Combination immunization drug, temperature-sensitive gel composition, chemokine particle, and use thereof

Abstract

The application discloses a combined immunization drug, a temperature-sensitive gel composition, a chemotactic factor particle and application thereof, and uses a mixed solution of an injectable temperature-sensitive polymer PLGA-PEG-PLGA, a chemotactic factor particle and an immune checkpoint inhibitor PD-1 monoclonal antibody to form a temperature-sensitive hydrogel beside a solid tumor at body temperature, and long-term controllable release of the chemotactic factor and aPD1, so as to enhance the targeting of adoptive T cells to the solid tumor and the resistance to immunosuppression in the tumor microenvironment. Through combination of the immunoregulatory temperature-sensitive gel and a tumor-penetrating peptide iRGD, the infiltration of the adoptive T cells to the solid tumor is enhanced, so that the solid tumor is efficiently treated. The chemotactic factor particle is constructed by using a distearoyl phosphoethanolamine-polyethylene glycol-N-hydroxysuccinimide modified chemotactic factor and an albumin carrier.

Description

TECHNICAL FIELD

[0001] The present application relates to a combination immunotherapy drug, a temperature-sensitive gel composition, a chemokine particle and its application. BACKGROUND

[0002] Adoptive cell therapy (ACT) represented by chimeric antigen receptor T cells (CAR-T) has achieved gratifying results in the treatment of hematological malignancies, but the efficacy of ACT in solid tumors such as lung cancer, breast cancer, liver cancer, etc. is still not satisfactory. In CAR-T therapy, T cells extracted from the peripheral blood of patients are genetically modified to enable them to specifically recognize tumor cells, and after activating and proliferating these cells in vitro, they are reinfused into the patient's body. The main factors restricting the use of adoptive T cells in the treatment of solid tumors are: 1) poor targeting of adoptive T cells to solid tumors; 2) adoptive T cells that reach the lesion cannot effectively infiltrate solid tumors; 3) adoptive T cells that infiltrate tumors are immunosuppressed by cancer cells in the tumor microenvironment (TME), resulting in reduced function and reduced number.

[0003] Some studies have attempted to use chemokines to enhance the targeting of adoptive T cells to solid tumors. Chemokines can stimulate immune cell migration through G protein-coupled receptors on the surface of immune cells. Cytotoxic CD8+ T lymphocytes (CTLs) are the main cells that eliminate tumor cells, and there is a high expression of CXC-chemokine receptor 3 (CXCR3) on the surface of activated CD8+ T cells and memory CD8+ T cells. Therefore, CXCR3-mediated immune cell migration is one of the key factors in anti-tumor immunity. The main ligands of CXCR3 are CXCL9, CXCL10 and CXCL11. CXCL9, also known as Monokine induced by gamma interferon (MIG), is located on human chromosome 4 and is induced by IFN-γ. However, solid tumors have low expression of chemokines, which cannot effectively attract T cells to migrate to solid tumors. SUMMARY

[0004] The present application provides a combination immunotherapy drug, a temperature-sensitive gel composition, a chemokine particle and its application. The present application injects a mixed solution of CXCL9 chemokine particles, PD1 monoclonal antibodies and temperature-sensitive polymers around a solid tumor, which forms an immunomodulatory temperature-sensitive hydrogel loaded with CXCL9 chemokine particles and PD1 monoclonal antibodies at body temperature, allowing the loaded drugs to be released controllably around the tumor for a long period of time, thereby improving the tumor targeting of immune cells and the anti-tumor killing power. Tumor penetrating peptide iRGD can be loaded into the temperature-sensitive gel or injected intravenously, and used in combination with the immunomodulatory temperature-sensitive gel to enhance the tumor infiltration ability of immune cells.

[0005] To achieve the above objectives, the present invention provides the following technical solution: chemokine particles, comprising an amphiphilic polymer, a chemokine, and an albumin carrier, wherein the amphiphilic polymer comprises molecules that bind to albumin, hydrophilic spacer groups, and functional groups that react with the chemokine;

[0006] The molecules that bind to albumin include lipophilic diacyl chains, lipophilic acyl chains, alkyl chains, fatty acids, vitamin E, and polypeptides containing the sequence AVGALEGPRNQDWLGVPRQL.

[0007] Preferably, the structure of the hydrophilic spacer group is as follows:

[0008]

[0009] Where X is a carbon chain containing multiple carbon atoms with ether bonds;

[0010] Preferably, the functional group that reacts with the chemokine includes a functional group capable of reacting with the amino, thiol, or disulfide bond of the chemokine.

[0011] Preferably, the functional groups that react with the protein are as follows:

[0012]

[0013] Wherein, Y is oxygen, or a carbon chain containing multiple carbon atoms with ether bonds.

[0014] Preferably, the functional groups that react with the protein are as follows:

[0015]

[0016] Where Z represents a carbon chain containing multiple carbon atoms.

[0017] Preferably, the chemokine includes one or any combination of two or more of CXCL9, CXCL10 and CXCL11.

[0018] Preferably, the albumin carrier has a particle size of 1–1400 nm.

