Oncolytic agent-loaded STING activated microgel as well as preparation method and application thereof

Abstract

The invention discloses an oncolytic agent-loaded STING activated microgel (OSAM) as well as a preparation method and application thereof. The oncolytic agent-loaded STING activated microgel (OSAM) is prepared by simply mixing hyaluronic acid microgel, an STING agonist and an oncolytic agent in a solution. The OSAM can continuously release an oncolytic agent and an STING agonist for 4 weeks, can induce significant up-regulation of MHC I and continuous activation of dendritic cells for more than one week, and can induce long-term stable activation of an STING pathway and direct lysis of tumor cells, so that various tumor specific antigens are generated, and long-acting and strong specific anti-tumor immunity is realized. Infiltration of cytotoxic T lymphocytes and natural killer cells can be remarkably promoted by injecting OSAM into a tumor for a single time. When the OSAM and the anti-CTLA-4 loaded microgel (C4M) are used in a combined manner, an excellent treatment effect is shown, and the cure rate reaches 71%. The oncolytic agent loaded STING activated microgel disclosed by the invention has the advantages of high safety, simplicity and convenience in preparation, strong immune activation capability and the like, and opens up a new way for the development of in-situ therapeutic tumor vaccines.

Description

Technical Field

[0001] This invention relates to a STING-activated microgel loaded with oncolytic peptides, a microgel-based drug delivery system, a method for preparing the STING-activated microgel loaded with oncolytic peptides, and its application in tumor immunotherapy. Background Technology

[0002] Cancer vaccines, by enhancing the release, processing, and presentation of exogenous tumor antigens, replenish the host with tumor-reactive T cells, thereby regaining control of tumor growth and demonstrating great potential in cancer treatment. To avoid the expensive and time-consuming antigen identification process, in situ tumor vaccines utilize the tumor's own endogenous antigens to elicit a broad immune response, and have received increasing attention in recent years. However, weak antigen presentation and the immunosuppressive tumor microenvironment pose significant challenges to achieving clinical efficacy (S. Jhunjhunwala, Nat. Rev. Cancer 2021, 21, 298). Currently, various treatment modalities, including chemotherapy, X-ray radiation, ablation, photothermal therapy, and photodynamic therapy, have been explored to induce immunogenic cell death (ICD) and the release of tumor-specific antigens to generate in situ cancer vaccines. However, the adverse effects of these methods on normal cells and immune cells can lead to serious side effects (Y. Huang, Adv. Mater. 2024, 36, e2407914). Furthermore, even after multiple booster vaccinations, weak and transient immune activation often leads to a transient anticancer immune response. Tumor-specific antigen processing and presentation in dendritic cells (DCs) can be improved by activating pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), nucleotide oligomer domain-like receptors (NLRs), and interferon gene stimulating factor (STING) (B. Pulendran, Nat. Rev. Drug Discov. 2021, 20, 454). STING agonists can activate the STING pathway, triggering the production of type I interferon and initiating cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells), showing great promise as adjuvants for cancer vaccines. However, despite the potential therapeutic effects of STING agonists (such as typical cyclic dinucleotides (CDNs)), their application is limited by low stability, restricted intracytoplasmic delivery, strong inflammatory responses at the injection site, and systemic toxicity caused by off-target activation (X. Shi, Adv. Sci. 2023, 10, e2204890). Therefore, there is a need to develop a delivery system that can efficiently encapsulate STING agonists and oncolytic peptides to improve drug bioavailability, reduce side effects, and thus effectively enhance cancer immunotherapy. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing STING-activated microgels capable of loading oncolytic agents. The microgels prepared using this method can effectively achieve efficient drug loading and controlled release. Furthermore, the STING-activated microgels loaded with oncolytic peptides prepared by this method show great promise for application in the field of tumor immunotherapy.

[0004] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A STING-activated microgel loaded with oncolytic agent includes a microgel and a STING agonist and oncolytic agent loaded on the microgel.

[0005] In this invention, the STING agonist is a noncyclic dinucleotide agonist; furthermore, the STING agonist includes diaminobenzimidazole (diABZI), GSK3745417, SR717, and Mn. 2+ Zn 2+ and Co 2+ .

[0006] In this invention, the oncolytic agent includes oncolytic peptides such as LTX-315, LTX-401, or Melittin.

[0007] As an example, this invention discloses a STING-activated microgel (OSAM) based on the oncolytic peptide LTX-315; the microgel is loaded with both the STING agonist and the oncolytic peptide LTX-315. Furthermore, this invention also relates to the application of this microgel loaded with an oncolytic agent and a STING agonist in tumor immunotherapy.

[0008] This invention discloses a method for preparing the above-mentioned STING-loaded oncolytic microgel, comprising the following steps: mixing STING agonist, microgel and oncolytic agent in a solvent to prepare STING-loaded oncolytic microgel.

[0009] Preferably, the present invention discloses a method for preparing the above-mentioned STING-activated microgel loaded with oncolytic agent, wherein a STING agonist solution and a microgel solution are mixed to prepare a STING agonist-loaded microgel (SAM); then an oncolytic agent is added to prepare an oncolytic agent-loaded STING-activated microgel (OSAM).

[0010] This invention discloses a method for preparing the above-mentioned microgel (MG), wherein the MG is a medical polymer microgel, including hyaluronic acid, sodium alginate or heparin microgel; the MG is prepared by microfluidic technology and free radical photopolymerization technology, and has biodegradability and uniform size.

[0011] This invention discloses a method for preparing STING-loaded microgels, comprising the following steps: adding a STING agonist solution to an MG solution and continuously shaking to mix, thereby preparing a STING agonist-loaded microgel (SAM); subsequently, adding an oncolytic peptide LTX-315 solution and continuing to shake to mix, thereby preparing an oncolytic peptide-loaded SAM (OSAM).

