Inhibitor-loaded oncolytic microgel as well as preparation method and application thereof

Abstract

The invention discloses an inhibitor-loaded oncolytic microgel as well as a preparation method and application thereof, and the inhibitor-loaded oncolytic microgel is an oncolytic microgel (OMG) with similar activity and immune activation capability to an oncolytic peptide, and can efficiently entrap an immune checkpoint inhibitor and realize adjustable drug sustained release so as to enhance the cancer immunotherapy effect. The oncolytic microgel can effectively maintain the biological activity of entrapped drugs and antibodies, and has wide application prospects in the fields of drug controlled release and the like. Meanwhile, single injection of OMG (P1C4-OMG) loaded with anti-PD-1 and anti-CTLA-4 in a tumor can effectively reverse an inhibitory tumor microenvironment, enhance anti-tumor immune response and realize a remarkable tumor inhibition effect and outstanding survival benefits, and the oncolytic microgel with the adjustable antibody release function is a safe and unique cancer immunotherapy platform.

Description

TECHNICAL FIELD

[0001] The present application relates to a tumor-inhibitor-loaded oncolytic microgel, a preparation method of an immune checkpoint inhibitor-loaded oncolytic microgel and its application. BACKGROUND

[0002] Cancer immunotherapy has become an important way to treat malignant tumors in various indications by inhibiting immune checkpoints, activating immune stimulation cascades, and reprogramming immune cells. Using antibodies to inhibit immune checkpoints including cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death protein 1 ligand 1 (PD-L1) is one of the most successful immunotherapy strategies (Y. Shiravand. Curr. Oncol.2022, 29, 3044-3060). However, it is worth noting that immune checkpoint inhibitors (ICIs) usually require multiple intravenous injections of antibodies, which can induce excessive activation of the immune system, leading to serious side effects and even patient death in some cases (Q. Sun. Signal. Transduct. Target. Ther. 2023, 8, 320). ICI therapy also has the general problem of clinical response rate, which is related to the immunosuppressive tumor microenvironment (TME) (S. Zhang. Adv. Mater.2022, 34, 2206851). Chemotherapy, radiotherapy, immunoadjuvants, and cancer vaccines that can modulate the TME have shown varying degrees of improvement in the effectiveness of ICI therapy. Oncolytic peptides that can directly lyse tumor cells, induce immunogenic cell death (ICD), and activate dendritic cells provide an effective immunotherapy strategy. Oncolytic peptide LTX-315 has shown potential in treating patients with advanced melanoma and sarcoma in clinical trials (M. Nielsen. Oncoimmunology 2024, 13, 2290900). Unfortunately, oncolytic peptides can cause necrosis at the injection site and trigger severe systemic toxicity effects (H.M.Liu. Bioact. Mater. 2024, 31, 206-230). Therefore, it is necessary to develop a delivery system that can efficiently load immune checkpoint inhibitors and oncolytic peptides to improve the bioavailability of drugs, reduce side effects, and effectively enhance cancer immunotherapy. SUMMARY

[0003] The purpose of the present application is to provide a preparation method of an immune checkpoint inhibitor-loaded oncolytic microgel. The drug-loaded microgel prepared by the method can effectively achieve efficient loading and controlled release of drugs. In addition, the immune checkpoint inhibitor-loaded oncolytic microgel prepared by the method has good application prospects in the field of tumor immunotherapy.

[0004] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: The oncolytic microgel loaded with inhibitors comprises a microgel and inhibitors and oncolytic agents loaded in the microgel.

[0005] In the present application, the inhibitors are located inside and / or outside the microgel, and the oncolytic agents are located inside and / or outside the microgel; preferably, the outer surface of the microgel is provided with an oncolytic agent coating.

[0006] In the present application, the inhibitors include immune checkpoint inhibitors, as examples, the immune checkpoint inhibitors include one or more of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-LAG-3 antibody; the oncolytic agents include oncolytic peptides such as LTX-315, LTX-401, and Melittin.

[0007] Specifically, the present application discloses an oncolytic microgel (OMG) coated with oncolytic peptide LTX-315, which is loaded with immune checkpoint inhibitors and used to regulate the release behavior of immune checkpoint inhibitors (ICIs). In addition, the present application also relates to the application of the oncolytic microgel loaded with immune checkpoint inhibitors in tumor immunotherapy.

[0008] The present application discloses a preparation method of the above-mentioned oncolytic microgel loaded with inhibitors, comprising the following steps: mixing the inhibitors, the microgel, and the oncolytic agents to prepare the oncolytic microgel loaded with inhibitors; further, mixing the inhibitor solution, the microgel solution, and the oncolytic agent solution to prepare the oncolytic microgel loaded with inhibitors.

[0009] Preferably, the present application discloses a preparation method of the above-mentioned oncolytic microgel loaded with inhibitors, the inhibitor solution and the microgel solution are added to a dialysis bag, and dialysis is performed to obtain the microgel loaded with inhibitors (such as ICI@MG); then the oncolytic agent is added to prepare the oncolytic microgel loaded with inhibitors.

[0010] Further, the release behavior of the immune checkpoint inhibitors can be regulated by adjusting the concentration of the oncolytic agent solution. In the present application, the microgel (MG) includes hyaluronic acid, sodium alginate, or heparin microgel.

[0011] The present application discloses a preparation method of the above-mentioned microgel (MG), which is prepared by microfluidic technology and free radical photopolymerization technology, and has biodegradability and uniform size.

[0012] As an example, the application discloses a preparation method of a solubly oncolytic microgel loaded with an immune checkpoint inhibitor (ICI), comprising the following steps: adding an ICI solution and an MG solution into a dialysis bag, dialyzing with a buffer solution to prepare an ICI-loaded microgel (ICI@MG); and then adding a solubly oncolytic peptide LTX-315 solution, oscillating and mixing to prepare an ICI-loaded OMG (ICI@OMG), wherein the solubly oncolytic peptide LTX-315 can regulate the release behavior of the inhibitor loaded by the microgel.