[0019] The preparation method of the above-mentioned chemokine particles includes the following steps:

[0020] 1) The chemokine and the amphiphilic polymer are mixed and reacted to obtain the chemokine-modified body;

[0021] 2) Prepare BSA and DTSSP solutions with PBS. Mix BSA and DTSSP at a molar ratio of 1:(10-300), stir and react. After the reaction is complete, centrifuge and wash with PBS to obtain NP.

[0022] 3) The chemokine modifier is added to the albumin carrier NP to carry out the reaction, thereby obtaining chemokine particles.

[0023] The method for preparing the chemokine particles described above includes the following steps:

[0024] 1) The chemokine and the amphiphilic polymer are mixed and reacted to obtain the chemokine-modified body;

[0025] 2) Mix BSA solution and DTSSP solution to react, and centrifuge the reacted sample solution. Resuspend the solution in PBS to prepare SMP.

[0026] 3) The chemokine modifier is added to the albumin carrier SMP to react and obtain chemokine particles.

[0027] Thermosensitive gel composition includes the chemokine particles, PD-1 monoclonal antibody, and thermosensitive hydrogel described above.

[0028] Thermosensitive gel composition includes the chemokine particles, PD-1 monoclonal antibody, iRGD peptide or peptide sequence containing c(CRGDKGPDC) as described above, and thermosensitive hydrogel.

[0029] Combined immunotherapy drugs, including the above-described thermosensitive gel composition applied at the tumor site and intravenously administered iRGD or a polypeptide sequence containing c(CRGDKGPDC).

[0030] The above-mentioned chemokine particles, the above-mentioned thermosensitive gel composition, and the above-mentioned combined immunotherapy drugs are used in adoptive cell therapy and non-adoptive cell immunotherapy; wherein, adoptive cells include CAR-T cells, CAR-NK cells, CAR-M cells, TCR-T cells, and neutrophils; non-adoptive cells include endogenous CD8+ cells. + T cells, endogenous CD4 + T cells, endogenous NK cells, endogenous macrophages, and endogenous neutrophils.

[0031] iRGD can enhance the targeting and infiltration capabilities of drugs to tumors. iRGD is a cyclic peptide composed of 9 amino acid residues (CRGDKGPDC). During systemic circulation, it can specifically bind to integrins and neurocilia protein-1 (NRP-1) receptors overexpressed on tumor cells, activating NRP-1 and increasing the permeability of tumor blood vessels and tissues.

[0032] In tumor cell ejaculation (TME), tumor cells typically upregulate the expression of PD-L1 on their cell surface. When the T cell surface receptor PD-1 binds to its ligand PD-L1, the immune checkpoint signaling pathway is activated, thereby inhibiting cell activity and reducing the anti-cancer immunity of T cells. Immune checkpoint inhibitors such as PD-1 antibodies (aPD1) and PD-L1 antibodies can effectively block the PD-1 signaling pathway, thereby restoring T cell activity.

[0033] Injectable hydrogel systems offer significant potential for localized, targeted drug delivery and controlled sustained-release, demonstrating great promise in improving drug efficacy and reducing systemic toxicity. Their high biocompatibility also allows for wide application in the biomedical field. Thermosensitive gels are a broad category of supramolecular hydrogels, forming a gel through hydrophobic interactions. They are liquid within a certain temperature range but transform into a gel with temperature changes.

[0034] Distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) block copolymers have good biocompatibility. In application, DSPE-PEG ends are usually modified with carboxyl, amino, maleimide or N-hydroxysulfosuccinimide groups to link other functional molecules to one end.

[0035] In summary, the present invention has the following beneficial effects:

[0036] This invention utilizes an injectable thermosensitive polymer, PLGA-PEG-PLGA, which forms a hydrogel around solid tumors at body temperature. This hydrogel allows for the long-term, controllable release of chemokine particles and the immune checkpoint inhibitor PD-1 monoclonal antibody (aPD1), enhancing the targeting of adoptive T cells to solid tumors and their resistance to immunosuppression in the tumor microenvironment (TME). Furthermore, by combining this with the tumor-penetrating peptide iRGD, the infiltration of adoptive T cells into solid tumors is further enhanced, resulting in highly effective treatment of solid tumors. The chemokine particles are constructed from CXCL9 (CXCL9-DP) modified with distearate ethanolamine-polyethylene glycol-N-hydroxysuccinimide (DSPE-PEG-NHS, DP-NHS) and an albumin submicron particle carrier (SMP) (CXCL9-DP@SMP). In a B16-OVA subcutaneous solid tumor mouse model, a thermosensitive gel (CMP / aPD1@Gel) co-loaded with CXCL9-DP@SMP (CMP) and aPD1, combined with iRGD, increased the number of adoptive T cells in the tumor by 21 times compared to an equal amount of free drug. These adoptive T cells highly expressed functional proteins such as IFN-γ and Granzyme B, exhibiting tumor cell killing activity. This technology is applicable to various adoptive T cell therapies, including chimeric antigen receptor T cell (CAR-T) and T cell receptor T cell (TCR-T) therapies, providing a new platform and approach for enhancing adoptive T cell therapy. Attached Figure Description

[0037] Figure 1 (A) Flowchart for the preparation of immunomodulatory thermosensitive hydrogels and (B) Schematic diagram of in vivo enhancement of adoptive T cell antitumor efficacy;