[0012] In this invention, the concentration of MG is 10-100 mg / mL, the concentration of STING agonist is 0.5-5 mg / mL, and the concentration of oncolytic peptide is 5-50 mg / mL.

[0013] In this invention, the oscillation conditions for preparing SAM are 900 rpm and 25 ℃; the oscillation conditions for preparing OSAM are 900 rpm and 25 ℃.

[0014] In this invention, the STING-activated microgel loaded with the oncolytic agent can efficiently load the STING agonist (diABZI) and the oncolytic peptide (LTX-315) and control their release behavior.

[0015] The STING-activated microgel loaded with the oncolytic agent in this invention can induce a significant upregulation of MHC I and achieve highly efficient activation of dendritic cells for more than a week.

[0016] This invention discloses the application of the above-mentioned oncolytic agent STING activated microgel in the preparation of drugs.

[0017] This invention discloses the application of the above-mentioned oncolytic agent STING activated microgel in the preparation of immunotherapeutic drugs.

[0018] This invention discloses the application of the above-mentioned oncolytic agent STING activated microgel in the preparation of antitumor drugs.

[0019] In this invention, medical macromolecules are used as raw materials to prepare microgels. Specifically, medical macromolecules are used to construct microgels through microfluidic technology and free radical polymerization technology. As an example, hyaluronic acid derivatives are used as raw materials to construct microgel MG through microfluidic technology and free radical polymerization technology.

[0020] This invention utilizes a microchannel system to achieve free radical polymerization, crosslinking, and solidification of an aqueous phase material containing medical macromolecules and an oil phase material, resulting in a microgel. In this invention, the aqueous phase material includes medical macromolecules, a crosslinking agent, a buffer solution, and an initiator; the oil phase material includes a surfactant and an oil solvent; and the microchannel system is a pre-existing product.

[0021] This invention discloses a drug whose active ingredient includes the aforementioned oncolytic agent STING activated microgel. Furthermore, the drug may also include other components that can exert therapeutic effects, such as other antitumor drugs.

[0022] This invention discloses the application of the above-mentioned STING-loaded microgels, specifically including: the application of OSAM microgels loaded with diABZI and LTX-315 in tumor treatment; and the application of OSAM and anti-CTLA-4 loaded microgels (C4M) in tumor treatment.

[0023] In this invention, a single intratumoral injection of OSAM significantly promotes the infiltration of cytotoxic T lymphocytes and natural killer cells, inhibits tumor growth, and prolongs survival time.

[0024] In this invention, the combined use of OSAM and C4M has shown excellent therapeutic effects in various mouse tumor models, with a cure rate as high as 71%.

[0025] In this invention, MG serves as a safe and efficient long-acting delivery platform for the delivery of STING agonists and oncolytic peptides, and has great potential in enhancing cancer immunotherapy.

[0026] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: 1. The method for preparing STING-activated microgels loaded with oncolytic agents disclosed in this invention is simple and efficient, and requires no complex process conditions or procedures. This STING-activated microgel can continuously release the oncolytic peptide LTX-315 and the STING agonist diABZI (for approximately 4 weeks). This characteristic not only achieves on-demand drug release but also avoids the problems associated with frequent dosing.

[0027] 2. The oncolytic STING activated microgel disclosed in this invention not only has antitumor activity similar to the oncolytic peptide LTX-315, but also can induce long-lasting and powerful immune activation by continuously releasing diABZI and LTX-315, thus significantly enhancing the antitumor immune response.

[0028] 3. The oncolytic STING-activated microgel disclosed in this invention can effectively reverse the immunosuppressive tumor microenvironment through a single intratumoral injection, promote the infiltration of cytotoxic T lymphocytes and natural killer cells, significantly inhibit tumor growth, and prolong survival time. Simultaneously, the combined use of OSAM and C4M has shown unique therapeutic effects in several different mouse tumor models, with cure rates reaching 40-71%. This oncolytic agent-loaded STING-activated microgel, as a safe and effective platform, can be widely used in cancer immunotherapy. Attached Figure Description

[0029] Figure 1 The image shows the 1H NMR spectrum of HA-AMA in the synthetic example (400 MHz, D2O / DMSO-d6, 2 / 1, v / v).

[0030] Figure 2 This is a characterization of MG in Example 1, where A is the size and size distribution of MG measured under a fluorescence inverted microscope, inset: microscope image (scale bar: 100 µm), and B is a representative scanning electron microscope image of MG (scale bar: 50 µm).

[0031] Figure 3 This is the construction and characterization of OSAM in Example 2, where A is the DLE and DLC of diABZI in MG, B is the DLE and DLC of LTX-315 and diABZI in MG, and C is the in vitro release curve of diABZI and LTX-315 in OSAM at pH 6.5.

[0032] Figure 4 This refers to the immunostimulatory effect of OSAM on B16F10 tumor cells in Example 3. RT-qPCR was used to detect type I IFN and inflammation-related genes in B16F10 cells 24 hours after OSAM treatment. Ifnα (A) Ifnβ (B) Cxcl10 (C) Tnfα (D) and Il6 (E) mRNA expression levels. Results are expressed as fold changes relative to the corresponding levels in the PBS group.

[0033] Figure 5 The average fluorescence intensity of MHC I on the cell surface after 24 hours of interaction with B16F10 tumor cells using LTX-315 (A), diABZI (B), and OSAM (C) in Example 3 is the average fluorescence intensity of MHC I on the cell surface.

