Cypate and melittin loaded self-repairing temperature-sensitive hydrogel and application thereof

Abstract

The invention discloses a self-repairing temperature-sensitive hydrogel loaded with Cypate and melittin and application of the self-repairing temperature-sensitive hydrogel. Cypate is wrapped with apoferritin, and obtained Cypate recombinant apoferritin nanoparticles are uniformly dispersed in chitosan / beta-sodium glycerophosphate hydrogel, so that the self-repairing temperature-sensitive hydrogel is constructed. Oxidized chondroitin sulfate is introduced, and the oxidized chondroitin sulfate and amino in chitosan can form Schiff base, so that the hydrogel can be endowed with a self-repairing characteristic, the mechanical strength of the hydrogel is improved, and the sustained release of a therapeutic agent is better controlled. Experimental results show that when the self-repairing temperature-sensitive hydrogel is injected to the periphery of a tumor, the self-repairing temperature-sensitive hydrogel shows excellent retention capacity, deep and continuous release of FC nanoparticles in the tumor is promoted, the photo-thermal treatment effect is improved, meanwhile, melittin is slowly and continuously released, the expression level of M1 macrophages can be specifically reduced, the M2 / M1 proportion is promoted, and the self-repairing temperature-sensitive hydrogel can be used for treating tumor tumors. And the immunosuppressive microenvironment is regulated and controlled, and the synergistic effect of the two shows a remarkable breast tumor resisting effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a self-repairing temperature-sensitive hydrogel loaded with Cypate and melittin and applications thereof. Background Art

[0002] Breast cancer is characterized by abnormal cell metabolism, rapid proliferation and invasive metastasis, and has become one of the major diseases threatening human health. Currently, mainstream clinical treatments include surgical resection, chemotherapy and radiotherapy, but there are still significant limitations: surgical treatment is only applicable to local solid tumors and cannot eliminate micrometastases; although chemotherapy drugs can kill cancer cells systemically, they have problems such as poor tumor tissue permeability, high drug resistance and systemic toxic side effects, resulting in incomplete elimination and easy recurrence; although radiotherapy reduces damage to normal tissues through precise dose control, tumor radiation resistance and recurrence risk still restrict its efficacy. Therefore, there is an urgent need to develop new treatment strategies based on multimodal synergistic enhancement.

[0003] Photothermal therapy (PTT) has become a hot topic in tumor treatment research due to its non-invasiveness, high spatiotemporal precision, and low systemic toxicity. Its core mechanism is that the photothermal converter generates local high heat (42-48°C) under near-infrared light excitation, inducing thermal ablation of tumor cells. However, existing PTT technology faces two major bottlenecks: (1) traditional photothermal agents (such as indocyanine green) are prone to thermal damage to surrounding normal tissues during high-temperature treatment; (2) hydrophobic photothermal agents (such as Cypate) have poor water solubility, resulting in low in vivo delivery efficiency, which limits their clinical application. To address the above problems, protein nanocarriers, specifically adenosine (HFn), have shown their unique advantages: HFn can form hollow nanocages through pH-responsive self-assembly, the inner cavity of which can efficiently encapsulate hydrophobic drugs and achieve tumor-targeted delivery through enhanced permeability retention (EPR). Previous studies have shown that HFn-encapsulated curcumin can significantly improve drug solubility and bioavailability [CUTRIN J C., et al., 2013, Molecular Pharmaceutics, 10(5):2079-85], suggesting its potential value in cypate delivery.

[0004] The tumor microenvironment (TME), a complex ecosystem composed of stromal cells, immune cells and extracellular matrix, plays a key role in tumor immune escape and treatment resistance. Among them, the M2 polarization of tumor-associated macrophages (TAMs) is the core driver of TME immunosuppression: M2 TAMs suppress anti-tumor immune responses by secreting cytokines such as IL-10 and TGF-β, and promote angiogenesis and metastasis. Therefore, reversing the immunosuppressive state of TME has become an important strategy to enhance the effect of tumor treatment. It is worth noting that the heat shock proteins produced by simple PTT may further activate immunosuppressive pathways, and there is an urgent need to develop new combination therapies that synergize PTT and TME regulation.