[0013] As a control example, the application discloses a preparation method of a solubly oncolytic microgel, comprising the following steps: dissolving a solubly oncolytic peptide LTX-315 in deionized water, adding an MG solution, oscillating and mixing to prepare a microgel coated with the solubly oncolytic peptide (OMG).

[0014] In the application, the concentration of the MG solution is 10-80 mg / mL, the concentration of the ICI solution is 2-10 mg / mL, and the concentration of the LTX-315 solution is 5-40 mg / mL.

[0015] In the application, when dialysis is performed, the molecular weight cut-off of the dialysis bag is 5 kDa-50 kDa.

[0016] The application discloses the application of the above-mentioned inhibitor-loaded solubly oncolytic microgel, and specifically comprises the following: the application of anti-CTLA-4-loaded microgel in tumor treatment; the application of anti-CTLA-4-loaded solubly oncolytic microgel in tumor treatment; and the application of anti-CTLA4 and anti-PD-1-loaded solubly oncolytic microgel in tumor treatment.

[0017] In the application, a single intratumoral injection of anti-PD-1 and anti-CTLA-4-loaded microgel (P1C4@OMG) significantly promotes the infiltration of CD8 + and CD4 + T cells and the secretion of proinflammatory cytokines, inhibits tumor growth and prolongs survival time.

[0018] In the application, the OMG serves as a safe and efficient long-acting delivery platform for the delivery of immune checkpoint antibodies and has great potential in enhancing cancer immunotherapy.

[0019] Thanks to the above technical scheme, the application has the following advantages compared with the prior art: 1. The preparation method of the solubly oncolytic microgel loaded with an immune checkpoint inhibitor disclosed by the application is simple and does not require complex processes and procedures. The solubly oncolytic microgel not only has similar antitumor activity and immune activation capacity to the solubly oncolytic peptide LTX-315, but also can continuously release the immune checkpoint inhibitor, thereby significantly enhancing the antitumor immune response.

[0020] 2. The oncolytic microgel disclosed in the present application can control the release rate of the immune checkpoint inhibitor loaded therein by adjusting the coating amount of the surface oncolytic peptide LTX-315. This feature not only realizes the on-demand release of the antibody, but also avoids the problems caused by frequent administration.

[0021] 3. The immune checkpoint inhibitor-loaded oncolytic microgel disclosed in the present application can effectively reverse the immunosuppressive tumor microenvironment, promote T cell infiltration and pro-inflammatory cytokine secretion, significantly inhibit tumor growth and prolong survival time after a single dose injection. At the same time, the microgel does not cause obvious side effects and dysfunction in vivo, and has good in vivo safety. The oncolytic microgel as a safe and effective platform can be widely used in cancer immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is the characterization of the microgel in Example 1, wherein A is the determination of the size and size distribution of the MG under a fluorescence inverted microscope, and the inset is a microscope image (scale bar: 100 µm), and B is a representative scanning electron microscope image of the MG (scale bar: 50 µm).

[0023] Figure 2 Figure 2 is the antibody loading and oncolytic microgel construction in Example 2, wherein A is the drug loading efficiency of anti-PD-1, anti-PD-L1 and anti-CTLA-4 in the MG at pH 5.0, B is the adsorption curve of the MG adsorbing LTX-315 at different theoretical drug loading amounts, and C is the drug loading efficiency and drug loading amount of LTX-315 and IgG in IgG@OMG.

[0024] Figure 3 Figure 3 is the in vitro release curve of IgG and LTX-315 in Example 2.

[0025] Figure 4 Figure 4 is the in vitro anti-tumor effect of the OMG in Example 3, A is the cytotoxicity of the OMG after incubation with B16F10 cells for 48 h, and B is the quantitative analysis of the apoptosis of the B16F10 cells after incubation with the OMG for 48 h.

[0026] Figure 5 Figure 5 is the immunogenic cell death of the B16F10 tumor cells induced by the OMG in Example 4, the CLSM images of the B16F10 cells stained with the APC-labeled CRT antibody (A) or the HMGB1 antibody (B) after incubation with PBS, free LTX-315 and the OMG for 24 h, and the levels of CRT (C), HSP70 (D), ATP (E) and HMGB1 (F) in the B16F10 cells after treatment for 24 h.

[0027] Figure 6is the CD11c + expression in BMDCs stimulated by OMG in Example Five + CD86 + quantitative analysis of DCs.

[0028] Figure 7 is the retention time of IgG@OMG at B16F10 tumor-bearing mice in Example Six, A is the fluorescence imaging of mice administrated with free LTX-315, IgG and IgG@OMG, B is the semi-quantitative analysis of Cy7-IgG, C is the semi-quantitative analysis of Cy5-LTX-315.

[0029] Figure 8 is the in vivo therapeutic effect of C4@MG on B16F10 tumor-bearing mice in Example Seven, A is the body weight of mice, B is the average tumor growth curve of mice, C is the survival curve of mice.

[0030] Figure 9 is the in vivo therapeutic effect of C4@OMG on B16F10 tumor-bearing mice in Example Eight, B16F10 tumor-bearing mice in vivo therapeutic effect.

[0031] Figure 10 is the quantitative analysis of CTLA-4 expression on CD8 + T cells in tumor (A), lymph node (B), spleen (C) and peripheral blood (D) in Example Eight.

[0032] Figure 11 is the in vivo therapeutic effect of P1C4@OMG on B16F10 tumor-bearing mice in Example Nine, A is the average tumor growth curve of mice, B is the survival curve of mice.

[0033] Figure 12 is the blood biochemical indicators such as alanine aminotransferase (A), aspartate aminotransferase (B), urea (C) and creatinine (D) of C57BL / 6J female mice sacrificed on the 7th day after different treatments in Example Nine.

[0034] Figure 13 is the blood cell count including total red blood cells (A), hemoglobin (B) and red blood cell mass (C) of C57BL / 6J female mice sacrificed on the 7th day after different treatments in Example Nine.