[0034] Figure 6 This describes the immune activation effect of OSAM on BMDCs in Example 4. After treating B16F10 cells with OSAM for 24 hours, the supernatant was added to BMDCs and incubated for another 24 hours. Here, A represents CD80 in BMDCs. + CD86 + Quantitative analysis of cells, where B represents the average fluorescence intensity of MHC I in BMDCs. From days 1 to 7, B16F10 cells were treated with OSAM for 24 hours, and their supernatant was added to BMDCs for further incubation for 24 hours. C represents the CD80 concentration in BMDCs. + CD86 + Quantitative analysis of cells, where D is the mean fluorescence intensity of MHC I in BMDCs. Results are expressed as fold changes relative to the PBS group.

[0035] Figure 7 This describes the immune activation effect of OSAM on BMDM in Example 4. After treating B16F10 cells with OSAM for 24 hours, the supernatant was added to BMDCs and incubated for another 24 hours. Here, A represents the F4 / 80 ratio in BMDM. + CD86 + Quantitative analysis of M1 / M2, where B is the quantitative analysis of the M1 / M2 ratio.

[0036] Figure 8 This is the anti-tumor effect of OSAM in the subcutaneous B16F10 melanoma model in Example 5, where A is the change in mouse body weight, B is the average tumor growth curve of mice, and C is the survival curve of mice.

[0037] Figure 9 This is the effect of OSAM on long-term immune memory in B16F10 mice in Example 5. A represents the tumor growth curve of the B16F10 melanoma model after re-attack (PBS and OSAM / C4M groups: n = 5; OSAM group: n = 3), B represents the tumor weight in mice, and C represents the CD3+ level in peripheral blood. + CD8 + Quantitative analysis of TEM cells in T cells, where D represents CD3+ in peripheral blood. + CD8 + Quantitative analysis of TCM cells in T cells, E represents CD3 in the spleen. + CD8 + Quantitative analysis of TEM cells in T cells, F represents CD3 in the spleen + CD8 + Quantitative analysis of TCM cells in T cells.

[0038] Figure 10 This describes the immunomodulatory effect of OSAM in a subcutaneous B16F10 melanoma model as shown in Example 6, where A represents the average fluorescence intensity of MHC I in tumor cells, and B represents mature dendritic cells (CD11c) in lymph nodes. + CD80 + CD86 + C represents the average fluorescence intensity of MHC I in dendritic cells of lymph nodes, and CD8 in tumors. + T cells (D) and CD4 + Quantitative analysis of T cells (E) and NK1.1 in tumor cells. + Cells (F), M2 macrophages (G), M1 / M2 ratio (H), and regulatory T cells (CD4+) in tumors. + CD25 + FoxP3 + Quantitative analysis of (I).

[0039] Figure 11 This describes the antitumor effect and immunomodulatory effect of OSAM / C4M in a subcutaneous 4T1 triple-negative breast tumor model in Example 7. In this model, A represents the mouse tumor growth curve, B represents the mouse survival curve, and C represents the CD8+ level in peripheral blood. + Quantitative analysis of T cells, D represents CD4+ in peripheral blood. + Quantitative analysis of T cells.

[0040] Figure 12 This is the anti-tumor effect of OSAM / C4M in the subcutaneous MC38 colon cancer model and the tumor re-attack in Example 8. In this example, A is the mouse tumor growth curve, B is the mouse survival curve, C is the mouse tumor growth curve after re-inoculation with MC38 cells, and D is the mouse survival curve. Detailed Implementation

[0041] This invention specifically discloses a method for preparing STING-activated microgels loaded with oncolytic agent and its application. The method includes the following steps: using hyaluronic acid derivatives as raw materials, microgels MG are constructed through microfluidic technology and free radical polymerization technology; then, by simply mixing with MG, the STING agonist diABZI can be easily loaded into MG; and then, the oncolytic peptide LTX-315 is added to prepare the oncolytic STING-activated microgel.

[0042] In the above technical solution, the hyaluronic acid derivative is a hyaluronic acid-2-aminoethyl methacrylate derivative.

[0043] In the above technical solution, MG is prepared using microfluidic technology and photocontrolled free radical polymerization technology. 50-200 mg of hyaluronic acid-2-aminoethyl methacrylate derivative (HA-AMA), 10-30 mg of 2-acrylamide-2-methylpropanesulfonic acid (AMPS), and 4-10 mg of photoinitiator I2959 are dissolved in 1 mL of PB buffer solution (pH 8.5, 10 mM) as the aqueous phase of the microfluidic device. Simultaneously, mineral oil containing 5-10 wt.% Span 80 is prepared as the oil phase. Subsequently, microdroplets are formed by shearing the aqueous phase in the oil phase through the microchannels, followed by photocrosslinking under 365 nm ultraviolet light irradiation to obtain an MG emulsion. Finally, the MG is washed with excess PB, isopropanol, and n-hexane to remove the mineral oil, Span 80, and I2959 initiator, ultimately obtaining a PB solution of MG.

[0044] In the above technical solution, the STING agonist is loaded into the microgel (SAM) through electrostatic and hydrogen bonding interactions between the STING agonist and MG using a vortex mixing method. Specifically, an MG suspension is mixed with an equal volume of STING agonist solution to obtain a STING agonist-loaded microgel.

[0045] In this invention, a microgel (OSAM) loaded with oncolytic peptide LTX-315 and STING agonist diABZI is prepared by mixing a SAM suspension with an equal volume of oncolytic peptide LTX-315 solution.

[0046] This invention discloses an oncolytic STING-activated microgel, its preparation method, and its applications. This microgel, as an in situ cancer vaccine, can continuously release the oncolytic peptide LTX-315 and the STING agonist diABZI, thereby triggering a highly efficient and sustained anti-tumor immune response. Utilizing the strong electrostatic and hydrogen bonding interactions between the drug and HA, the microgel (MG) can efficiently co-load the oncolytic peptide LTX-315 and the STING agonist diABZI, achieving long-term controlled release of both drugs. The continuously released LTX-315 can directly lyse tumor cells, generating various tumor-specific antigens. Simultaneously, the combined continuous release of diABZI can induce long-term stable activation of the STING pathway, thus achieving a powerful specific anti-tumor immune response. Furthermore, a single intratumoral injection of OSAM significantly promotes the infiltration of CTLs and NK cells. Combined with C4M, it further inhibits the growth of mouse tumor models of melanoma, triple-negative breast cancer, and colon cancer, with cure rates of 71%, 43%, and 40%, respectively. The oncolytic STING-activated microgel designed in this invention opens up a new avenue for enhancing tumor immunotherapy.