[0038] Figure 2 For the preparation, characterization, and drug loading determination of BSA carriers. (A) Particle size and PDI of BSA:DTSSP nanoparticles (NPs) obtained with different reaction molar ratios. (B) Particle size distribution of BSA NPs (1:200). (C) Schematic diagram of different preparation methods of submicron particles (SMPs). (D) Particle size and PDI of SMPs (n=3) under different preparation methods. (E) Particle size distribution of BSA SMPs. (F) Zeta potential of NPs and SMPs (n=3). (G) TEM image of NPs. (H) TEM image of SMPs. (I) Binding of DP-FITC to SMPs under fluorescence microscopy. (J) Drug loading of DP-FITC on BSA carriers. (K) Drug loading of DP-Cytochrome C-Cy5 on BSA NP / SMPs;

[0039] Figure 3 Preparation of thermosensitive hydrogels and determination of phase transition temperature and drug loading. (A) Phase transition diagram of hydrogels. (B) Phase transition temperatures of thermosensitive copolymers with different mass ratios. (C) Cytochrome C loading capacity per mg of PLGA-PEG-PLGA hydrogel. (D) Loading efficiency of hydrogels for different drugs and carriers;

[0040] Figure 4 To investigate the in vitro degradation and drug release kinetics of thermosensitive hydrogels, and the in vitro migration of chemokine carriers. (A) Degradation of thermosensitive hydrogels in a physiological environment at 37°C in vitro. (B) Release kinetics of CC-Cy5 in various carrier systems. (C) Release kinetics of aPD1-FITC in hydrogels. (D) Effect of different reaction molar ratios of DSPE-PEG-NHS on the activity of CXCL9. (E) Schematic diagram of Transwell migration assay. (F) Number of CD8+ T cells migrating to the lower chamber at different time points (n=3, ****, p<0.0001);

[0041] Figure 5 Biodegradation of thermosensitive gel in mice. (A) Balb / c mice without tumor grafts. (B) Edge of B16-OVA subcutaneous melanoma in C57Bl / 6 mice;

[0042] Figure 6 For the in vivo release of the thermosensitive gel. (A) In Vitro IVI fluorescence imaging in mice at different time points. (B) Quantitative analysis of fluorescence intensity kinetics (n=5, *, p<0.05, **, p<0.01, ***, p<0.001);

[0043] Figure 7 The expression of CD8 and CXCR3 in spleen cells of OT-1 mice during activation. (A) Flow cytometry histogram of CD8+ T cell percentage. (B) Quantitative analysis of CD8+ T cell percentage. (C) Flow cytometry histogram of CXCR3 expression in cells. (D) Quantitative analysis of the percentage of cells expressing CXCR3 (n=4, *, p<0.05, **, p<0.01, ****, p<0.0001);

[0044] Figure 8 Chemokines-based biogels, in synergistic effect with free aPD1 and iRGD, significantly inhibited the growth of subcutaneous melanoma. (A) Experimental schedule. (B) Tumor volume changes in tumor-bearing mice during treatment. (C) Relative changes in body weight. (D) Survival curves (n=5, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001);

[0045] Figure 9 To evaluate the in vivo tumor-suppressive effect of a co-loaded drug combination biogel. (A) Experimental schedule. (B) Tumor volume changes in tumor-bearing mice during treatment. (C) Relative changes in body weight. (D) Survival curves (n=6, **, p<0.01, ***, p<0.001, ****, p<0.0001);

[0046] Figure 10 For the in vivo immunological evaluation of the drug-coated biogel. (A) Experimental schedule. (B) Tumor volume changes in tumor-bearing mice during treatment. (C) Images of ex vivo tumor tissue from mice (n=7, *, p<0.05, ***, p<0.001, ****, p<0.0001);

[0047] Figure 11 Flow cytometry analysis of immune cells in tumors. (A) Representative flow cytometry plots for each group showing the proportion of CD8+ T cells in lymphocytes. (B) Quantitative analysis of the proportion of CD8+ T cells in lymphocytes. (C) Representative flow cytometry plots for each group showing the proportion of adoptive T cells (CD8+Thy1.1+) in CD8+ T cells. (D) Quantitative analysis of the proportion of adoptive T cells in lymphocytes. (E) Quantitative analysis of the proportion of adoptive T cells (CD8+Thy1.1+) in CD8+ T cells. (F) Quantitative analysis of adoptive T cells (CD8+Thy1.1+) per mg of tumor. (G) Quantitative analysis of endogenous CD8+ T cells per mg of tumor (n=7, ns, no significant difference, *, p<0.05, ***, p<0.001, ****, p<0.0001). Endo, endogenous; ACT, adoptive T cells;

[0048] Figure 12 This section describes the expression of functional proteins in immune cells within tumors. (A) Representative flow cytometry plots of IFN-γ expression percentage in CD8+ T cells. (B) Quantitative analysis of IFN-γ expression in CD8+ T cells. (C) Representative flow cytometry plots of Granzyme B expression percentage in CD8+ T cells. (D) Quantitative analysis of Granzyme B expression in CD8+ T cells (n=7, ns, no significant difference, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001). Endo, endogenetic; ACT, adoptive T cells;