[0035] Figure 14 is the immune analysis of P1C4@OMG in B16F10 tumor-bearing mice in Example Nine, the quantitative graph of lymph node mature DCs (A), CD8 + T cells (B) and CD4 + T cells (C) in tumor, the quantitative graph of CD49b +Quantitative analysis of NK cells (D), M2 macrophages (E), MDSCs (F) and Tregs (G), splenic CD8 + T (H) and CD4 + Quantitative determination of T (I) cells.

[0036] Figure 15 It is a structural schematic diagram of the microfluidic chip adopted in the application. DETAILED DESCRIPTION

[0037] The application specifically discloses a solerotic microgel capable of carrying an immune checkpoint inhibitor and a preparation method and application thereof, and the method comprises the following steps: taking a hyaluronic acid derivative as raw material, constructing MG through microfluidic technology and free radical polymerization technology; then, different antibodies (including anti-PD-1, anti-PD-L1 and anti-CTLA-4, etc.) can be easily loaded into the MG through simple mixing with the MG; and then, a solerotic peptide LTX-315 is coated to obtain the solerotic microgel capable of regulating the controlled release of antibodies.

[0038] In the technical scheme, the hyaluronic acid derivative is a hyaluronic acid-2-aminoethyl methacrylate derivative.

[0039] In the technical scheme, the antibodies are loaded into the microgel through the electrostatic interaction and hydrogen bond interaction between the antibodies and the MG by using a dialysis replacement method. Specifically, the antibodies are loaded into the MG through the dialysis replacement method. As an example, 20-40 mg / mL of MG solution is mixed with an antibody solution in a PBS solution, and the dialysis is performed using a PB solution with a pH of 5.0, and the antibodies are loaded into the MG.

[0040] In the technical scheme, the solerotic microgel (OMG) coated with the solerotic peptide on the surface is prepared through the strong electrostatic interaction between the solerotic peptide LTX-315 and the MG. Specifically, the MG suspension is mixed with an equal volume of LTX-315 solution to obtain the solerotic microgel loaded with LTX-315 on the surface.

[0041] In the application, the ICI@MG is mixed with LTX-315 to prepare ICI@OMG, and the IgG@MG is also mixed with LTX-315 to prepare IgG@OMG.

[0042] The application discloses a solerotic microgel capable of regulating the controlled release of antibodies and prepared based on the above method, which can be used as a cancer immunotherapeutic agent.

[0043] The application discloses a tumor-inhibitor-loaded oncolytic microgel as well as a preparation method and application thereof, and is an oncolytic microgel (OMG) with similar activity and immune activation capacity to oncolytic peptides, which can efficiently load immune checkpoint inhibitors and realize adjustable drug sustained release, thereby enhancing the effect of cancer immunotherapy. The oncolytic microgel can effectively maintain the bioactivity of the loaded drugs and antibodies, and has wide application prospects in the field of drug controlled release. The application can quantitatively load immune checkpoint inhibitors including anti-PD-1, anti-PD-L1 and anti-CTLA-4, and realize slow release for several weeks in vitro and in tumors. In a B16F10 melanoma model, a single intratumoral injection of the anti-CTLA-4-loaded microgel has a better tumor inhibition effect than multiple intravenous injections and intratumoral injections of free antibodies. Meanwhile, a single intratumoral injection of the anti-PD-1 and anti-CTLA-4-loaded OMG (P1C4@OMG) can effectively reverse the inhibitory tumor microenvironment, enhance the anti-tumor immune response, realize a significant tumor inhibition effect and outstanding survival benefit. The oncolytic microgel with adjustable antibody release function is a safe and unique cancer immunotherapy platform.

[0044] The application is further described below in combination with the drawings and examples; the raw materials used are all existing products, the specific preparation operation and performance test are conventional technologies, the animal experiment meets the relevant requirements of Suzhou University, the data statistics adopts a conventional statistical method. The anti-PD-1, anti-PD-L1 and anti-CTLA-4 are purchased from the Bioxcell Company in the United States, the LTX-315 (KKWWKKW(Dip)K-NH2) is purchased from the Shanghai Qiangyao Biological Technology Company Limited, and the IgG is purchased from the Wuhan Aimeijie Technology Company Limited, the hyaluronic acid is purchased from the Huaxi Biological Technology Company Limited.

[0045] Synthesis of hyaluronic acid methacrylic acid derivative (HA-AMA) Under nitrogen protection, 100 mL two-necked bottles are added with hyaluronic acid (3.0 g), deionized water 150 mL and dimethyl sulfoxide 10 mL, and after stirring to be dissolved, 2-aminoethyl methacrylate (AMA, 200-600 mg) is added to the solution, stirring is conducted for 10 minutes, then coupling agent 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 400-1200 mg) is added, an oil bath reaction is conducted at 35 DEG C for 24 hours, after the reaction is completed, dialysis is conducted through deionized water, and finally freeze-drying is conducted to obtain the hyaluronic acid derivative, and the yield is 86.9 %; HA-AMA nuclear magnetic resonance characterization 1¹H NMR (D₂O / DMSO-d₆): HA: δ 1.98, 3.15–4.05, 4.38–4.65; AMA: δ 1.90, 5.72, 6.12. The degree of substitution of the methacrylate groups (the number of methacrylate groups per 100 sugar units) can be calculated from the peak areas of the signal peaks, and is found to be 10–20.