[0005] Melittin, the main active ingredient in bee venom, can reshape the TME immune microenvironment by specifically reducing the proportion of M1 macrophages. However, its clinical application is limited by two major defects: (1) it causes serious toxic side effects such as hemolysis of red blood cells when administered systemically; and (2) it lacks targeting, leading to damage to normal tissues.

[0006] Thermosensitive hydrogels offer a novel approach to addressing these challenges: they can achieve localized sustained release via in situ injection, avoiding systemic exposure. Their dynamic cross-linked network structure can be loaded with a variety of therapeutic components (e.g., drugs, nanoparticles), and their release kinetics can be controlled through temperature-responsive sol-gel transitions. For example, thermosensitive hydrogels loaded with paclitaxel significantly increased local drug concentration in tumors while reducing systemic toxicity [SHEU MT, et al., Colloids Surface B: Biointerfaces, 2016, 143:260-270], suggesting their potential application in melittin delivery.

[0007] In view of the above-mentioned characteristics of breast cancer, the present invention designs the use of in situ injection of hydrogel as a carrier for delivering Cypate and melittin, and develops a multifunctional synergistic treatment system that can integrate targeted delivery, photothermal ablation and immune microenvironment remodeling, thereby improving the pathological state of breast cancer. Summary of the Invention

[0008] In view of the technical problems existing in the background technology, the purpose of the present invention is to provide a self-repairing temperature-sensitive hydrogel loaded with Cypate and melittin and its application.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides a self-repairing thermosensitive hydrogel loaded with Cypate and melittin, comprising the following components in parts by weight: 30-50 parts of chitosan, 5 parts of sodium β-glycerophosphate, 1-4 parts of oxidized chondroitin sulfate, 1-4 parts of Cypate recombinant apoferrin nanoparticles, 1-4 parts of melittin, and the remainder being water.

[0011] Preferably, the Cypate-recombinant apoferritin nanoparticles comprise a recombinant apoferritin shell and Cypate encapsulated in the recombinant apoferritin shell, wherein the recombinant apoferritin shell is recombinant apoferritin HFn.

[0012] A second aspect of the present invention provides a method for preparing the above-mentioned self-repairing thermosensitive hydrogel loaded with Cypate and melittin, comprising the steps of: dropwise adding a sodium β-glycerophosphate solution to a chitosan solution, then adding oxidized chondroitin sulfate and melittin, and finally adding Cypate recombinant apoferritin nanoparticles to obtain a self-repairing thermosensitive hydrogel.

[0013] The third aspect of the present invention provides the use of the above-mentioned self-repairing temperature-sensitive hydrogel loaded with Cypate and melittin in the preparation of drugs for treating breast cancer.

[0014] The present invention has the following beneficial effects:

[0015] (1) Since the existing thermosensitive hydrogel composed of chitosan and β-sodium glycerophosphate (β-GP) has poor gel strength and is easily affected by external factors, resulting in drug leakage. In view of this, the present invention designs the introduction of dynamic imine chemical bonds to construct a thermosensitive self-healing hydrogel. Specifically, the present invention first introduces oxidized chondroitin sulfate (OCS) into the thermosensitive hydrogel composed of chitosan and β-sodium glycerophosphate (β-GP) (i.e., chitosan / β-sodium glycerophosphate hydrogel) (chondroitin sulfate is a natural glycosaminoglycan present in the extracellular matrix, and its D-glucuronic acid unit contains an O-dihydroxy group, which can be selectively oxidized by sodium periodate (NaIO4) to generate an active aldehyde group, thereby forming oxidized chondroitin sulfate (OCS)). Since oxidized chondroitin sulfate can form a Schiff base with the amino group in chitosan, it can give the hydrogel self-healing properties, improve its mechanical strength, and better control the sustained release of the therapeutic agent. This design aims to achieve in situ injection (the hydrogel is injected in situ into a gel at the tumor site and its pharmacodynamics is evaluated in animals) and to give the hydrogel the ability to self-repair, thereby making drug release more controllable. This not only improves the performance of the hydrogel, but also provides a new way to control drug release.

