin nanoparticles is about 100-200 nm, the hydrogel is composed of type A gelatin nanoparticles and manganese phosphate nanoparticles, and the hydrogel loaded with cisplatin has good injectability.

[0090] Example 2

[0091] A method for preparing a hydrogel comprises the following steps:

[0092] Without adding cisplatin, the other steps were the same as those in Example 1.

[0093] The hydrogel loaded with cisplatin in Example 1 (i.e., drug-loaded hydrogel) and the hydrogel in Example 2 (i.e., non-drug-loaded hydrogel) were subjected to fluid dynamics testing using an MCR 302e rotational rheometer at a test temperature of 25°C, a test gap of 1000 μm, and a frequency of 0.1-100 Hz. Figure 4 shown.

[0094] Figure 4 Fluid mechanics of cisplatin-loaded hydrogel and unloaded hydrogel.

[0095] Depend on Figure 4 It can be seen that the un-drug-loaded hydrogel has good stability at different shear frequencies, and the introduction of cisplatin into the hydrogel does not have a significant effect on the rheological properties of the hydrogel.

[0096] Example 3

[0097] Take the drug-loaded hydrogel and the non-drug-loaded hydrogel to detect the cisplatin and manganese ion release curves under different pH conditions. The specific detection method is as follows:

[0098] Cisplatin release assay: A cisplatin-loaded hydrogel was prepared according to the method of Example 1, and 2.5 mg of cisplatin was loaded in each 500 μL of hydrogel. Other operations were the same as in Example 1;

[0099] Take three portions of the above hydrogel, 500 μL each, place them in a dialysis bag, immerse them in 45 mL of deionized water buffer with pH values ​​of 5.5, 6.5 and 7.4, respectively, and place them on a shaker. Take 1 mL of buffer as a sample at regular intervals, and add the same volume of deionized water buffer with the same pH value, and use a UV spectrophotometer to measure the UV absorption value of the sample at 347 nm. Use a UV spectrophotometer to measure the UV absorbance of standard cisplatin solutions with different pH values ​​at 347 nm, draw a standard curve, and determine the content of cisplatin in the sample according to the standard curve.

[0100] Manganese ion release assay: A hydrogel loaded with cisplatin was prepared according to the method of Example 1, and each 500 μL of hydrogel contained 2.5 mg of manganese phosphate nanoparticles. Other operations were the same as in Example 1;

[0101] Take three portions of the above hydrogel, 500 μL each, put them in dialysis bags, immerse them in 45 mL of ammonium acetate buffer with pH values ​​of 5.5, 6.5 and 7.4 respectively and place them on a shaker. Take 1 mL of the buffer as a sample at regular intervals, and add the same volume of ammonium acetate buffer with the same pH value. After the sample is diluted to 10 mL, the manganese ion concentration is determined by ICP-MS.

[0102] The results are as follows Figure 5 As shown, Figure 5 Cumulative release curves of cisplatin and manganese ions in cisplatin-loaded hydrogels under different pH conditions.

[0103] Depend on Figure 5 It can be seen that cisplatin can be stably released in the hydrogel and is not affected by pH value; manganese phosphate can be slowly released in the hydrogel, and the release rate is faster and the drug amount is larger under acidic conditions.

[0104] Example 4

[0105] A hydrogel loaded with cisplatin was prepared according to the method of Example 1, so that 1 mL of the hydrogel contained 0.5 mg of manganese phosphate nanoparticles, 100 mg of type A gelatin nanoparticles, and 0.8 mg of cisplatin. Other operations were the same as in Example 1.

[0106] On the 7th, 14th and 28th days after the hydrogel was implanted in mice, the hearts, livers, spleens, lungs and kidneys of the mice were dissected and stained with HE. The results were as follows: Figure 6 shown.

[0107] Figure 6 Biosafety of cisplatin-loaded hydrogels.

[0108] Depend on Figure 6 It can be seen that the internal organs of the mice did not show any related pathological symptoms and the tissues were intact, indicating that the hydrogel loaded with cisplatin has good biosafety.