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments. All raw materials used are existing products; the specific preparation operations and performance tests are all conventional techniques; animal experiments meet the relevant requirements of Soochow University; and data statistics are performed using conventional statistical methods. diABZI was purchased from Shanghai Taoshu Biotechnology Co., Ltd.; LTX-315 (KKWWKKW(Dip)K-NH2) was purchased from Shanghai Qiangyao Biotechnology Co., Ltd.; anti-CTLA-4 was purchased from Bioxcell, Inc. (USA); hyaluronic acid was purchased from Bloomage Biotechnology Co., Ltd.; and 2-acrylamide-2-methylpropanesulfonic acid was purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0048] Synthesis of Hyaluronic Acid Methacrylate Derivative (HA-AMA) Under nitrogen protection, HA (3.00 g, 7.92 mmol carboxyl group) was dissolved in a mixed solution of 140 mL deionized water and 10 mL dimethyl sulfoxide (DMSO) to obtain HA solution; AMA (2-aminoethyl methacrylate, 566 mg, 3.41 mmol) was dissolved in 6.30 mL of DMSO solution, and then 1.50 mL of deionized water was added to dissolve it to obtain an AMA solution. AMA solution was added to HA solution, and after stirring for 10 minutes, the pH was adjusted to 6.5 with 5 M sodium hydroxide solution. Then, the coupling agent DMTMM (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1.43 g, 5.12 mmol) was added, and the reaction was carried out at 35 °C for 24 hours. After the reaction was complete, the reaction solution was transferred to a dialysis bag (MWCO 3500 Da), and then dialyzed sequentially with PBS buffer and deionized water (volume ratio 1:3). Finally, the final product HA-AMA was obtained by freeze-drying. The characteristic peak of the methyl group on AMA was clearly observed in the 1H NMR spectrum. δ 1.89) and the characteristic peaks of double bonds ( δ (5.70, 6.10), which confirms that AMA has been successfully grafted onto the HA mainchain ( Figure 1 Furthermore, by comparing the integral values ​​of the double bond characteristic peak on AMA and the methyl characteristic peak on HA, the degree of substitution of AMA on HA can be calculated to be 22.0. By changing the feed ratio of AMA to HA, HA-AMA with different degrees of substitution can be prepared (Table 1).

[0049] Table 1 Characterization of HA-AMA

[0050] a Depend on 1 Measured by H NMR.

[0051] Example 1: Preparation of Hyaluronic Acid Microgel (MG) Hyaluronic acid microgels (MG) were prepared using hyaluronic acid-2-aminoethyl methacrylate derivative (HA-AMA) as raw material via microfluidic technology and UV-controlled free radical polymerization. Refer to CN2025107464448, "A biodegradable, elastic, and uniformly sized embolic microsphere and its preparation method and application." Briefly, the process involved loading 100 mg / mL HA-AMA, 25 mg / mL 2-acrylamide-2-methylpropanesulfonic acid (AMPS), and 6 mg / mL I2959 into a syringe as the aqueous phase of the microfluidic device in PB buffer solution (pH 8.5, 10 mM). 10 wt.% Span 80 mineral oil was added as the oil phase (oil phase flow rate 6.5 μL / min, aqueous phase flow rate 1.5 μL / min). Subsequently, microdroplets were formed in the microchannels by the lateral fluid shearing action of the oil phase through the aqueous core flow, and photocrosslinking was performed under 365 nm UV irradiation to obtain an MG emulsion. Then, mineral oil, Span 80, and I2959 initiators were removed using excess PB buffer, isopropanol, and n-hexane, followed by conventional washing and purification steps to obtain a PB solution of MG. Morphological characterization revealed that the prepared MG exhibited a regular spherical shape. Specifically, the purified MG was dispersed at a concentration of 25 mg / mL in PB buffer (pH 7.4), and samples were placed on glass slides. Observation under a fluorescence inverted microscope showed that the microgels had a uniform spherical morphology with an average diameter of 70 μm and a narrow particle size distribution (CV < 2%). Figure 2 A). Further characterization using scanning electron microscopy (SEM) revealed that the MG surface was smooth and without obvious defects. Figure 2 B). The above results indicate that hyaluronic acid microgels with uniform morphology and intact structure were successfully prepared.

[0052] Example 2: Preparation and Characterization of Oncolytic STING Activated Microgel (OSAM) Utilizing the electrostatic and hydrogen bonding interactions between diABZI and hyaluronic acid microgels, diABZI-loaded microgels (SAMs) can be easily constructed by simply mixing an MG suspension (PB solution, 25 mg / mL) with an equal volume of diABZI aqueous solution for 30 minutes on a shaker at 900 rpm and 37°C. The unloaded diABZI in the supernatant was measured by high-performance liquid chromatography (HPLC), and the drug loading efficiency (DLE) and drug loading capacity (DLC) of diABZI were calculated. Experimental results show that when the theoretical drug loading capacity of diABZI is in the range of 2-6 wt.%, its encapsulation efficiency is high, with a DLE exceeding 90%. Figure 3 A), demonstrating MG's efficient load-bearing capacity for diABZI.