[0049] Figure 13 Analysis of immune cells in lymphoid organs and peripheral blood. (A) Representative flow cytometry plot of ACT(CD8+Thy1.1+) percentage in lymph node cells. (B) Quantitative analysis of ACT(CD8+Thy1.1+) percentage in lymph node cells. (C) Representative flow cytometry plot of ACT(CD8+Thy1.1+) percentage in spleen lymph cells. (D) Quantitative analysis of ACT(CD8+Thy1.1+) percentage in spleen lymph cells. (E) Representative flow cytometry plot of ACT(CD8+Thy1.1+) percentage in peripheral blood CD8+ T cells. (F) Quantitative analysis of ACT(CD8+Thy1.1+) percentage in peripheral blood lymph cells (n=7, ns, no significant difference, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001). ACT, adoptive T cells;

[0050] Figure 14 To evaluate the systemic antitumor effect of a co-loaded drug combination biogel. (A) Experimental schedule. (B) Changes in primary tumor volume in tumor-bearing mice during treatment. (C) Changes in distal tumor volume. (D) Relative changes in body weight. (E) Survival curves (n=8, **, p<0.01, ***, p<0.001, ****, p<0.0001);

[0051] Figure 15 To further challenge the experiment in curing tumors in mice. (A) Experimental schedule. (B) Tumor volume changes in tumor-bearing mice. (C) Images of isolated tumor tissue from mice (n=8, ****, p<0.0001). Detailed Implementation

[0052] The invention will be further described with reference to the accompanying drawings.

[0053] This embodiment discloses chemokine particles, including an amphiphilic polymer, a chemokine, and an albumin carrier. The amphiphilic polymer includes molecules that bind to albumin, hydrophilic spacer groups, and functional groups that react with the chemokine.

[0054] Molecules that bind to albumin include lipophilic diacyl chains, lipophilic acyl chains, alkyl chains, fatty acids, vitamin E, and polypeptides containing the sequence AVGALEGPRNQDWLGVPRQL.

[0055] The chemokines include one or any combination of two or more of CXCL9, CXCL10, and CXCL11. The particle size of the albumin carrier is 1–1400 nm.

[0056] In some embodiments, the structure of the hydrophilic spacer group is shown below:

[0057]

[0058] Where X is a carbon chain containing multiple carbon atoms with ether bonds; and n is 1 to 200.

[0059] Furthermore, the structure of the hydrophilic spacer group is shown below:

[0060]

[0061] In some embodiments, the functional groups that react with the chemokine include functional groups capable of reacting with the amino, thiol, or disulfide bonds of the chemokine.

[0062] In some implementations, the functional groups that react with the protein are as follows:

[0063]

[0064] Wherein, Y is oxygen, or a carbon chain containing multiple carbon atoms with ether bonds.

[0065] Furthermore, the functional groups that react with proteins are shown below:

[0066]

[0067] In some implementations, the functional groups that react with the protein are as follows:

[0068]

[0069] Where Z represents a carbon chain containing multiple carbon atoms.

[0070] Furthermore, the functional groups that react with proteins are shown below:

[0071]

[0072] In some embodiments, the amphiphilic polymers include DP-NHS and DP-MAL.

[0073] The method for preparing the chemokine particles described above includes the following steps:

[0074] 1) The chemokine and the amphiphilic polymer are mixed and reacted to obtain the chemokine-modified body;

[0075] 2) Prepare BSA and DTSSP solutions using PBS. Mix BSA and DTSSP at a molar ratio of 1:(10-300), stir and react. After the reaction, centrifuge and wash with PBS to obtain NP. Specifically, the molar ratio of BSA to DTSSP is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, 1:250, 1:260, 1:270, 1:280, 1:290 and 1:300.

[0076] 3) The chemokine modifier is added to the albumin carrier NP to carry out the reaction, thereby obtaining chemokine particles.

[0077] The method for preparing the chemokine particles described above includes the following steps:

[0078] 1) The chemokine and the amphiphilic polymer are mixed and reacted to obtain the chemokine-modified body;

[0079] 2) Mix BSA solution and DTSSP to react, centrifuge the reacted sample solution, resuspend it in PBS to prepare SMP;

[0080] 3) The chemokine modifier is added to the albumin carrier SMP to react and obtain chemokine particles.

[0081] The above technical solution involves three different methods for preparing SMP, as described below:

[0082] (1) One-step method: Mix 1 part by weight of BSA solution and 2 parts by weight of DTSSP and react for 3 hours;

[0083] (2) Two-step method: Mix 0.5 parts by weight of BSA and 1 part by weight of DTSSP and react for 1.5 h; then add 0.5 parts by weight of BSA and 1 part by weight of DTSSP to the reaction system and continue to react for 1.5 h.

[0084] (3) Three-step method: Mix 0.5 parts by weight of BSA and 1 part by weight of DTSSP and react for 1 hour; then add 0.5 parts by weight of BSA to the reaction system and continue to react for 1 hour; finally add 1 part by weight of DTSSP and react for 1 hour.

[0085] The sample solution after the reaction was centrifuged and resuspended in PBS to prepare SMP.

[0086] A thermosensitive gel composition comprising the chemokine particles, PD-1 monoclonal antibody, and thermosensitive hydrogel described above. The chemokine particles, PD-1 monoclonal antibody, and thermosensitive polymer are mixed in a PBS solution, and the mixture is injected adjacent to the tumor. At body temperature, an immunomodulatory thermosensitive hydrogel loaded with chemokine particles and PD-1 monoclonal antibody is formed adjacent to the tumor.