[0109] Example 5

[0110] Determination of the killing effect of hydrogel extracts of different concentrations on 4T1 cells:

[0111] A hydrogel loaded with cisplatin was prepared according to the method of Example 1, so that 1 mL of the hydrogel contained 1 mg of manganese phosphate nanoparticles, 100 mg of type A gelatin nanoparticles, and 0.2 mg of cisplatin. Other operations were the same as in Example 1.

[0112] The hydrogel was added to 4 mL of RPMI 1640 culture medium and incubated in a 37°C incubator for 12 h to obtain a hydrogel extract;

[0113] 4T1 cells were inoculated into 96-well plates at a number of 20,000 / well and cultured. After adherence, different proportions of hydrogel extract were added and incubated for 24 hours. Then the old culture medium was aspirated, washed twice with PBS, 200 μL of MTT solution containing 5 mg / mL was added to the sample well, and 200uL of PBS was added to the control well. The plates were incubated at 37°C for 4 hours, the supernatant was aspirated, 150 μL of DMSO was added, and the plates were shaken on a shaker for 15-20 minutes in the dark. The absorbance at 570 nm was measured using an enzyme reader to calculate the cell survival rate. Cell survival rate (%) = (absorbance value of sample well - absorbance value of control well) / absorbance value of blank well × 100%. The results are as follows: Figure 7 shown.

[0114] Figure 7 This is the MTT cytotoxicity experiment diagram of the hydrogel extract loaded with cisplatin.

[0115] Depend on Figure 7 It can be seen that when 70% of the hydrogel extract was added, the survival rate of 4T1 cells was 48.25%, and the hydrogel was able to effectively inhibit the proliferation of breast cancer cells in vitro.

[0116] Example 6

[0117] Determination of the activation of cGAS-STING pathway in RAW264.7 cells by 7% hydrogel extract:

[0118] The hydrogel was prepared according to the method of Example 2, so that 1 mL of the hydrogel contained 1 mg of manganese phosphate nanoparticles and 100 mg of type A gelatin nanoparticles. The other operations were the same as those in Example 2, and were recorded as Gela NPs / Mn3(PO4)2;

[0119] A hydrogel loaded with cisplatin was prepared according to the method of Example 1, so that 1 mL of the hydrogel contained 1 mg of manganese phosphate nanoparticles, 100 mg of type A gelatin nanoparticles, and 0.2 mg of cisplatin. The other operations were the same as in Example 1, and were recorded as GelaNPs / Mn3(PO4)2 / CP;

[0120] The two hydrogels were added to 4 mL of RPMI 1640 culture medium and incubated in a 37°C incubator for 12 h to obtain two groups of 7% hydrogel extracts;

[0121] RAW264.7 cells were seeded into 6-well plates at a number of 1 million per well and cultured. After adherence, 2 mL of RPMI 1640 medium (recorded as con group), 0.56 μg / mL cisplatin aqueous solution (recorded as CP group), 14 μg / mL Mn3(PO4)2 aqueous solution (recorded as Mn3(PO4)2 group), 5.55 μg / mL MnCl2 aqueous solution (recorded as MnCl2 group), 7% Gela NPs / Mn3(PO4)2 hydrogel extract (recorded as Gela NPs / Mn3(PO4)2 group), and 7% Gela NPs / Mn3(PO4)2 / CP hydrogel extract (recorded as Gela NPs / Mn3(PO4)2 / CP group) were added to each well and incubated for 24 h respectively;

[0122] After discarding the old culture medium, wash twice with PBS, add 100 μL protein lysis buffer (RIPA: protease inhibitor: phosphatase inhibitor = 100:1:2) to each well, lyse in a shaker at 4°C for 1 h, scrape off the lysed cells with a cell scraper, grind in a multi-sample tissue grinder at 60 Hz for 60 seconds, centrifuge at 4°C and 12000 rpm for 10 min, take the supernatant, add 5x loading buffer according to the amount of supernatant, and cook in a metal bath at 95°C for 10 min to denature the protein to obtain a protein sample; prepare 10% SDS-PAGE gel and apply to the gel The protein sample was added to the gel wells, and the upper gel was electrophoresed at a constant voltage of 80mV for 30min, then changed to a constant voltage of 120mV until bromophenol blue ran out. The gel was then transferred to a 0.45μM PVDF membrane using a wet transfer method at a constant current of 230mA for 1.5h, and blocked with 3% BSA for 30min; the blocked PVDF membrane was then placed in a primary antibody solution containing GAPDH, TBK1, pTBK1, IRF3 and pIRF3 and incubated overnight at 4°C; finally, the PVDF membrane was washed with TBST and placed in a secondary antibody solution for incubation at room temperature for 1h, and developed with a developer. The results are shown in Figure 8 shown.