[0016] (2) The present invention uses apoferritin to encapsulate Cypate, and the resulting FC (HFn@Cypate) nanoparticles can be evenly dispersed in the OCGM hydrogel to construct the OCGM-FC hydrogel. When injected around the tumor, the OCGM-FC hydrogel exhibits excellent retention ability and promotes the deep and sustained release of FC nanoparticles inside the tumor, thereby improving the photothermal therapy effect. At the same time, the slow and sustained release of bee venom peptide can specifically reduce the expression level of M1 macrophages, promote the M2 / M1 ratio, and regulate the immunosuppressive microenvironment. The experimental results show that the combined use of the above two methods exhibits a significant anti-tumor effect.

[0017] (3) Chitosan is one of the main biomacromolecules in the hydrogel. This polymer has been approved by the FDA for its high biocompatibility and safety and is widely used in pharmaceutical production. Chondroitin sulfate is also recognized for its excellent biocompatibility and is commonly used in clinical practice. Therefore, the hydrogel developed by the present invention provides a safe, reliable, and cost-effective option for the clinical treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Characterization results of HFn protein carrier: Figure 1 A is the SDS-PAGE protein analysis of HFn; Figure 1 B is the spectrum image of HFn in the range of 200-600 nm; Figure 1 C is the hydrated particle size image of HFn; Figure 1 D is the transmission electron microscopy (TEM) image of HFn after negative staining with 2% phosphotungstic acid;

[0020] Figure 2 Characterization results of FC nanoparticles: Figure 2 A is the TEM image of FC nanoparticles; Figure 2 B is the hydrated particle size distribution of FC nanoparticles; Figure 2 C is the fluorescence spectrum of FC nanoparticles;

[0021] Figure 3 : Figure 3 A is the image of OCGM-FC hydrogel before and after incubation at 37°C; Figure 3 B is the self-healing property of OCGM-FC hydrogel; Figure 3 C is the SEM image of OCGM-FC hydrogel; Figure 3 D is the compressive strength of thermosensitive hydrogel, self-healing hydrogel and thermosensitive self-healing hydrogel;

[0022] Figure 4 : Figure 4 A shows the effects of different treatment groups on 4T1 cell migration at different incubation times; Figure 4 B is the result of semi-quantitative analysis of cell migration using Image J;

[0023] Figure 5 The internal penetration of Cypate and FC into 4T1 tumor spheres;

[0024] Figure 6 The apoptosis of 4T1 cells after treatment in different treatment groups;

[0025] Figure 7 The fluorescence degradation in mice injected subcutaneously with OCGM-FC hydrogel;

[0026] Figure 8 : Figure 8 A is the tumor volume of different treatment groups; 8B is the in vitro tumor tissues of mice in different treatment groups on the 10th day of treatment. DETAILED DESCRIPTION

[0027] In the following description, specific details such as specific system structures and technologies are provided for the purpose of illustration rather than limitation to facilitate a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0028] Example 1

[0029] 1. Preparation of H subunit recombinant apoferritin (HFn)

[0030] Referring to the method of Chinese patent CN110075087A, H subunit recombinant apoferritin (HFn) was prepared by recombinant genetic engineering technology. The recombinant plasmid and engineering bacteria were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0031] Specifically, a trace amount of ampicillin-resistant engineered bacteria was inoculated into 10 mL of LB-Amp+ medium containing ampicillin (LB-Amp+) and shaken at 37°C overnight. 5 mL of the activated bacterial solution was inoculated into 100 mL of LB-Amp+ medium and shaken at 37°C for 3-4 hours. IPTG was then added to induce HFn protein expression, and the shaking was continued. The solution was centrifuged to collect the precipitated cells, which were lysed using an ultrasonic disruptor to obtain a crude protein extract. Finally, the crude protein solution was purified using nickel column affinity chromatography with a gradient imidazole solution to obtain the target protein HFn.

[0032] The molecular weight of the prepared ferritin was verified by SDS-PAGE. Figure 1 As shown in A.

[0033] Figure 1 A results showed that no miscellaneous bands appeared in the 300 mM imidazole eluate, and the molecular weight was around 29.0 KD, which was consistent with the literature reports, indicating that HFn was successfully extracted and purified.