[0087] A thermosensitive gel composition comprising the aforementioned chemokine particles, PD-1 monoclonal antibody, iRGD peptide or peptide containing the c(CRGDKGPDC) sequence, and a thermosensitive hydrogel. The chemokine particles, PD-1 monoclonal antibody, iRGD peptide or peptide containing the c(CRGDKGPDC) sequence, and a thermosensitive polymer are mixed in a PBS solution. The mixture is injected adjacent to the tumor, and an immunomodulatory thermosensitive hydrogel loaded with the chemokine particles, PD-1 monoclonal antibody, iRGD peptide or peptide containing the c(CRGDKGPDC) sequence is formed adjacent to the tumor at body temperature.

[0088] Combined immunotherapy drugs, including the above-described thermosensitive gel composition applied at the tumor site and intravenously administered iRGD peptide or peptide sequence containing c(CRGDKGPDC).

[0089] The above-described chemokine particles, the above-described thermosensitive gel composition, and the above-described combined immunomodulatory drugs are used in adoptive and non-adoptive cell therapy; wherein, adoptive cells include CAR-T cells, CAR-NK cells, CAR-M cells, TCR-T cells, and neutrophils. Non-adoptive cells include endogenous CD8+ cells. + T cells, endogenous CD4 + T cells, endogenous NK cells, endogenous macrophages, and endogenous neutrophils.

[0090] This technology cleverly combines the chemokine CXCL9, the immune checkpoint inhibitor PD-1 monoclonal antibody (aPD1), and the tumor-penetrating peptide iRGD with a hydrogel to enhance the targeting, infiltration, and intratumoral immune response of adoptive T cells to solid tumors, thereby achieving highly efficient treatment of solid tumors. This technology loads CXCL9 and aPD1 onto a hydrogel and injects the thermosensitive hydrogel into the solid tumor, allowing for long-term, controllable release of the loaded drugs near the tumor. Because the chemokine CXCL9 has a small molecular weight (only 12 kDa), it is rapidly released from the hydrogel. Therefore, we modified CXCL9 with DSPE-PEG(DP) to obtain CXCL9-DP, and then linked CXCL9-DP to BSA submicron particles (SMPs) through the specific binding of DSPE to BSA, resulting in CXCL9-DP@SMP. CXCL9 released at the tumor margin recruits adoptive T cells to solid tumors; iRGD binds to αvβ integrin specifically expressed on tumor vascular endothelium, activating neuropilin NRP-1, increasing tumor vascular and tissue permeability, thereby enhancing the infiltration of adoptive T cells into solid tumors; aPD1 released in the hydrogel helps intratumoral adoptive T cells resist immunosuppression of cancer cells, improving therapeutic efficacy. Figure 1 (B) This method is applicable to various adoptive T-cell therapies such as CAR-T and TCR-T, providing a new approach for the treatment of solid tumors. The system is simple to prepare, uses biodegradable raw materials, and has high clinical translatability. Specific implementation examples:

[0092] 1. Materials and Methods

[0093] 1.1 Materials

[0094] Mouse-derived CXCL9, IL-2, and IL-7 were purchased from Peprotech (Rocky Hill, NJ, USA). Anti-PD1 (clone RMP1-14), anti-CD3 (clone 145-2C11), and anti-CD28 (clone 37.51) antibodies were purchased from Bioxcell (Lebanon, NH, USA). Ovalbumin peptide (257-264) was purchased from InvivoGen (San Diego, CA, USA). EasySep TMMouse CD8+ T cell isolation kit was purchased from StemCell Technologies (Vancouver, BC, Canada). Ficoll-Paque Plus was purchased from Cytiva (Pharmacia, Uppsala, Sweden). LIVE / DEADFixable Dead Cell Stain Kit was purchased from Invitrogen (Carlsbad, CA, USA). Anti-mouse CD183 (CXCR3) APC, CD8a FITC / PE, Thy1.1 APC / FITC, CD45 FITC, CD4 APC-Cy7, CD69 BV421, IFN-γ APC, Ki67 BV421, and Granzyme B PE were purchased from Biolegend (San Diego, CA, USA). 3,3'-Dithiobis(sulfosuccinimide propionate) (DTSSP) was purchased from Abcam (Cambridge, UK). Distearate phosphatidylethanolamine (DSPE), including DSPE-PEG-NHS (MW of PEG=2000) and DSPE-PEG-FITC (MW of PEG=2000), was purchased from Ponsure Biotech (Shanghai, China). Cy5 NHS ester (non-sulfonated) was purchased from APExBIO (Houston, Texas, USA). PLGA1640-PEG1500-PLGA1640 was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Shanxi, Xian, China).