[0123] Figure 8 After incubating RAW264.7 cells with cisplatin-loaded hydrogels and hydrogel extracts for 24 hours, Western Blot was used to detect the activation of the cGAS-STING pathway in RAW264.7.

[0124] Depend on Figure 8 It can be seen that the manganese ions in the cisplatin-loaded hydrogel can effectively activate related proteins in the cGAS-STING pathway.

[0125] Example 7

[0126] After incubating bone marrow-derived dendritic cells (BMDC) with cisplatin-loaded hydrogel extract for 24 hours, flow cytometry was used to detect the maturation of BMDC:

[0127] Extraction of BMDC: After the bone marrow of the femur and tibia of C57BL / 6 male mice was washed with PBS, the red blood cells were lysed with red blood cell lysis buffer and centrifuged to obtain bone marrow cells; then, the cells were seeded into 6-well plates and cultured with RPMI 1640 culture medium containing 20ng / mL GM-CSF and 10ng / mL IL-4; every 2 days, half of the culture medium was replaced with RPMI 1640 culture medium containing 20ng / mL GM-CSF and 10ng / mL; on the 8th day of culture, the suspension and semi-suspension cells were harvested to obtain BMDC, and seeded in 24-well plates;

[0128] The hydrogel was prepared according to the method of Example 2, so that 1 mL of the hydrogel contained 1 mg of manganese phosphate nanoparticles and 100 mg of type A gelatin nanoparticles. The other operations were the same as those in Example 2, and were recorded as Gela NPs / Mn3(PO4)2;

[0129] A hydrogel loaded with cisplatin was prepared according to the method of Example 1, so that 1 mL of the hydrogel contained 1 mg of manganese phosphate nanoparticles, 100 mg of type A gelatin nanoparticles, and 0.2 mg of cisplatin. The other operations were the same as in Example 1, and were recorded as GelaNPs / Mn3(PO4)2 / CP;

[0130] The two hydrogels were added to 4 mL of RPMI 1640 culture medium and incubated in a 37°C incubator for 12 h to obtain two groups of 7% hydrogel extracts;

[0131] RPMI 1640 culture medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4 (denoted as PBS group), 0.56 μg / mL cisplatin aqueous solution (denoted as CP group), 14 μg / mL Mn3(PO4)2 aqueous solution (denoted as Mn3(PO4)2 group), 5.55 μg / mL MnCl2 aqueous solution (denoted as MnCl2 group), 7% Gela NPs / Mn3(PO4)2 hydrogel extract (denoted as Gela NPs / Mn3(PO4)2 group), and 7% Gela NPs / Mn3(PO4)2 / CP hydrogel extract (denoted as Gela NPs / Mn3(PO4)2 / CP group) were added to BMDCs, and the cells were collected after incubation for 24 hours. The cells were centrifuged, blocked, stained, and membrane-filtered, and the expression of CD80 and CD86 was analyzed by flow cytometry. The results are shown in Fig. 9 shown.

[0132] Fig. 9 The data of flow cytometry detection of maturation of bone marrow-derived dendritic cells (BMDC) after incubation of cisplatin-loaded hydrogel extract on BMDC for 24 hours.

[0133] Depend on Fig. 9 It can be seen that the hydrogel loaded with cisplatin can effectively promote the maturation of BMDC.