[0034] In addition, the prepared HFn was scanned in the wavelength range of 200 to 600 nm, and the results were as follows Figure 1 As shown in B.

[0035] Figure 1 B. The result shows that the ultraviolet absorption peak at 280 nm indicates that the purified substance is a protein.

[0036] The hydrated particle size of HFn was measured by Malvern particle size analyzer. Figure 1 As shown in C.

[0037] Figure 1 The results showed that the hydrated particle size of HFn was 14.79±1.86 nm and the polydispersity index (PDI) was 0.106±0.03, indicating that it had good dispersibility.

[0038] In addition, the morphology of HFn was characterized by transmission electron microscopy (TEM), and the results were as follows: Figure 1 As shown in D.

[0039] Figure 1 The results showed that HFn exhibited a spherical morphology with uniform and consistent particle size distribution, approximately 15 nm. This is consistent with the morphology of ferritin reported in the literature, confirming that HFn has been successfully prepared using recombinant genetic engineering technology.

[0040] 2. Preparation and characterization of HFn@Cypate (FC) nanoparticles

[0041] FC nanoparticles were prepared by pH depolymerization-reconstitution method.

[0042] Specifically, 0.1M HCl solution was first added dropwise to 1.0mL of 5mg / mL HFn solution until the pH dropped to 2.0, causing HFn to disaggregate into subunits. 1.0mL of 10mmol / mL Cypate solution was then added to the mixture, and the mixture was incubated at room temperature for 30min. The pH was then raised to 8.0 using 0.1M NaOH solution, allowing the disaggregated HFn subunits to reassemble into a cage-like structure, thereby encapsulating Cypate. The mixture was then dialyzed using a 100kDa molecular weight cutoff dialysis bag to remove unencapsulated Cypate, resulting in FC nanoparticles.

[0043] The morphology of FC nanoparticles was characterized by TEM. Figure 2 As shown in A.

[0044] Figure 2 AThe results show that FC nanoparticles also present a spherical morphology structure with uniform particle size and consistent distribution.

[0045] The hydrated particle size of FC nanoparticles was measured by Malvern particle size analyzer. Figure 2 As shown in B.

[0046] Figure 2 B The results showed that the hydrated particle size of FC nanoparticles was 19.04±2.36nm and the PDI was 0.206±0.07, indicating that they had good dispersibility.

[0047] To confirm that Cypate was effectively loaded into HFn, fluorescence spectrophotometry was used to investigate it. The results are as follows: Figure 2 As shown in C.

[0048] Figure 2 The results of C showed that the excitation wavelength of Cypate and FC nanoparticles was 783 nm, and the emission wavelength was 815 nm. The fluorescence spectrum results showed that the fluorescence properties of Cypate were not changed after encapsulation.

[0049] 3. Preparation and characterization of thermosensitive self-healing hydrogels of oxidized chondroitin sulfate-chitosan-melitin-HFn@Cypate (abbreviated as OCGM-FC)

[0050] (1) Preparation and characterization of oxidized chondroitin sulfate (OCS)

[0051] Accurately weigh 0.8 g of chondroitin sulfate and prepare a 2% (w / v) aqueous solution. Add 0.4 g of sodium periodate at a mass ratio of 1:1 and allow to react in the dark for 6 h. Then, add 600 μL of ethylene glycol to terminate the reaction. After 30 min, add approximately 0.5 g of sodium chloride and dissolve thoroughly. Then, add approximately 50 mL of anhydrous ethanol at a volume ratio of 1:2 and stir for 5 min to obtain a flocculent precipitate. Then, vacuum filter the mixture to obtain a white precipitate. Dissolve the precipitate in 50 mL of water and place it in a dialysis bag (MW: 8,000-14,000). Dialyze the mixture at 4°C for 2 days, followed by reverse dialysis for 3-5 days. Finally, freeze-dry the product to obtain oxidized chondroitin sulfate (OCS).

[0052] Fourier transform infrared spectroscopy (FTIR) was used to characterize the changes of the obtained chondroitin sulfate before and after oxidation. Figure 3 As shown in A.