[0053] Furthermore, by mixing the prepared SAM (PB solution, 25 mg / mL) with an equal volume of oncolytic peptide LTX-315 aqueous solution for 30 minutes on a shaker at 900 rpm and 25 ℃, a microgel (OSAM) simultaneously loaded with diABZI and LTX-315 was successfully constructed. The unloaded diABZI and LTX-315 in the supernatant were measured by high-performance liquid chromatography and ultraviolet spectrophotometry, respectively. Encapsulation with LTX-315 further improved the diABZI loading in the SAM, with both diABZI and LTX-315 loading efficiencies exceeding 90%. Figure 3 B).

[0054] Following the method described above, LTX-315-loaded microgels (OMs) can be easily constructed by simply mixing an MG suspension (PB solution, 25 mg / mL) with an equal volume of LTX-315 aqueous solution for 30 minutes.

[0055] Following the method described above, anti-CTLA-4 loaded microgels (C4M) can be easily constructed by simply mixing an MG suspension (PB solution, 25 mg / mL) with an equal volume of anti-CTLA-4 solution (PB solution) for 30 minutes.

[0056] Furthermore, the dosage can be controlled by adjusting the theoretical drug loading of LTX-315 and diABZI (Table 2).

[0057] Table 2 Characterization of OSAM

[0058] a Measured by fluorescence spectroscopy, b Measured by HPLC.

[0059] To evaluate the release behavior of diABZI and LTX-315 from OSAM, in vitro release experiments of diABZI and LTX-315 from OSAM (preparation scheme group 6 in Table 2) were conducted in PBS buffer (pH 6.5, 10 mM). 100 μL of OSAM (MG concentration of 25 mg / mL) was placed in the inner chamber of a Transwell plate. Then, 1.0 mL of PBS buffer was added to submerge the OSAM, and the plate was placed on a shaker at 100 rpm and 37 °C. At predetermined time points, the release medium was collected and replaced with 1.0 mL of PBS buffer. The concentrations of diABZI and LTX-315 in the release medium were measured using high-performance liquid chromatography (HPLC) and UV spectrophotometry, respectively. The results showed that diABZI and LTX-315 could be released from OSAM in a controlled and sustained manner, with a release time of up to four weeks (…). Figure 3 C).

[0060] Example 3: Study on the immunostimulatory effect of OSAM in in vitro tumor cells The effect of OSAM on the mRNA expression levels of type I interferon (IFNs) and other pro-inflammatory cytokine-related genes in B16F10 tumor cells was investigated using RT-qPCR. 1.0 mL of B16F10 tumor cells (2 × 10⁻⁶ cells) were used. 5 Cells were seeded in 12-well plates with a 1 μM / well suspension, and PBS, OM, SAM, or OSAM (diABZI dose 1 μM, LTX-315 dose 20 μg / mL) were added separately. After incubation for 24 h, cells were collected. RNA was then extracted from B16F10 cells using RNA-easy precipitation, followed by reverse transcription and qPCR using SYBR Green qPCR. The reaction conditions were: 95 °C pre-denaturation for 2 min, followed by 40 cycles of 95 °C denaturation for 5 s + 60 °C annealing / extension for 30 s, and then storage at 4 °C. The fold change in gene expression was calculated using the comparative threshold cycle method. The results showed that SAM significantly upregulated interferon-α (…) by activating the STING pathway through the diABZI agonist. Ifnα ), interferon-β ( Ifnβ OSAM further increased the expression level of related gene mRNAs by more than 3.5 times. Figure 4 (A, B) This may be because partial DNA damage caused by LTX-315 is recognized and bound by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), thereby further synergistically activating the cGAS-STING pathway. Furthermore, OSAM also significantly induced the high expression of inflammation-related genes, such as CXC motif chemokine ligand 10 (…). Cxcl10 ), tumor necrosis factor-α ( ) Tnfα ) and interleukin-6 ( Il6 () Figure 4 CE).

[0061] In addition, B16F10 cells (2×10) 5After being seeded in 12-well plates and cultured for 12 hours, tumor cells were treated with PBS, OM, SAM, or OSAM (diABZI at 2 μM, LTX-315 at 40 μg / mL) for 24 hours. Tumor cells were then collected, blocked on ice with anti-mouse CD16 / 32 for 30 minutes, and then stained with anti-H-2Kb / H-2Db-PE (clone: ​​28-8-6) antibody for 30 minutes. After washing with PBS, the mean fluorescence intensity corresponding to MHC I expression on tumor cells was analyzed by flow cytometry. Interestingly, both LTX-315 and diABZI upregulated MHC I expression in B16F10 tumor cells in a concentration-dependent manner, and OSAM induced significantly higher MHC I expression compared to OM and SAM. Figure 5 MHC I plays a crucial role in antigen presentation, and its upregulation may promote the direct presentation of antigens from tumor cells to T cells, thereby activating T cell-mediated anti-tumor immune responses.

[0062] Example 4: Study on OSAM-stimulated immune cells in vitro To investigate the interaction between the damage-related molecular patterns of immunogenic cell death induced by the oncolytic peptide LTX-315 in OSAM and STING agonists, and their ability to stimulate the maturation of tumor-associated antigen-presenting cells (such as dendritic cells). Bone marrow-derived dendritic cells were extracted from the leg bones of healthy female C57BL / 6J mice to study the activation of dendritic cells in vitro. B16F10 cells (2 × 10⁻⁶) were used. 5 (1 / well) were treated with PBS, OM, SAM, or OSAM (diABZI dose 1 μM, LTX-315 dose 20 μg / mL) for 24 hours, and then the supernatant was added to BMDC (1×10⁶) pre-seeded in 12-well plates. 6 / well). After incubation for 24 hours, BMDC immune cells were collected, blocked on ice with anti-mouse CD16 / 32 for 30 minutes, and then incubated with anti-CD11c-FITC, anti-CD80-APC, anti-CD86-PE-Cy7, and anti-H-2Kb / H-2Db-PE antibodies for 30 minutes. After washing with PBS, the proportion of mature BMDCs (CD80⁺CD86⁺, MHC I) was assessed by flow cytometry. The results showed that OM, SAM, and OSAM all significantly induced an increase in CD80⁺CD86⁺ expression in BMDCs, with OSAM showing the strongest activation effect. Compared with the PBS group, the number of mature dendritic cells in OSAM-treated BMDCs increased by more than 11-fold ( Figure 6A) indicates that OSAM can significantly promote the maturation of BMDCs. Furthermore, the expression level of MHC I in OSAM-treated BMDCs increased more than twofold (A). Figure 6 B). The upregulation of MHC I expression, combined with the maturation of BMDCs, will significantly enhance the efficiency of tumor antigen transmission from dendritic cells to T cells, thereby initiating a stronger anti-tumor immune response.