[0134] Example 8

[0135] Determining the effect of cisplatin-loaded hydrogels in the treatment of mouse breast cancer models:

[0136] One million 4T1-Luc cells were implanted subcutaneously in 5-6 week old Balb / c female mice and the tumors were grown to about 50 mm. 3 Afterwards, the mice were divided into 9 groups, and each group of mice was injected with drugs. The specific drug administration method for each group was:

[0137] Control group: Normal saline was injected peritumorally on day 0 and day 6, with an injection volume of 0.1 mL / mouse;

[0138] CP group: cisplatin aqueous solution was injected peritumorally on day 0 and day 6, with the injection dose of cisplatin being 4 mg / kg / mouse;

[0139] MnP group: Mn3(PO4)2 aqueous solution was injected peritumorally on days 0 and 6, with the injection dose of Mn3(PO4)2 being 2.5 mg / kg / mouse;

[0140] MnCl2 group: MnCl2 aqueous solution was injected peritumorally on days 0 and 6, with the injection dose of 2.5 mg / kg / mouse based on MnCl2;

[0141] GelaNPs / MnP group: hydrogel was prepared according to the method of Example 2, so that 1 mL of hydrogel contained 0.5 mg of manganese phosphate nanoparticles and 100 mg of type A gelatin nanoparticles. Other operations were the same as in Example 2. The above hydrogel was injected peritumorally on day 0 and day 6, and the injection amount was 2.5 mg / kg / mouse based on Mn3(PO4)2.

[0142] GelaNPs / MnP / CP group: a hydrogel loaded with cisplatin was prepared according to the method of Example 1, so that 1 mL of the hydrogel contained 0.5 mg of manganese phosphate nanoparticles, 100 mg of type A gelatin nanoparticles, and 0.8 mg of cisplatin. The other operations were the same as those in Example 1. The hydrogel loaded with cisplatin was injected peritumorally on days 0 and 6. The injection amount of Mn3(PO4)2 was 2.5 mg / kg / mouse, and the injection amount of cisplatin was 4 mg / kg / mouse.

[0143] aTIGIT group: anti-TIGIT monoclonal antibody injection was intraperitoneally administered on days 1, 5, 9, and 13, with an injection dose of 100 μg / kg / mouse based on anti-TIGIT monoclonal antibody;

[0144] GelaNPs / MnP / CP+aTIGIT group: administration was performed according to the GelaNPs / MnP / CP group and aTIGIT group;

[0145] The length and width of the tumors of each group of mice were measured with a vernier caliper every 2 days, and the tumor volume was calculated. The tumor volume of the mouse = length * width * width / 2. Fig.10 shown.

[0146] Fig.10 Tumor volume inhibition curve of cisplatin-loaded hydrogel in the treatment of mouse breast cancer model.

[0147] Depend on Fig.10 It can be seen that the tumor volume of mice in the cisplatin-loaded hydrogel group was significantly smaller than that in the saline group and also smaller than that in the cisplatin group, showing good efficacy in treating breast cancer tumors; and when the cisplatin-loaded hydrogel group was used in combination with the checkpoint anti-TIGIT monoclonal antibody, the tumor volume hardly increased, showing good efficacy of combined drug treatment for breast cancer tumors.

[0148] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A hydrogel, characterized in that: The hydrogel is obtained by cross-linking gelatin nanoparticles with manganese phosphate nanoparticles.

2. The hydrogel according to claim 1, characterized in that The hydrogel is an injectable hydrogel; preferably, cross-linking is performed under acidic or neutral conditions; preferably, cross-linking is performed by electrostatic action under acidic or neutral conditions; preferably, the pH of the acidic or neutral conditions is 6-7; preferably, the gelatin is type A gelatin; preferably, the particle size of the manganese phosphate nanoparticles is 50-100nm; preferably, the particle size of the gelatin nanoparticles is 100-200nm; preferably, 1mL of the hydrogel contains 0.1-5mg of manganese phosphate nanoparticles and 70-150mg of gelatin nanoparticles; preferably, cross-linking is performed in water; preferably, the concentration of manganese phosphate nanoparticles in water is 0.1-5mg / mL.

3. A method for preparing the hydrogel according to claim 1 or 2, characterized in that: The method comprises the following steps: mixing manganese phosphate nanoparticles, gelatin nanoparticles and alkaline aqueous solution to obtain a suspension; and then using an acidifier to adjust the pH of the suspension to 6-7 to obtain a hydrogel.