[0053] Figure 3 A results showed that compared with chondroitin sulfate (CS), oxidized chondroitin sulfate (OCS) at 1725 cm -1 A characteristic aldehyde peak was shown at , confirming the occurrence of oxidation reaction.

[0054] (2) Preparation and characterization of OCGM-FC hydrogel

[0055] First, 2 mL of 6% (w / v) sodium β-glycerophosphate solution was added dropwise to 3 mL of 2% (w / v) chitosan solution under continuous stirring, and stirring was continued for 15 minutes to prepare a chitosan / sodium β-glycerophosphate thermosensitive hydrogel solution (CG). Then, 0.02 g of OCS powder and 1 mL of 10 mmol / L melittin were added to the CG hydrogel under continuous stirring. After complete dissolution, stirring was continued for 15 minutes to obtain an OCS / chitosan / β-GP / melittin thermosensitive self-healing hydrogel solution (OCGM).

[0056] Characterization:

[0057] (1) The temperature sensitivity of the obtained hydrogel was examined by the inversion method using a 37±1℃ water bath. Figure 3 As shown in A.

[0058] Figure 3 A The results showed that the OCGM-FC solution was liquid at room temperature and immediately formed a hydrogel after incubation at 37°C for 1 minute, indicating its good temperature sensitivity and ability to quickly form a hydrogel in vivo.

[0059] (2) By cutting a complete OCGM-FC hydrogel into two halves, staining the two halves with yellow and purple dyes, and then putting the two halves back together to observe whether they can gradually fuse back into a complete hydrogel, thus demonstrating self-healing ability; this process was repeated to determine whether the hydrogel can undergo multiple self-repair cycles. The results are shown in Figure 2. Figure 3 As shown in B.

[0060] Figure 3 B The results showed that when the cut hydrogel was recombinated, the cut surface gradually blurred and the cut parts were recombinated, demonstrating repeated self-healing properties, which also confirmed the successful synthesis of OCS.

[0061] (3) Scanning electron microscopy (SEM) was used to examine the morphology of the hydrogel. The mechanical properties of the hydrogel were evaluated using a TA-XT2 texture analyzer. Figure 3 As shown in C.

[0062] Figure 3 The C results show its three-dimensional network structure and relatively dense configuration.

[0063] (4) Mechanical properties are crucial when gels are used as drug delivery systems. Conventional thermosensitive hydrogels are mechanically weak and easily damaged, so the mechanical strength of the hydrogels was evaluated. Figure 3 As shown in D.

[0064] Figure 3 The results showed that the mechanical strength of the CS / β-GP hydrogel was relatively low, while the mechanical strength of the OCS / CS hydrogel prepared by introducing OCS was significantly improved. This strength improvement was attributed to the chemical crosslinking between OCS and chitosan through dynamic amide bonds, which made the hydrogel denser and significantly improved its mechanical strength, overcoming the fragility of the CS / β-GP thermosensitive hydrogel. Furthermore, the addition of FC nanoparticles did not affect the mechanical strength of the OCS hydrogel.

[0065] In summary, the present invention successfully synthesized OCS and prepared OCGM-FC hydrogel with temperature sensitivity and self-healing ability.

[0066] Test Example 1

[0067] Cell experiments

[0068] 1. Cell migration

[0069] The cell scratch assay was used to evaluate the anti-tumor cell migration effect of OCGM-FC hydrogel in vitro.

[0070] First, 4T1 cells were plated into a 24-well plate (1 mL per well) at a density of 2×104 cells / well, and the cells were cultured to adhere to the wall and reach the required density. Next, a sterile pipette tip was used to draw a straight line on the monolayer of cells to form a scratch, and then gently rinsed with PBS to remove the detached cells. Then, 1 mL of the drug solution diluted with complete culture medium was added to the well plate and placed in the incubator for continued culture. The plates were observed under a microscope at 0, 12, and 24 hours and photographed. Finally, ImageJ was used to measure the change in scratch width and calculate the cell migration rate. The results are shown in the figure below. Figure 4 As shown in A and 4B.