[0046] Example 1: Preparation of Microgels (MG) Hyaluronic acid microgels were successfully prepared using a hyaluronic acid derivative (HA-AMA, degree of substitution 20) as the raw material via microfluidic technology and free radical polymerization. 100 mg of HA-AMA and 6 mg of I2959 photoinitiator were dissolved in 1 mL of PB buffer solution (pH 8.5, 10 mM) and loaded into an aqueous phase syringe as the aqueous phase of the microfluidic device. Simultaneously, mineral oil containing 10 wt.% Span 80 was loaded into an oil phase syringe as the oil phase. The aqueous and oil phases were then injected into a microfluidic chip (chip structure shown in [link to chip structure]). Figure 15 The process itself, along with microfluidic technology and free radical polymerization technology, is an existing technology. The oil phase flow rate is 10 μL / min, and the aqueous phase flow rate is 2.5 μL / min. After the two liquid phases enter the chip, the aqueous core flow forms uniform droplets under the continuous laminar shearing action of the lateral oil phase in the microchannel, and photocrosslinking is performed under 365 nm ultraviolet light irradiation to obtain MG emulsion. Then, mineral oil, Span 80 and I2959 initiators are removed using PB buffer, isopropanol and n-hexane to obtain MG PB solution. Morphological characterization revealed that the prepared MG exhibits a regular spherical shape. Specifically, the purified MG was dispersed in PB buffer (pH 7.4) at a concentration of 30 mg / mL, and samples were placed on glass slides. Observation under an inverted fluorescence microscope showed that the microgel had a uniform spherical morphology with an average diameter of 90 μm and a narrow particle size distribution (CV < 3.0%). Figure 1 A). Further characterization using scanning electron microscopy (SEM) revealed that the MG surface was smooth and without obvious defects. Figure 1 B). The above results indicate that hyaluronic acid microgels with uniform morphology and intact structure were successfully prepared.

[0047] Example 2: Antibody Loading and Oncolytic Microgel Construction The immune checkpoint antibodies were encapsulated in the MG by dialysis replacement method to prepare the ICI@MG. When the theoretical loading amount of the antibody was 5 wt.%, 150 μL of phosphate buffer solution (PBS, pH 7.4, 10 mM) containing 3.2 mg / mL of the antibody was added into 300 μL of PB buffer (pH 7.4) of 30 mg / mL of the MG. Subsequently, the mixture was dialyzed six times using 45 mL of PB solution (pH 5.0), and the dialysis medium was replaced every hour to obtain the ICI@MG. The un-loaded antibody in the supernatant was detected by using the Micro BCA kit. When the theoretical DLC (drug loading capacity) of the anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies was 5 wt.%, the drug DLE (encapsulation efficiency) was more than 95%, which was attributed to the strong electrostatic interaction and hydrogen bond interaction between the MG and the antibody. Figure 2 A), the ICI@MG of the anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies were respectively referred to as C1@MG, CL1@MG and C4@MG.

[0048] The LTX-315 was mixed with the blank MG in the PB solution to coat the LTX-315 on the MG to obtain the oncolytic microgel (OMG). Specifically, 300 μL of PB buffer (30 mg / mL) of the MG was mixed with an equal volume of the LTX-315 solution (PB buffer) to load the LTX-315 under the condition of 25 °C and 900 rpm, and the theoretical drug loading amount of the LTX-315 was in the range of 10-30 wt.%. It was known from the adsorption curve that even when the theoretical drug loading amount was increased to 30 wt.%, the positively charged LTX-315 could be quickly loaded into the MG within 5 minutes, the DLE of the LTX-315 was as high as more than 98%, and the LTX-315 was always stable on the MG subsequently Figure 2 B).

[0049] The preparation process of the ICI@MG was described by taking the preparation of the C4@OMG as an example. The specific steps were as follows: first, 150 μL of phosphate buffer solution (PBS, pH 7.4, 10 mM) containing 3.2 mg / mL of the antibody was added into 300 μL of PB buffer (pH 7.4) of 30 mg / mL of the MG to prepare the C4@MG solution by dialysis replacement method, and the total volume of the solution was 450 μL at this time. Subsequently, 150 μL of the supernatant was taken out. Then, 300 μL of the C4@MG solution was mixed with 300 μL of the LTX-315 solution (PB buffer) to perform the shock loading under the condition of 25 °C and 900 rpm to prepare the C4@OMG solution.

[0050] In addition, to achieve different doses of anti-CTLA-4 in C4@MG mice in vivo treatment, the theoretical drug loading of anti-CTLA-4 can be adjusted (Table 1). The specific doses are as follows: theoretical loading of 1.4 wt.% anti-CTLA-4 corresponds to 2 mg / kg; theoretical loading of 2.7 wt.% anti-CTLA-4 corresponds to 4 mg / kg; theoretical loading of 5.2 wt.% anti-CTLA-4 corresponds to 8 mg / kg. Further, to achieve the doses of 45 mg / kg and 4 mg / kg of LTX-315 and anti-CTLA-4 in C4@OMG mice in vivo treatment, by adjusting the theoretical drug loading of LTX-315 and anti-CTLA-4, the precise control of the administration dose can be achieved under the conditions of item three (Table 2).

[0051] Table 1 Characterization of C4@MG

[0052] a Measured by fluorescence spectrum.

[0053] Table 2 Characterization of C4@OMG

[0054]

[0055] a Measured by fluorescence spectrum.

[0056] The co-loading of MG with dual antibodies (anti-PD1 and anti-CTLA-4) was also achieved by adding 150 μL of different concentrations of dual antibodies in PBS solution to 300 μL of MG in PB buffer (30 mg / mL), and the preparation process is briefly described as follows: 75 μL of anti-PD1 antibody-containing phosphate buffer solution (PBS, pH 7.4, 10 mM) and 75 μL of anti-CTLA-4 antibody-containing phosphate buffer solution (PBS, pH 7.4, 10 mM) were added to 300 μL of 30 mg / mL MG in PB buffer (pH 7.4). Subsequently, the mixture was dialyzed six times using 45 mL of PB solution (pH 5.0), and the dialysis medium was replaced every hour to obtain P1C4@MG. By adjusting the theoretical loading amount of anti-PD-1 and anti-CTLA-4, the results showed that when the theoretical DLC of anti-CTLA-4 and anti-PD-1 was set to 2.70 wt.% and 2.75 wt.% respectively, the actual DLC reached 2.63 wt.% (Table 3). The loading amount of the two antibodies in P1C4@MG was the same, and the dose of the dual antibody in mice was 4 mg / kg. In addition, using a similar preparation method as C4@OMG, OMG (P1C4@OMG) loaded with anti-CTLA-4 and anti-PD-1 was prepared. When the theoretical DLC of LTX-315, anti-CTLA-4 and anti-PD-1 was set to 23.1 wt.%, 2.72 wt.% and 2.77 wt.% respectively (Table 4), the doses of LTX-315, anti-CTLA-4 and anti-PD-1 in the P1C4@OMG treatment group were 45 mg / kg, 4 mg / kg and 4 mg / kg, respectively.