4. The method for preparing the hydrogel according to claim 3, characterized in that: The pH of the suspension is 10-12; preferably, the acidifier is D-glucono-δ-lactone; preferably, the dosage ratio of the acidifier to the suspension is 1-5.5 mg:1 mL; preferably, the concentration of the manganese phosphate nanoparticles in the suspension is 0.1-5 mg / mL; Preferably, in the process of preparing manganese phosphate nanoparticles, oleylamine, oleic acid, a phosphorus source, toluene, a manganese source and water are mixed, hydrothermally reacted, and solid-liquid separation is performed to obtain manganese phosphate nanoparticles; preferably, in the process of preparing manganese phosphate nanoparticles, the hydrothermal reaction is carried out at 170-190° C. for 8-10 hours; preferably, in the process of preparing manganese phosphate nanoparticles, the phosphorus source is triethyl phosphate; preferably, in the process of preparing manganese phosphate nanoparticles, the manganese source is manganese (II) acetylacetonate; preferably, in the process of preparing manganese phosphate nanoparticles, the molar ratio of phosphorus element to manganese element is 2-2.5:1; preferably, in the process of preparing manganese phosphate nanoparticles, the volume ratio of oleylamine, oleic acid, toluene and water is 1.4-1.6:0.8-1.2:4-5:0.1; preferably, in the process of preparing manganese phosphate nanoparticles, the amount ratio of oleic acid to manganese element is 1.6-2.4mL:1mmol; Preferably, in the process of preparing gelatin nanoparticles, an aqueous gelatin solution is added dropwise into an organic solvent, cooled to room temperature, a cross-linking agent is added for solidification, and solid-liquid separation is performed to obtain gelatin nanoparticles; preferably, in the process of preparing gelatin nanoparticles, the temperature of the aqueous gelatin solution is 40-45°C; preferably, in the process of preparing gelatin nanoparticles, the concentration of gelatin in the aqueous gelatin solution is 0.02-0.025 g / mL; preferably, in the process of preparing gelatin nanoparticles, the pH of the aqueous gelatin solution is 2-2.5; preferably, in the process of preparing gelatin nanoparticles, the organic solvent is acetone; preferably, in the process of preparing gelatin nanoparticles, the cross-linking agent is glutaraldehyde; preferably, in the process of preparing gelatin nanoparticles, a neutralizing agent is added to allow the remaining cross-linking agent to react completely before solid-liquid separation is performed; preferably, in the process of preparing gelatin nanoparticles, the neutralizing agent is guanidine hydrochloride.

5. Use of the hydrogel as claimed in claim 1 or 2 as a carrier of a drug; preferably, use of the hydrogel as a carrier of an anti-tumor drug.

6. A hydrogel loaded with anti-tumor drugs, characterized in that: include: An anti-tumor drug and a hydrogel as claimed in claim 1 or 2; preferably, the anti-tumor drug comprises: at least one of cisplatin and carboplatin; preferably, the hydrogel loaded with the anti-tumor drug is an anti-tumor implant.

7. A method for preparing a hydrogel loaded with anti-tumor drugs, characterized in that: The method comprises the following steps: preparing the hydrogel according to the method for preparing the hydrogel according to claim 3 or 4, wherein the anti-tumor drug is first added to the suspension, and then the pH of the suspension is adjusted to 6-7 with an acidifier to obtain a hydrogel loaded with the anti-tumor drug.

8. A cisplatin-loaded hydrogel, characterized in that: include: Cisplatin and the hydrogel as claimed in claim 1 or 2; preferably, the dosage ratio of cisplatin to the hydrogel is 0.2-5 mg:1 mL.

9. A method for preparing a cisplatin-loaded hydrogel as claimed in claim 8, characterized in that: The method comprises the following steps: preparing the hydrogel according to the preparation method of the hydrogel according to claim 3 or 4, wherein cisplatin is first added to the suspension, and then the pH of the suspension is adjusted to 6-7 with an acidifier to obtain a hydrogel loaded with cisplatin; preferably, the dosage ratio of cisplatin to the suspension is 0.2-5 mg:1 mL.

10. An anti-breast cancer drug, characterized in that: The anti-breast cancer drug is the cisplatin-loaded hydrogel according to claim 8; Or the anti-breast cancer drug comprises: the cisplatin-loaded hydrogel according to claim 8 and an anti-TIGIT monoclonal antibody.

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