[0071] Figure 4Results A and 4B show that, without laser irradiation, OCGM hydrogel and OCGM-FC hydrogel administration exhibited an inhibitory effect on cell migration. However, with laser irradiation, OCGM-FC hydrogel treatment significantly inhibited 4T1 cell migration. This result suggests that the combined treatment group has a stronger ability to inhibit cell migration. This result is attributed to the strong positive charge of bee venom peptide in OCGM, which can induce apoptosis in tumor cells, thereby inhibiting their migration. OCGM-FC shares the same principle as OCGM, but when OCGM-FC is exposed to light, the cypate in FC produces a photothermal effect, synergizing with cypate to produce a stronger tumor inhibitory effect.

[0072] 2. Tumor sphere penetration experiment

[0073] The tumor penetration effect of FC nanoparticles was investigated by tumor sphere culture. First, agarose was dissolved in serum-free high-glucose DMEM culture medium, and sterilized after being prepared into a 1.5% agarose solution (sterilized for 20 minutes). The agarose was transferred to a sterile operating table while hot (over 90°C) and added to a 96-well plate (50 μL / well). Then, after the agarose cooled and solidified, the cells were seeded into a 96-well plate at a density of 5000 cells / well and incubated for one week. During this period, the cell growth and density were continuously observed and complete culture medium was supplemented. Then, after one week, cell spheres of uniform size were selected as tumor sphere models, 100 μL of drug solution was added to each well, and the cells were placed in an incubator and incubated for 6 hours. Finally, the tumor spheres were gently rinsed with PBS and transferred to a confocal dish, and the fluorescence distribution in the tumor spheres was observed by confocal microscopy.

[0074] The tumor sphere permeability experiment was conducted to investigate whether HFn as a drug delivery carrier can promote drug penetration into the tumor. Laser confocal microscopy was used to perform slices of the tumor spheres at different depths to observe the drug penetration. Figure 5 shown.

[0075] Figure 5 The results showed that compared with simple Cypate, encapsulating it in HFn can significantly penetrate into the tumor spheres. On the basis of improving the hydrophobicity of Cypate, it promotes Cypate to penetrate deep into the tumor, thereby better exerting the photothermal therapeutic effect.

[0076] 3. Apoptosis

[0077] Cell apoptosis was used to investigate the anti-tumor effect of OCGM-FC hydrogel in vitro.

[0078] First, place a sterile coverslip in a 24-well plate in advance and plate the cells at a density of 2×104 cells / well (1 mL per well) to adhere to the wall and reach the required density. Next, remove the culture medium, add 1 mL of PBS, rinse gently, and remove by aspiration. Add 1 mL of a drug solution diluted with complete culture medium to the plate. After incubation for 24 hours, remove the drug solution from the plate and add 1 mL of PBS, rinse gently, and remove by aspiration. Then, add a cell fixative (4% paraformaldehyde) to the plate and fix it at room temperature for 10 minutes. After removing the fixative, rinse gently with PBS, and then add a strong immunostaining permeabilization solution to the plate and incubate at room temperature for 5 minutes. Finally, prepare TUNEL detection solution according to the TUNEL kit instructions (prepare and use immediately), incubate in the dark at 37°C for 60 minutes, rinse gently with PBS three times and remove by aspiration, then add anti-fluorescence quenching sealing solution containing DAPI and store for later use. Place the side containing cells face down on the slide and seal with sealing agent, observe and take pictures with a fluorescence microscope. The apoptosis of 4T1 tumor cells in different drug groups was observed by fluorescence microscopy. The results are shown in Figure 2. Figure 6 shown.

[0079] Figure 6 The results showed that compared with the control group, there was no obvious cell apoptosis after the administration of OCG hydrogel alone. In the absence of laser irradiation, the effects of melittin, OCGM, and OCGM-FC on cell apoptosis were similar. This is mainly because melittin plays a role in reshaping the immunosuppressive tumor microenvironment and cannot exert a photothermal effect in the absence of laser irradiation. However, in the presence of laser irradiation, the OCGM-FC treatment group had a significantly stronger effect on cell apoptosis than the FC and OCG-FC treatment groups, which also shows that the combined treatment has a better anti-tumor effect.