[0063] In addition, to further investigate the sustained stimulatory effect of OSAM on BMDCs, B16F10 cells (2×10⁻⁶) were used. 5 (2 × 10⁹ / well) cells were seeded in the lower chamber of a Transwell plate for 12 hours, then treated with OSAM placed in the upper chamber for 24 hours. Subsequently, the OSAM in the upper chamber of the Transwell plate was transferred to cells pre-seeded with B16F10 cells (2 × 10⁹ / well). 5 ( / well) A new Transwell plate was added, and the supernatant from the old Transwell plate was added to BMDC (1×10⁶) pre-inoculated in a 12-well plate. 6 Cells were incubated in wells ( / well). After 24 hours of incubation, immune cells were collected and stained. This process was repeated 7 times to investigate the long-term immunostimulatory effect of OSAM on BMDCs. Notably, the sustained release of LTX-315 and diABZI from OSAM provided long-term (>7 days) immunostimulation of BMDCs. Figure 6 (C, D) This long-lasting activation effect provides an important advantage for the application of OSAM in tumor immunotherapy.

[0064] Macrophages play a crucial role in the tumor immune microenvironment, and their polarization state (M1 or M2) directly influences the effectiveness of anti-tumor immune responses. Bone marrow-derived macrophages were extracted from the leg bones of healthy female C57BL / 6J mice to investigate the effect of OSAM on macrophage polarization. B16F10 cells (2 × 10⁻⁶) were used. 5 After treating each well with PBS, OM, SAM, or OSAM (diABZI dose 1 μM, LTX-315 dose 20 μg / mL) for 24 h, the upper culture medium was added to BMDM cells (1×10⁶ cells / well) pre-seeded in 12-well plates. 6 Incubation was performed in wells. After 24 h of incubation, immune cells were collected, blocked on ice with anti-mouse CD16 / 32 for 30 min, and then incubated with anti-CD11b-FITC, anti-F4 / 80-PE, anti-CD86-PE-Cy7, and anti-CD206-APC antibodies for 30 min. After washing with PBS, the macrophage polarization ratio (M1:CD11b) was detected by flow cytometry. + F4 / 80 + CD86 + M2: CD11b+ F4 / 80 + CD206 + Notably, the proportion of M1 macrophages in BMDM treated with SAM and OSAM significantly increased from 13.8% to 56.3% and 60.9%, respectively. Figure 7 A). Furthermore, the OSAM-treated groups had the highest M1 / M2 ratio, reaching 1.5, approximately six times that of the PBS group ( Figure 7 B). This indicates that OSAM can effectively promote the polarization of BMDM to M1 macrophages, thereby enhancing its anti-tumor activity.

[0065] Example 5: OSAM for Immunotherapy in B16F10 Melanoma Mice First, a mouse B16F10 melanoma subcutaneous tumor model was established. B16F10 cells (3 × 10⁻⁶) were then used. 5 Each mouse was resuspended in PBS containing 20% ​​matrigel and injected subcutaneously at a dose of 50 μL per mouse above the right hind leg. The tumors grew to approximately 75 mm in volume after about 10 days. 3 The experiment began on day 0 with a single intratumoral injection (50 μL) of different formulations: PBS, MG-loaded anti-CTLA-4 (C4M), MG-loaded LTX-315 (OM), MG-loaded diABZI (SAM), OSAM, and OSAM / C4M. The doses of LTX-315 were 45 mg / kg, diABZI 2 mg / kg, and anti-CTLA-4 2 mg / kg. Tumor volume, body weight, and status were monitored every two days. Mice were assessed for tumor volume and status based on natural death, weight loss exceeding 15% of initial body weight, or tumor volume greater than 1500 mm². 3 If diagnosed as deceased, a survival curve is plotted accordingly. Tumor volume is calculated using the formula V = W. 2 The value is calculated as ×L×0.5 (L and W are the distances at the widest and narrowest points of the tumor as measured by calipers). Figure 8 A showed that none of the microgel-based formulations caused a significant decrease in mouse body weight after intratumoral administration; instead, they caused a gradual increase in mouse body weight over time, indicating that these formulations have good biocompatibility. Compared with the rapid tumor growth in the PBS control group, a single intratumoral injection of OM, SAM, or OSAM significantly inhibited tumor growth. Figure 8 B). Among them, OSAM showed the strongest anti-tumor effect, with significant tumor regression in mice treated with OSAM and a MST prolonged to 37 days. Furthermore, 3 out of 7 mice achieved complete tumor remission within 90 days, and no recurrence occurred during the observation period. Figure 8(C) indicates that OSAM can induce a durable anti-tumor immune response. To further enhance the therapeutic effect, OSAM was combined with C4M. Observations showed that OSAM / C4M combined treatment significantly improved the cure rate in mice (71%), and no tumor recurrence was observed within 90 days. This indicates that OSAM / C4M combined treatment can not only effectively inhibit tumor growth, but also induce a strong and durable anti-tumor immune response.