[0095] Materials in Chinese and English abbreviations

[0096]

[0097]

[0098] Drug delivery system (abbreviation)

[0099]

[0100] 1.2 Mice and cell lines

[0101] Female C57BL / 6 mice (6 weeks old) and female BALB / C mice (6 weeks old) were purchased from Cavens (Changzhou, China). OT-1 mice were purchased from the Shanghai Model Organisms Center (Shanghai, China) and bred in-house. Pmel-1 mice were purchased from Jackson Laboratory (Maine, USA) and bred in-house. All mice were housed in SPF-grade rooms, 5 mice per cage, with ample food and drinking water. The housing was kept in a light-dark cycle for 12 hours each (7:00 AM to 7:00 PM), with a room temperature of 25 ± 1°C. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Soochow University. The animal experimental protocols were conducted according to the NIH Guidelines for Laboratory Animal Care and Use (NIH Publication No. 85-23 Rev. 1985). The B16-OVA cell line was purchased from ATCC (Rockville, MD, USA) and cultured in DMEM medium containing 10% fetal bovine serum (FBS).

[0102] 1.3 Preparation and characterization of BSA nanoparticles (NP) and submicron particles (SMP)

[0103] BSA (1 mg / mL) and DTSSP (10 mg / mL) solutions were prepared using PBS. BSA and DTSSP were mixed at molar ratios of 1:10, 1:20, 1:50, 1:100, 1:200, and 1:300, respectively. After adding a stir bar, the mixture was reacted at 600 rpm for 1 h. The resulting solution was then transferred to a 50 kDa ultrafiltration tube, centrifuged (4000 rpm, 4 min), and washed twice with PBS to obtain NP.

[0104] SMP was prepared according to three different methods: (1) one-step method: 1 mg BSA solution and 2 mg DTSSP solution were reacted for 3 h; (2) two-step method: 0.5 mg BSA solution and 1 mg DTSSP solution were reacted for 1.5 h, and then 0.5 mg BSA solution and 1 mg DTSSP solution were added and reacted for another 1.5 h; (3) three-step method: 0.5 mg BSA solution and 1 mg DTSSP solution were reacted for 1 h, then 0.5 mg BSA solution was added and reacted for another 1 h, and finally 1 mg DTSSP solution was added and reacted for another 1 h. The reacted sample solution was centrifuged (23000 rpm, 10 min), resuspended in PBS and SMP was obtained.

[0105] The hydration size and zeta potential of the prepared NP and SMP were measured using a laser nanoparticle size and potential analyzer (90Plus Particle Size Analyzer, Brookhaven), as shown in Tables 1 and 2 below. 20 μL of NP and SMP at appropriate concentrations were dropped onto a copper mesh, dried at room temperature, and their morphology was observed using a transmission electron microscope (HT7700, Hitachi).

[0106] Table 1: Molar ratio of albumin to DTSSP and particle size of the prepared albumin NP carrier

[0107]

[0108] Table 2: Particle size of albumin SMP carriers obtained by different preparation methods

[0109]

[0110]

[0111] 1.4 BSA NP / SMP DSPE-PEG-FITC (DP-FITC) loading

[0112] Five 1mg aliquots of prepared NP and SMP were taken, and 10, 20, 40, 60, and 80μg of DP-FITC were added respectively. The reaction volumes were then uniformly increased to 1mL of PBS, and the mixtures were placed in a four-dimensional rotating microplate (BE-1100, Kylin-Bell) for 0.5h. The NP reaction solution was ultrafiltered using a 50kDa ultrafiltration tube (4500rpm, 4min), and the lower layer filtrate was collected. The SMP reaction solution was centrifuged (23000rpm, 10min), and the supernatant was collected. The fluorescence intensity of FITC in each group of samples was detected using a full-wavelength multi-mode microplate reader (M1000Pro, TECAN) (Ex=470nm, Em=530nm), and the amount of DP-FITC not loaded onto BSA particles and the loading amount of DP-FITC on BSA particles were calculated accordingly.

[0113] 1.5 Preparation of DP-modified chemokine CXCL9 (CXCL9-DP) and DP-modified model fluorescent protein CC-Cy5 (CC-Cy5-DP)

[0114] CXCL9 was mixed with DP-NHS (reaction molar ratios of 1:1, 1:2, 1:5, and 1:10) in PBS solution and reacted overnight at room temperature on a four-dimensional rotator to prepare CXCL9-DP.

[0115] Cytochrome C (CC), a protein with a similar molecular weight and isoelectric point to CXCL9, was used as a model protein. CC-Cy5, which is linked to the fluorescent molecule Cy5, was mixed with DP-NHS (molar ratio 1:5) in PBS solution and reacted overnight on a four-dimensional rotator to prepare CC-Cy5-DP.

[0116] 1.6 Combination of CXCL9-DP, CC-Cy5-DP with NP and SMP

[0117] Add CXCL9-DP or CC-Cy5-DP prepared in Method 2.5 to the NP or SMP prepared by Method 2.3, respectively, and mix at room temperature in a four-dimensional rotator for 0.5 h to obtain CXCL9-DP@NP, CXCL9-DP@SMP, CC-Cy5-DP@NP, and CC-Cy5-DP@SMP.

[0118] 1.7 Drug loading capacity of DP-modified proteins in BSA NP / SMP

[0119] Take five 1 mg NP or SMP aliquots and add 10, 20, 40, and 80 μg of CC-Cy5 or CC-Cy5-DP containing the corresponding mass of CC-Cy5, respectively. Combine the reaction volume with 1 mL of PBS and mix in a four-dimensional gyroscope for 0.5 h. Ultrafilter the NP reaction solution using a 50 kDa ultrafiltration tube (4500 rpm, 4 min) and collect the lower filtrate. Centrifuge the SMP reaction solution (23000 rpm, 10 min) and collect the supernatant. Detect the Cy5 fluorescence intensity (Ex = 620 nm, Em = 670 nm), and calculate the mass of CC-Cy5 in the filtrate and the mass of CC-Cy5 linked to the NP / SMP.