[0057] Table 3 Characterization of P1C4@OMG

[0058] a Measured by fluorescence spectrum.

[0059] Table 4 Characterization of P1C4@OMG

[0060] a Measured by fluorescence spectrum.

[0061] Comparative Example In Reference Example 2, IgG was used instead of antibodies to prepare IgG@MG and IgG@OMG.

[0062] LTX-315 was added to IgG@MG to prepare microgels co-loaded with LTX-315 and IgG protein (IgG@OMG). With different amounts of LTX-315 coating, IgG-loaded MG showed excellent loading capacity. It was found that the DLE of LTX-315 and IgG could still reach more than 95% and 90% (C), respectively, indicating that MG could efficiently load two different drugs at the same time, and the drug loading efficiency was not affected. Figure 2

[0063] In addition, the release behavior of IgG and LTX-315 in IgG@OMG was explored. 100 μL of IgG@OMG (containing 30 mg / mL MG) was placed in the inner chamber of a Transwell plate. Then, 1.0 mL of PBS (pH 6.5, 10 mM) was added to the plate to immerse the IgG@OMG. The plate was placed in a shaking bed at 37 °C and 100 rpm. At the predetermined time point, the release medium was collected and replaced with 1.0 mL of PBS. The content of IgG and LTX-315 in the release medium was measured, and the cumulative drug release curve of the relative drug was made. The results showed that compared with the burst release phenomenon of IgG in the MG without LTX-315 coating (IgG@MG) (about 60% released within one day), the MG with LTX-315 coating (IgG@OMG) achieved sustained release of antibodies and LTX-315 (Fig. 4B). Figure 3 The increase of LTX-315 loading from 0 to 25 wt.% could extend the release time of antibodies to four weeks, while further increasing the LTX-315 loading led to faster release of IgG, which might be due to the excessive LTX-315 weakening the interaction between antibodies and MG. Therefore, the MG coated with 25 wt.% LTX-315 achieved the best balance between drug loading capacity and long-term drug release, and was used for subsequent studies.

[0064] Example Three In Vitro Anti-tumor Effect of OMG B16F10 tumor cells were used as a model to study the in vitro toxicity of free LTX-315 and OMG on tumor cells. B16F10 cells were seeded in a 96-well plate (3000 cells per well) and cultured for 12 hours. After adding 20 μL of oncolytic peptide LTX-315 or OMG with a concentration range of 0.1 to 100 μg / mL per well, the cells were incubated for another 48 hours. Then, 10 μL of thiazolyl blue (MTT) solution (5 mg / mL) was added to react with the DNA in the living cells for 4 hours to generate formazan crystals, which were then dissolved in 150 μL of dimethyl sulfoxide (DMSO). The absorbance (OD) at 492 nm was detected using a multifunctional enzyme label instrument. Six replicate wells were set for each concentration group, and the relative survival of the cells was calculated. The half-inhibitory concentration (IC​50 ) were calculated by non-linear regression in cell viability curves. It is worth noting that, similar to free oncolytic peptide LTX-315, OMG showed comparable cytotoxicity against B16F10 cells, with IC 50 of 19.30 μg / mL ( Figure 4 A).

[0065] In addition, the effect of LTX-315 on B16F10 cell apoptosis was studied by flow cytometry. 1 mL of B16F10 cell suspension was inoculated in a 12-well plate (2 x 10 5 / well), and then 20 μL of PBS, free LTX-315 and OMG (LTX-315 concentration: 20 μg / mL) was added to each well. After incubation in an incubator for 48 hours, the cells were collected, washed twice with PBS, resuspended with 300 μL of binding buffer, and 5 μL of Annexin V-APC and 10 μL of 7-AAD staining solution were added according to the kit instructions, and incubated at 37°C for 5 minutes in the dark, and then the apoptosis of the cells was detected by FACS flow cytometry. The results showed that OMG had a significant pro-apoptotic ability, and the ability to promote cell apoptosis was comparable to free LTX-315 ( Figure 4 B).

[0066] Example Four Study on OMG Inducing Immunogenic Cell Death of B16F10 Tumor Cells To observe the expression of calreticulin (CRT) and high mobility group box 1 protein B1 (HMGB1) by confocal microscopy (CLSM), B16F10 cells were inoculated on 18 mm glass coverslips in a 12-well plate for 12 hours, and then treated with PBS, free LTX-315 or OMG (LTX-315 concentration: 20 μg / mL) for 24 hours. After 24 hours, the upper culture medium was removed and washed twice with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, then incubated with anti-CRT antibody or anti-HMGB1 antibody overnight, and then incubated with Alexa Flour 647-conjugated secondary antibody for 1 hour. DAPI staining for 3 minutes, and washed with PBS for 3-5 times between each step, and then mounted with glycerol and observed by CLSM (20x). The CLSM images showed that B16F10 cells treated with OMG for 24 hours showed obvious CRT expression ( Figure 5 A), and the HMGB1 signal was significantly reduced ( Figure 5 B) due to the escape of HMGB1 from the nucleus during cell death.