[0080] Test Example 2

[0081] Animal experiments

[0082] 1. In vivo degradation experiment

[0083] First, an OCGM-FC hydrogel precursor solution was prepared and subcutaneously injected into mice. Cypate degradation rates were observed and recorded at predetermined time points using a small animal in vivo imaging device. Furthermore, mice were sacrificed on days 1, 2, 4, 8, 12, and 24, and skin tissue from the injection site was removed and photographed to assess the in vivo degradation of the OCGM-FC hydrogel.

[0084] In order to analyze the degradation rate of Cypate in vivo, a small animal in vivo imaging system was used to observe its luminescence in vivo. The results are as follows Figure 7 shown.

[0085] Figure 7 The results showed that although the fluorescence intensity of Cypate weakened over time, weak fluorescence could still be observed after the 12th day, which means that OCGM-FC hydrogel can exist stably in the body for at least 12 days.

[0086] 2. In vivo evaluation of anti-tumor effects

[0087] First, a subcutaneous breast tumor model in mice was established. The hair on the mouse abdomen was shaved with a razor, and then the depilatory cream was applied to the mouse abdomen. The depilatory cream was then applied to the area, and the depilatory cream was thoroughly removed after a few minutes to ensure that the skin surface was clean and free of residue. Then, 100 μL of a cell suspension containing approximately 2×106 4T1 cells was subcutaneously injected into the mouse mammary gland to establish a tumor-bearing mouse animal model. Then, 30 tumor-bearing mice were randomly assigned to six experimental groups (5 mice in each group), namely: PBS control group, OCG group, OCGM group, OCGM-FC group, OCG-FC+Laser group, and OCGM-FC+Laser group. When the tumor volume grew to about 100 mm 3 At the same time, each group of mice was given PBS and the corresponding hydrogel precursor solution by peritumoral injection. At the same time, the temperature changes of each treatment group during the treatment were monitored and recorded by infrared thermal imager to evaluate the effect of photothermal therapy. Starting from the start day of treatment (day 0), the length and width of the mouse tumor were measured and recorded with a vernier caliper every other day, and the weight of the mouse was recorded at the same time. The tumor volume was calculated according to the following formula: V (mm3) = length × width × width / 2 to evaluate the anti-tumor efficacy in vivo. The results are shown in Figure 8 shown.

[0088] Figure 8 The results showed that OCGM-FC hydrogel had a certain inhibitory effect on breast cancer, as evidenced by recording tumor growth curves and excising tumor tissue 10 days after treatment. Compared to the control group (PBS), the OCG hydrogel had no significant inhibitory effect on tumors. All other treatment groups significantly inhibited tumor growth, with the OCGM+Laser group exhibiting the smallest excised tumor tissue, demonstrating its greatest anti-tumor efficacy.

[0089] The present invention is not limited to the above-mentioned specific implementation methods. Various changes made by ordinary technicians in this field based on the above-mentioned concept without creative work are all within the scope of protection of the present invention.

Claims

1. A self-repairing thermosensitive hydrogel loaded with Cypate and melittin, characterized in that: The invention comprises the following components in parts by weight: 30-50 parts of chitosan, 5 parts of sodium beta-glycerophosphate, 1-4 parts of oxidized chondroitin sulfate, 1-4 parts of Cypate recombinant apoferritin nanoparticles, 1-4 parts of melittin, and the rest is water.

2. The self-repairing thermosensitive hydrogel loaded with Cypate and melittin according to claim 1, characterized in that: The Cypate-recombinant apoferritin nanoparticles include a recombinant apoferritin shell and Cypate encapsulated in the recombinant apoferritin shell, wherein the recombinant apoferritin shell is recombinant apoferritin HFn.

3. A method for preparing a self-repairing thermosensitive hydrogel loaded with cypate and melittin according to any one of claims 1 or 2, characterized in that: The method comprises the following steps: adding a sodium beta-glycerophosphate solution dropwise into a chitosan solution, then adding oxidized chondroitin sulfate and melittin, and finally adding Cypate recombinant apoferritin nanoparticles to obtain a self-repairing thermosensitive hydrogel.

4. Use of the self-repairing thermosensitive hydrogel loaded with cypate and melittin as claimed in any one of claims 1 or 2 in the preparation of a drug for treating breast cancer.

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