[0066] Furthermore, to further confirm the high efficacy of OSAM immunotherapy and explore whether cured mice possess immune memory, this experiment selected cured mice from the OSAM and OSAM / C4M treatment groups mentioned above. The study investigated whether cured mice could resist re-attack by B16F10 cells and measured the levels of various memory T cells in the peripheral blood and spleen of the mice. On day 120 of treatment, the cured mice in both groups were in good condition and received subcutaneous re-inoculation with the same number of B16F10 cells. Other C57BL / 6J mice that only received B16F10 cell inoculation served as the control group. The results showed that compared with the rapid tumor growth in the control group, tumor progression was slowed in the cured mice. Figure 9 A), and the tumor weight on day 18 further confirmed this. Figure 9 B). On day 18 (138 days) after revaccination, all mice were sacrificed, and peripheral blood and spleen were collected for flow cytometry analysis of memory T cells. Spleen was ground, and erythrocytes were lysed with ACK; peripheral blood was directly lysed. Corresponding staining antibodies were added to both single-cell suspensions, and flow cytometry was used to analyze central memory T cells (T cells). FlowJo_V10 software was used for analysis. CM CD44 + CD62L + ) and effector memory T cells (T EM CD44 + CD62L - The results showed that, compared with the PBS group, both treatments increased the number of memory T cells in mice. Figure 9 CF). Among them, CD8 in peripheral blood and spleen of the OSAM group + T EM and CD8 + T CM The levels of CD8 were significantly increased in the peripheral blood and spleen of the OSAM / C4M group; while the levels of CD8 in the peripheral blood and spleen of the OSAM / C4M group were significantly higher. + T EM and CD8 + T CM The levels of these substances were all higher. For example, compared to the PBS group, OSAM and OSAM / C4M reduced the levels of T in peripheral blood. EM The number of cells increased approximately threefold, resulting in a strong immune memory.

[0067] Example 6: Immunological Analysis of OSAM in B16F10 Melanoma Mice Fourteen days after transplantation of B16F10 melanoma cells, the tumor volume was approximately 150 mm. 3 B16F10 mice were randomly divided into six groups: PBS, C4M, OM, SAM, OSAM, and OSAM / C4. A single intratumoral administration was administered on day 0, and mice were sacrificed after 7 days of treatment. Inguinal lymph nodes were harvested, and the levels of mDCs and MHC I were analyzed. Tumors were harvested and ground into single-cell suspensions, stained with appropriate immune cell antibodies, and the content of immune cells was measured by flow cytometry. Data were analyzed using FlowJo_V10 software. The levels of MHC I, DCs, NK cells, macrophages, T cells, and Tregs in tumor cells were analyzed. Flow cytometry showed that OSAM significantly upregulated the expression of MHC I in tumor cells. In tumor tissues from mice treated with OSAM and OSAM / C4M, the MFI of MHC I was 1.8-fold and 2.3-fold higher than that in the PBS group, respectively. Figure 10 A). Considering that dendritic cells play a crucial role in adaptive immune responses by effectively presenting antigens and activating T cells, the number of mature DCs (CD11c⁺CD80⁺CD86⁺) in lymph nodes was first assessed. Compared with PBS-treated mice, both OM and SAM treatments promoted DC maturation, while OSAM treatment significantly increased the proportion of mature DCs (A). Figure 10 B) and activated DC (CD11c⁺ MHC I) MFI ( Figure 10 (C), which is consistent with in vitro study results. Furthermore, the number of mature DCs induced by OSAM / C4M was 4.2 times that of the PBS group.

[0068] To better evaluate the antitumor immune response, tumor-infiltrating cytotoxic T lymphocytes, natural killer cells, and tumor-associated macrophages were further examined. Given the important roles of CD4⁺ and CD8⁺ T cells in adaptive immune responses, T cell infiltration in mouse tumors and spleens was assessed. Anti-CTLA-4 treatment alone had minimal impact on T cell numbers; OSAM significantly increased the proportion of CD8⁺ effector T cells and CD4⁺ helper T cells in tumors; and by inhibiting the CTLA-4 immune checkpoint on T cells, the proportion of CD8⁺ T cells and CD4⁺ T cells was even higher in mouse tumor cells treated with OSAM / C4M. Figure 10 D, E). SAM and OSAM increased the proportion of NK1.1-positive natural killer cells (NK cells) by more than 3-fold, primarily due to the secretion of type I interferon following activation of the STING pathway. This trend was further enhanced upon administration of anti-CTLA-4. Figure 10 F). Simultaneously, OSAM significantly reduced the proportion of M2 type tumor-associated macrophages, promoted the polarization of tumor-associated macrophages towards the M1 type, and increased the M1 / M2 ratio. The M1 / M2 ratio of OSAM / C4M was 3.3 times that of the PBS group (F). Figure 10 G, H), confirmed its effective polarization effect on macrophages. Furthermore, compared with PBS-treated mice, OSAM-treated mice showed a significant reduction in regulatory T cells (Tregs), and the number of Tregs was further reduced after combining with anti-CTLA-4 antibody (G, H). Figure 10 I). In summary, OSAM / C4M can promote the maturation of dendritic cells (DCs) and enhance their antigen-presenting capacity, increase the infiltration of CD8⁺ / CD4⁺ T cells, promote TAM polarization towards the M1 type, and reduce Treg cells, effectively improving the immunosuppressive microenvironment and thus promoting the clearance of tumor cells.