[0067] In addition, B16F10 cells (2 x 10 5B16F10 cells were seeded in 12-well plates for 12 hours, and then treated with free LTX-315 or OMG (LTX-315: 20 pg / mL) for 24 hours (n = 3). The supernatant was collected and the concentrations of adenosine triphosphate (ATP) and HMGB1 were determined according to the kit instructions. Cells were trypsinized and washed twice with PBS; incubated with blocking antibody for 20 minutes at 4°C; incubated with anti-CRT antibody for 1 hour at 4°C, and washed twice with PBS; incubated with Alexa Fluor 647-labeled goat anti-rabbit / mouse antibody for 30 minutes at 4°C, and washed twice with PBS. The expression of CRT on the surface of B16F10 cells was determined by FACS flow cytometry. The expression of heat shock protein 70 (HSP70) was determined in a similar manner, but using an anti-HSP70 antibody. Quantitative analysis by flow cytometry found that the expression of CRT and heat shock protein 70 (HSP70) in OMG-treated cells increased by about 2.5 times, and the expression levels of extracellular ATP and HMGB1 also increased significantly Figure 5 C-F). These results show that OMG can effectively induce immunogenic cell death of B16F10 tumor cells, and significantly release damage-associated molecular patterns (DAMPs) that can act as tumor-associated antigens (TAAs) to initiate an immune response through antigen-presenting cells (APCs).

[0068] Example Five OMG Promotes Maturation of BMDCs DCs, as the most important APC cells, play a crucial role in immune responses. Next, we investigated whether the OMG-mediated ICD effect in cancer cells can stimulate the maturation of tumor-associated antigen-presenting cells, such as dendritic cells. We extracted bone marrow-derived dendritic cells (BMDCs) from the limbs of healthy C57BL / 6J mice, and investigated the effect of OMG-induced ICD produced by B16F10 tumor cells within 24 hours, and then co-incubated the supernatant with BMDC cells for 24 hours to mature the BMDCs (mDCs, CD11c + CD80 + CD86 + ). To study the activation of BMDCs by OMG incubated with B16F10 cells, which simulates the tumor environment, B16F10 cells were seeded in 12-well plates (2 x 10 5 / well) overnight to adhere, and then 20 μL of PBS, free LTX-315, or OMG (LTX-315 concentration: 20 pg / mL) was added for 24 hours. The supernatant of the B16F10 cells treated in each group was then transferred to the BMDCs (1 x 10 6BMDCs were co-incubated with B16F10 cells at a ratio of 1:1 (BMDCs: B16F10 cells) in a 12-well plate (n = 3). After 24 hours of co-incubation, immune cells were collected and blocked with anti-mouse CD16 / 32 for 30 minutes on ice. Then, BMDCs were incubated with anti-CD11c-FITC, anti-CD80-APC and anti-CD86-PE-Cy7 antibodies at 4 °C in the dark for 30 minutes to label the cells. After washing the cells with PBS, the cells were resuspended in 300 μL PBS and the proportion of mature BMDCs was detected by FACS flow cytometry. The results showed that the ICD effect induced by B16F10 cells with OMG could successfully activate the maturation of about 20% of BMDCs. The maturation of BMDCs induced by OMG was 7.7 times that of the PBS group ( Figure 6 ), confirming that OMG can induce strong immune activation.

[0069] Example Six Retention of IgG@OMG in Tumors To evaluate the retention of IgG@OMG in tumors in vivo. B16F10 melanoma models were established by subcutaneously injecting B16F10 cells (1 x 10 5 cells, 50 μL) into the right back of C57BL / 6J female mice (6-8 weeks old, 18-20 g). Cy5-labeled LTX-315 (Cy5-LTX-315) and Cy7-labeled rat IgG (Cy7-IgG) were used to monitor the retention of antibodies and oncolytic peptides after intratumoral administration. At different time points, mice treated with free LTX-315, IgG or IgG@OMG were imaged using an in vivo fluorescence imaging system (PerkinElmer IVIS Lumina III), and the retention of drugs was quantitatively analyzed by measuring the corresponding fluorescence density. The results showed that after intratumoral injection, free IgG and LTX-315 were rapidly cleared, and the fluorescence signal almost disappeared 2 days after administration ( Figure 7 A). In contrast, MG significantly prolonged the retention time of IgG and LTX-315 in the tumor, and about 75.1% of IgG and 55.9% of LTX-315 were detected at the tumor site on the first day of administration, and the enrichment could last for more than 14 days ( Figure 7 B, C). Long-term accumulation of anti-tumor drugs at the tumor site can generally reduce the required dose and provide sustained therapeutic effect.

[0070] Example Seven Anti-tumor Effect of C4@MG in Vivo To evaluate the therapeutic effect of anti-CTLA-4-loaded microgels (C4@MG) in vivo, to explore whether local delivery of anti-CTLA-4 can promote anti-tumor effect, and to reduce the toxic side effects of immune checkpoint therapy, a B16F10 subcutaneous tumor model was established according to the conventional method. C57BL / 6J female mice (6-8 weeks old, 18-20 g) were subcutaneously inoculated with B16F10 cells (3 x 105 The average tumor volume of mice was about 75 mm 3 After that, the mice were treated, which was set as day 0, and were given a single intratumoral injection of 100 µL of free anti-CTLA-4 (4 mg / kg), C4@MG (2 mg / kg), C4@MG (4 mg / kg), or C4@MG (8 mg / kg), and 4 intravenous injections of 100 µL of anti-CTLA-4 (1 mg / kg) on days 0, 2, 4, and 6. The tumor volume was calculated by the following formula: Tumor volume = width2x length x 0.5. The body weight of mice was recorded every other day. If a mouse died, lost more than 15% of the initial body weight, or the tumor volume reached 1500 mm 3 , the end point of the experiment was considered to be reached. During the course of this study, C4@MG did not cause the body weight of mice to decrease at anti-CTLA-4 antibody doses of 2 and 4 mg / kg, which confirmed its excellent safety profile (Fig. 2B). Figure 8 A). Compared with four intravenous (i.v.) and one intratumoral (i.t.) injections of free anti-CTLA-4 antibody, a single intratumoral injection of C4@MG showed better tumor inhibition and survival rate at an anti-CTLA-4 dose of 4 mg / kg (Fig. 2B, C). In addition, increasing the dose of anti-CTLA-4 in C4@MG to 8 mg / kg significantly improved the tumor inhibition effect and prolonged the median survival time (MST) to 20 days. Although C4@MG showed the best anti-tumor effect at an anti-CTLA-4 dose of 8 mg / kg, potential toxic side effects (such as loose stools, hair loss, etc.) were observed during the treatment. Therefore, in the subsequent study, an anti-CTLA-4 dose of 4 mg / kg was used. Figure 8 B, C). In addition, increasing the dose of anti-CTLA-4 in C4@MG to 8 mg / kg significantly improved the tumor inhibition effect and prolonged the median survival time (MST) to 20 days. Although C4@MG showed the best anti-tumor effect at an anti-CTLA-4 dose of 8 mg / kg, potential toxic side effects (such as loose stools, hair loss, etc.) were observed during the treatment. Therefore, in the subsequent study, an anti-CTLA-4 dose of 4 mg / kg was used.