[0120] 1.8 Preparation of Thermosensitive Hydrogel (Gel)

[0121] Five 10mg PLGA-PEG-PLGA polymer solutions were weighed and added to different volumes of PBS. The solutions were vortexed until the polymer was fully dissolved, preparing polymer solutions with concentrations of 14% w / v, 16% w / v, 18% w / v, 20% w / v, and 22% w / v. The polymer solutions of different concentrations were placed in a metal bath, and the temperature was increased by 2°C starting from room temperature (25°C). After each increase, the system was allowed to stabilize for 10 minutes. The state of the polymer solutions was observed, and the phase transition temperature was recorded.

[0122] Preparation methods of various in vitro hydrogels: Weigh PLGA-PEG-PLGA polymer and dissolve it in various prepared carrier solutions (w / v% = 16%). Stir with a pipette tip while vortexing until the polymer is fully dissolved. Place the culture dish on a metal bath at 37°C to prepare PLGA-PEG-PLGA polymer gel.

[0123] 1.9 Determination of Drug Loading Capacity of Thermosensitive Hydrogel

[0124] The absorbance of cytochrome C (CC) at 280 nm was measured at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, and 6 mg / mL (Nanodrop 2000, Eppendorf), and a standard curve was plotted. 8 mg of PLGA-PEG-PLGA polymer was weighed and mixed with 1 mg, 2 mg, 3 mg, 4 mg, and 6 mg of CC powder in PBS to prepare 16% w / v PLGA-PEG-PLGA solutions, which were then heated to 37°C to form a gel. The surface of the hydrogel was washed with 200 μL of PBS, and the absorbance of the washing solution at 280 nm was measured. The mass of CC not loaded into the temperature-sensitive gel in the washing solution was calculated using the standard curve, thus obtaining the final drug loading of the hydrogel.

[0125] 1 mg of NP (or SMP) was mixed with CC-Cy5-DP (containing 15 μg CC-Cy5) in PBS solution and reacted on a four-dimensional rotator for 0.5 h to obtain CC-Cy5-DP@NP and CC-Cy5-DP@SMP. CC-Cy5-DP@NP, CC-Cy5-DP@SMP, and 15 μg of free CC-Cy5 solution were then added to 8 mg of PLGA-PEG-PLGA polymer to prepare a 16% w / v PLGA-PEG-PLGA solution, which was heated to 37 °C to form a gel. The surface of the hydrogel was washed with PBS, and the fluorescence intensity of Cy5 in the washing solution was measured (Ex = 620 nm, Em = 670 nm). The mass of unloaded CC-Cy5 and the drug loading of the hydrogel were calculated. Similarly, 30 μg of free aPD1-FITC and 8 mg of PLGA-PEG-PLGA polymer were mixed in PBS solution and heated to 37 °C to form a gel. The drug loading of the thermosensitive gel on aPD1 was calculated using the same method.

[0126] 1.10 Release curve of proteins loaded on thermosensitive hydrogel

[0127] CC-Cy5 was reacted with DP-NHS at a molar ratio of 1:5 to synthesize CC-Cy5-DP. Different carriers (CC-Cy5-DP@NP, CC-Cy5-DP@SMP, CC-Cy5@Gel, CC-Cy5-DP@NP@Gel, and CC-Cy5-DP@SMP@Gel) containing equal amounts of 15 μg CC-Cy5 were prepared, with free CC-Cy5 serving as the control group.

[0128] Each group of samples (total volume 200 μL) was placed in a dialysis bag (MWCO = 300 kDa), and the dialysis bag was then placed in a centrifuge tube containing 20 mL of PBS. The centrifuge tube was placed in a constant-temperature shaker (37℃, 100 rpm) for 7 days (d) of release. 200 μL of solution was taken from the centrifuge tube at 2 h, 8 h, 1 d, 2 d, 3 d, 4 d, 6 d, and 7 d to measure the Cy5 fluorescence intensity, and 200 μL of PBS at the same temperature was added. The cumulative percentage of CC-Cy5 release for each group of samples was calculated using the following formula.

[0129]

[0130] Where Ern is the cumulative percentage of CC-Cy5 released at a certain time point; Ve is the replacement volume of the release medium, i.e., 200 μL; V0 is the total volume of the release medium, i.e., 20 mL; FCC-Cy5 is the total fluorescence of CC-Cy5 in the initial dialysis bag; and fn is the fluorescence reading of the sample taken at the nth replacement sampling.

[0131] 30 μg aPD1-FITC was mixed with 8 mg PLGA-PEG-PLGA in PBS solution and gelled at 37 °C. The aPD1-FITC-loaded hydrogel was released over 7 days using the same method. FITC fluorescence was detected at 2 h, 8 h, 1 d, 2 d, 3 d, 4 d, 6 d, and 7 days, with 200 μL of PBS at the same temperature added after each detection. The cumulative release percentage of aPD1-FITC was calculated.