[0069] Example 7: Immunotherapy of OSAM / C4M in a TNBC Mouse Model Encouraged by the high cure rate of 71% achieved with OSAM combined with anti-CTLA-4 immunotherapy, we further explored the therapeutic effect of this regimen in the triple-negative breast cancer (TNBC) model 4T1 mice. 4T1 cells (5 × 10⁻⁶) were used... 5 (Each mouse) was resuspended in PBS containing 20% ​​Matrigel, and 50 μL / mouse was injected subcutaneously into the area above the right hind leg of each mouse to establish a subcutaneous 4T1 triple-negative breast cancer model. The tumors grew to approximately 75 mm in volume after about 8 days. 3 The experiment began on day 0 with a single intratumoral injection (50 μL) of different formulations: PBS, OSAM, and OSAM / C4M. The doses were LTX-315 45 mg / kg, diABZI 2 mg / kg, and anti-CTLA-4 2 mg / kg. Results showed that compared to the PBS group, OSAM and OSAM / C4M treatment significantly delayed tumor growth, extending MST from 18 days to 38 days and 52 days, respectively. Figure 11 (A, B). Furthermore, 14% and 43% of mice in the OSAM and OSAM / C4M groups, respectively, were cured, and 3 out of 7 mice treated with OSAM / C4M experienced complete tumor disappearance within 90 days. Blood was collected from the orbital rimole on day 7 of the treatment process, and after erythrocyte sac rupture, T cells (CD8+) were analyzed. + T, CD4 + The levels of CD8⁺ T cells and CD4⁺ T cells in peripheral blood were measured. Flow cytometry analysis revealed that, compared to the PBS treatment group, OSAM / C4M increased the number of CD8⁺ T cells and CD4⁺ T cells in peripheral blood by approximately two-fold (…). Figure 11(C, D). Although the efficacy shown in this experiment was significantly worse than that in B16F10 mice, it still validated the potential use of OSAM combined with immune checkpoint inhibitors for the treatment of poorly immunogenic TNBC.

[0070] Example 8: Immunotherapy of OSAM / C4M in a Mouse Model of MC38 Colon Cancer MC38 cells (1×10) 6 (Each mouse) was resuspended in PBS containing 20% ​​matrigel, and 50 μL / mouse was injected subcutaneously into the area above the right hind leg of each mouse to establish a subcutaneous MC38 colon cancer model. The tumor grew to approximately 75 mm in volume after about 9 days. 3 The experiment began on day 0 with a single intratumoral injection (50 μL) of different formulations, namely PBS and OSAM / C4M. Tumor volume, body weight, and status were monitored in mice every two days. Compared with the PBS group, OSAM / C4M treatment significantly delayed tumor growth in mice, and tumors disappeared in 2 out of 5 mice, achieving a cure rate of 40%. No tumor recurrence was observed within 90 days. Figure 12 A, B). On day 150 of treatment, the cured mice in the OSAM / C4M group were in good condition and received a subcutaneous re-inoculation with the same number of MC38 cells. Other C57BL / J mice that only received MC38 cell inoculation served as the control group. Tumor growth, body weight, and status were monitored and recorded. Results showed that tumors grew rapidly in the control group mice, while all cured mice showed 100% resistance to the re-inoculation with MC38 cells and did not form tumors at all. Figure 12 (C, D) indicates that the OSAM / C4M combined treatment strategy also has good therapeutic effects in the MC38 mouse model.

[0071] This invention discloses a method for preparing a microgel loaded with the oncolytic agent interferon gene stimulating factor (STING) and its application. The invention involves a simple mixing of hyaluronic acid microgel, a STING agonist, and an oncolytic agent in solution. The STING-activated microgel (OSAM) developed in this invention can continuously release the oncolytic agent and the STING agonist for up to 4 weeks, while simultaneously inducing significant upregulation of major histocompatibility complex class I (MHC I) and sustained activation of dendritic cells for over a week. Furthermore, OSAM can effectively load and control the release of the oncolytic agent and the STING agonist. The sustained release of these two drugs within the tumor is expected to simultaneously induce long-term stable activation of the STING pathway and direct lysis of tumor cells, thereby generating multiple tumor-specific antigens and achieving long-lasting and potent specific anti-tumor immunity. Notably, a single intratumoral injection of OSAM significantly promotes the infiltration of cytotoxic T lymphocytes and natural killer cells. When OSAM is used in combination with an anti-CTLA-4 loaded microgel (C4M), it exhibits excellent therapeutic effects in various mouse tumor models, with a cure rate of 71%. The STING-activated microgel containing the oncolytic agent disclosed in this invention has advantages such as high safety, simple preparation, and strong immune activation ability, opening up new avenues for the development of in situ therapeutic tumor vaccines.

Claims

1. An oncolytic agent-loaded STING activating microgel, characterized in that, The microgel and the oncolytic agent and the STING agonist loaded in the microgel.

2. The oncolytic STING-activating microgel according to claim 1, characterized in that, The STING agonist is inside and / or outside the microgel, and the oncolytic agent is inside and / or outside the microgel.

3. The oncolytic STING-activating microgel according to claim 1, wherein, The STING agonist is a non-cyclic dinucleotide agonist, and the oncolytic agent includes an oncolytic peptide.

4. A process for the preparation of oncolytic agent loaded STING activating microgels as claimed in claim 1, wherein, The oncolytic agent-loaded STING-activating microgel is prepared by mixing the STING agonist, the microgel and the oncolytic agent.

5. A method of preparing oncolytic STING-activating microgels according to claim 4, wherein, The microgel is a medical polymer microgel.

6. A method of preparing oncolytic STING-activating microgels according to claim 4, wherein, The oncolytic agent-loaded STING-activating microgel is prepared by mixing the STING agonist, the microgel and the oncolytic agent in a solvent.

7. A medicine, wherein the active ingredient comprises the oncolytic agent-loaded STING-activating microgel according to claim 1.

8. Use of the oncolytic agent-loaded STING-activating microgel according to claim 1 in the preparation of a medicine.

9. Use of the oncolytic agent-loaded STING-activating microgel according to claim 1 in the preparation of an immunotherapy medicine.

10. Use of the oncolytic agent-loaded STING-activating microgel according to claim 1 in the preparation of an antitumor medicine.

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