[0071] Example Eight Anti-tumor effect of C4@OMG in vivo To enhance its immunotherapy effect, cancer combination therapy can usually provide a more optimal treatment effect by rationally selecting drugs and their combinations. LTX-315 was coated on C4@MG (C4@OMG) to regulate the release of the antibody and coordinate the oncolytic therapy and cancer immunotherapy. To evaluate the therapeutic effect of C4@OMG loaded with oncolytic peptide LTX-315, a B16F10 subcutaneous tumor model was constructed as described above. On day 0, 100 µL of free drugs (a mixture of anti-CTLA-4 and LTX-315), OMG, C4@MG, or C4@OMG was intratumorally injected, with the doses of LTX-315 and anti-CTLA-4 being 45 mg / kg and 4 mg / kg, respectively. Both C4@OMG and OMG caused the body weight of mice to gradually increase over time (Figure 9 (A) This indicates that the MG local delivery system can significantly improve the safety and therapeutic window of oncolytic peptides and immune checkpoint inhibitors. In contrast, free anti-CTLA-4 and LTX-315 led to a significant decrease in body weight in mice, possibly due to overactivation of the systemic immune system and toxicity to healthy tissues. Notably, C4@OMG almost completely inhibited tumor growth within 14 days, in stark contrast to the rapid tumor growth in other groups. Figure 9 B). Consistently, the survival rate of mice in the C4@OMG treatment group was significantly prolonged, with a MST of 28 days, much longer than that in the OMG and C4@MG groups ( Figure 9 C). This improved therapeutic effect can be attributed to the synergistic effect of anti-CTLA-4 immune checkpoint blockade with the oncolytic and ICD effects of LTX-315.

[0072] In addition, in the established B16F10 subcutaneous tumor model, 50 µL of PBS, OMG, or C4@OMG were injected intratumorally on day 0, with LTX-315 and anti-CTLA-4 doses of 45 mg / kg and 4 mg / kg, respectively. Three days after treatment, mice were sacrificed, and tumors, lymph nodes, spleens, and peripheral blood were collected. Single-cell suspensions were obtained by grinding and splitting red blood cells. CD45-PerCP / Cy5.5, CD3-FITC, CD8-PE / Cy7, and CTLA-4-PE staining were performed. Flow cytometry analysis was then performed to analyze the CD8+ staining. + The level of CTLA-4 expression on T cells. Results showed that LTX-315 induced CD8... + Increased CTLA-4 expression in T cells in tumors, lymph nodes, spleen, and peripheral blood was reversed by anti-CTLA-4. Figure 10 This underscores the importance of combination therapy. Specifically, LTX-315 induces immunogenic cell death, releasing tumor-associated antigens, activating antigen-presenting cells, and promoting CD8 activation. + T cell infiltration and activation. However, LTX-315 alone may lead to CD8 cell infiltration and activation. + Upregulation of CTLA-4 expression on the surface of T cells limits their anti-tumor activity. The introduction of anti-CTLA-4 antibodies effectively blocks the interaction between CTLA-4 and its ligands, relieving CD8+ inhibition. + The immunosuppressed state of T cells restored their ability to kill tumor cells. This synergistic effect not only significantly enhanced the anti-tumor immune response but also prolonged the therapeutic effect, providing a new approach to cancer immunotherapy.

[0073] Example 9: In vivo antitumor effect of P1C4@OMG Microgels (MGs) can efficiently co-load multiple immune checkpoint inhibitors and peptides. Here, a microgel (P1C4@OMG) loaded with LTX-315, anti-CTLA-4, and anti-PD-1 was constructed and used to treat B16F10 melanoma. Specifically, in an established B16F10 subcutaneous tumor model, on day 0, 100 µL of the free drug combination (anti-CTLA-4 and anti-PD-1 mixture), OMG, P1C4@MG, or P1C4@OMG was injected intratumorally, with LTX-315, anti-CTLA-4, and anti-PD-1 doses of 45 mg / kg, 4 mg / kg, and 4 mg / kg, respectively. Bispecific antibodies (anti-CTLA-4 and anti-PD-1) are widely used in the clinical treatment of melanoma patients (V. Giacco. Nat. Cancer 2025, 6, 407-407), therefore, this invention selected free bispecific antibody therapy as the control group. The experimental results showed that tumor growth was slow in mice treated with P1C4@OMG within 20 days, which contrasted sharply with the rapid tumor growth in the OMG and P1C4@MG groups. Figure 11 A). Furthermore, P1C4@OMG significantly prolonged the survival time of mice, with a median survival time (MST) of 35 days, and achieved complete tumor regression in one out of seven mice. Figure 11 B). Notably, compared to the MST (28 days) of the C4@OMG group mice in Example 8, P1C4@OMG further prolonged the survival time of the mice, confirming the significant advantage of the combined use of bispecific antibodies and oncolytic peptides. Blood samples were collected 7 days after treatment for routine blood tests and blood biochemistry analysis. Unlike healthy mice, mice injected with free anti-PD-1 and anti-CTLA-4 antibodies showed significantly elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), and creatinine (CREA) in their serum. The blood biochemical indicators of the P1C4@OMG group were similar to those of normal mice. Figure 12 Furthermore, P1C4@OMG had little effect on the total red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) in mouse blood, while these parameters were significantly reduced in mice treated with a combination of free anti-PD-1 and anti-CTLA-4 antibodies. Figure 13 These results indicate that the introduction of MG delivery systems can avoid the risks of anemia and liver and kidney dysfunction caused by free immune checkpoint inhibitors, thereby significantly broadening its therapeutic window.

[0074] Seven days after treatment, mice were sacrificed, and inguinal lymph nodes were harvested for mDC (CD45) analysis. + CD11c + CD80+ CD86 + The content of T cells (CD8+) was measured. Spleen and tumor cells were collected, weighed, and ground into single-cell suspensions. Appropriate immune cell antibody staining was performed, and the immune cell content was determined by flow cytometry. Data were analyzed using FlowJo_V10 software. Specifically, after erythropoiesis, the content of T cells (CD8+) in spleen cells was analyzed. + T(CD45) + CD3 + CD8 + ) and CD4 + T(CD45) + CD3 + CD4 + The content of T cells (CD8+) in tumor cells was analyzed. + T cells, CD4 + T cells), NK cells (CD45) + CD3 - CD49b + ), MDSC (CD45) + CD11b + Gr-1 + M2 macrophages (CD11b) + F4 / 80 + CD206 + ), Tregs (CD4) + CD25 + FoxP3 + Due to the generation of damage-associated molecular patterns (DAMPs) and tumor-specific antigens, the formulation containing LTX-315 (OMG) induced a significant increase in the number of mature dendritic cells (mDCs) in lymph nodes (LNs), and this number further increased upon the addition of ICIs. Figure 14 A). Compared to the slight increase in the number of CD4⁺ T cells, CD8⁺ T cells, and natural killer cells (NK cells) in tumors treated with free anti-PD-1 and anti-CTLA-4 antibodies, ICIs loaded in MG (P1C4@MG) induced an approximately two-fold increase in the infiltration of these cells. Figure 14 (BD), highlighting the crucial role of ICIs in long-acting delivery. Following the introduction of LTX-315 (P1C4@OMG), the infiltration of cytotoxic T cells and NK cells further increased. Furthermore, compared to the slight changes in immunosuppressive cells in the free ICIs group, both OMG and P1C4@MG significantly reduced the proportion of immunosuppressive cells in the tumor, including M2 macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). Figure 14E-G). In the spleen, the P1C4@OMG group also showed the most infiltration of CD8+ effector T cells and CD4+ helper T cells (Figs. Figure 14 H, I). Therefore, the MG provides a significant role of activating DCs, significantly increasing cytotoxic T cell and NK cell infiltration, and significantly down-regulating immunosuppressive cells for ICIs and oncolytic peptides, achieving long-term activation of immune response and strong anti-tumor efficacy.

[0075] In the present application, the microgel (MG) is a hyaluronic acid microgel, which is prepared by microfluidic technology and free radical photopolymerization technology, and has biodegradability and uniform size. The water phase and the oil phase are injected into a microfluidic chip (MG preparation device) Figure 15 , After the two-phase liquid enters the chip, the water phase core flow is subjected to continuous laminar shear of the lateral oil phase in the microchannel to form uniform droplets, and is subjected to photo-crosslinking under 365 nm ultraviolet light irradiation to obtain the MG. The ICI solution and the MG solution are added to a dialysis bag, and a buffer is used for dialysis to prepare the ICI-loaded microgel (ICI@MG); then the oncolytic peptide LTX-315 solution is added and shaken to mix uniformly to prepare the ICI-loaded OMG (ICI@OMG), and the oncolytic peptide LTX-315 can regulate the release behavior of the inhibitor loaded by the microgel. The oncolytic microgel loaded with the immune checkpoint inhibitor disclosed in the present application can effectively reverse the immunosuppressive tumor microenvironment, promote T cell infiltration and pro-inflammatory cytokine secretion, significantly inhibit tumor growth and prolong survival time after a single dose injection. At the same time, the microgel does not cause obvious side effects and dysfunction in vivo, and has good in vivo safety. The oncolytic microgel as a safe and effective platform can be widely used in cancer immunotherapy.

Claims

1. A tumor-inhibitor-loaded oncolytic microgel, comprising a microgel and an inhibitor and an oncolytic agent loaded on the microgel.

2. The inhibitor-loaded oncolytic microgel according to claim 1, characterized in that, The inhibitor is located inside and / or outside the microgel, and the oncolytic agent is located inside and / or outside the microgel.

3. The inhibitor-loaded oncolytic microgel according to claim 1, characterized in that, The inhibitor comprises an immune checkpoint inhibitor; and the oncolytic agent comprises an oncolytic peptide.

4. The method of claim 1, wherein the preparation of the oncolytic agent-loaded microgel is characterized by, The tumor-inhibitor-loaded oncolytic microgel is prepared by mixing the inhibitor, the microgel and the oncolytic agent.

5. The method for preparing the inhibitor-loaded oncolytic microgel according to claim 4, characterized in that, The microgel comprises a hyaluronic acid microgel, a sodium alginate microgel or a heparin microgel.

6. The method of claim 4, wherein the method further comprises the step of adding a cytostatic agent to the microgel. The tumor-inhibitor-loaded oncolytic microgel is prepared by mixing an inhibitor solution, a microgel solution and an oncolytic agent solution. ​ 7.A medicament, wherein the active ingredient comprises the tumor-inhibitor-loaded oncolytic microgel according to claim 1. 8.Use of the tumor-inhibitor-loaded oncolytic microgel according to claim 1 in the preparation of a medicament. 9.Use of the tumor-inhibitor-loaded oncolytic microgel according to claim 1 in the preparation of an antitumor medicament.

10. Use of oncolytic microgels for the preparation of antitumor medicaments, characterized in that, The oncolytic microgel comprises a microgel and an oncolytic agent loaded on the microgel.

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