[0132] 1.11 Evaluation of the effect of chemokine hydrogels on the in vitro migration of T cells

[0133] Take 20 μL of CXCL9 sample solution (@25 ng / mL) and mix it with DP-NHS (reaction molar ratio 1:1, 1:2, 1:5, 1:10) in PBS solution to a final volume of 1 mL. React overnight on a four-dimensional rotating apparatus. Detect CXCL9 activity in each sample according to the ELISA kit instructions.

[0134] A 0.1% gelatin solution was spread on an inverted Transwell chamber (3415, Costa), and MS1 endothelial cells (1×10⁵) were seeded and cultured upright in the chamber. After 2 days, TNF-α (20 ng / mL) was added to activate the MS1 cells in the chamber and upregulate the expression of their adhesion molecules. After 4 hours, 200 μL of RPMI 1640 complete medium containing IL-2 (10 ng / mL) and IL-7 (10 ng / mL) was added to each well of the upper chamber. The lower chamber was divided into 7 groups: ① No CXCL9; ② Free CXCL9; ③ CXCL9-DP; ④ CXCL9-DP@NP; ⑤ CXCL9-DP@NP@Gel; ⑥ CXCL9-DP@SMP; ⑦ CXCL9-DP@SMP@Gel (except for group ① which contained no CXCL9, the other groups contained an equal amount of 0.25 μg CXCL9). The liquid levels in the upper and lower chambers were kept equal. 10 μL of culture medium was taken from the lower chamber daily for cell counting. After sampling on the 4th day, the liquid in the upper chamber was aspirated, and fresh RPMI 1640 complete culture medium containing 2.5 × 10⁵ CD8+ T cells was added. Half of the liquid in the lower chamber was aspirated and replaced with fresh RPMI 1640 complete culture medium. Sampling continued on the 5th day.

[0135] 1.12 Degradation of thermosensitive hydrogels in in vitro physiological environments

[0136] 50 mg of PLGA-PEG-PLGA polymer was dissolved in Ponceau S staining solution (10%) to prepare a polymer solution with a mass concentration of 16% (w / v%). The polymer was placed in a petri dish and heated to 37°C to form a gel, thus preparing a thermosensitive hydrogel loaded with Ponceau S. The petri dish was immersed in PBS and placed on a shaker (37°C, 100 rpm), and the morphological changes of the hydrogel were recorded by photographing at different time points.

[0137] 1.13 In vivo biodegradation of thermosensitive gel

[0138] B16-OVA cells were dissolved in sterile PBS (4 × 10⁶ cells / mL) and added to a 1:1 volume ratio of matrix gel (356234, Costa). Two × 10⁵ B16-OVA cells were subcutaneously seeded into each C57BL / 6 female mouse. On day 7 post-seeding, 5 mg SMP and 60 μg CC-Cy5-DP were mixed and reacted in a four-dimensional rotating apparatus for 0.5 h. 40 mg PLGA-PEG-PLGA was slowly added to the reaction solution to prepare a gel solution (w / v % = 16%). 50 μL of the gel solution was subcutaneously injected into mice adjacent to tumors or without tumors, and gelation occurred at body temperature. Mice were euthanized 15 min, 2 d, 4 d, 6 d, and 8 d after injection, and the injection site and surrounding area were dissected and photographed.

[0139] 1.14 In vivo release of thermosensitive gel

[0140] BALB / c mice were randomly divided into the following 6 groups: ①CC-Cy5; ②CC-Cy5@Gel; ③CC-Cy5@NP; ④CC-Cy5@NP@Gel; ⑤CC-Cy5@SMP; ⑥CC-Cy5@SMP@Gel. 50 μL of different sample solutions (containing an equal volume of 44 μg CC-Cy5) were subcutaneously injected into the right back of each mouse. The fluorescence intensity of Cy5 on the mouse back was examined using a small animal in vivo imaging system (IVISL Minina II) at 0 h, 8 h, 1 d, 2 d, 3 d, 6 d, 7 d, and 8 d after administration (Ex = 620 nm, Em = 670 nm).

[0141] 1.15 Activation, culture and characterization of T cells from OT-1 / Pmel-1 mice

[0142] After euthanizing OT-1 (or Pmel-1) mice, the spleen was removed, ground and washed on a 70 μm cell filter, and centrifuged (700 g, 4 min). The cell pellet was treated with ACK erythrocyte lysis buffer, centrifuged again, and resuspended in RPMI 1640 medium containing OVA257-264 peptide (or gp100) (1 μg / mL), IL-2 (10 ng / mL), and IL-7 (1 ng / mL), adjusting the cell density to 2 × 10⁶ cells / mL. After incubation at 37°C for 3 days, lymphocytes were purified using the Ficoll-paque plus gradient centrifugation method. The collected cells were cultured in RPMI 1640 medium containing IL-2 (10 ng / mL) and IL-7 (10 ng / mL), with the medium changed every 2 days. Flow cytometry was used to stain 1×10⁶ cells at 0, 3, 4 and 5 days after cell activation to detect the proportion of CD8+ T cells and the expression level of CXCR3+ in OT-1 mice.

[0143] 1.16 Chemokine-Sensitive Gel's In Vivo Tumor-Suppressing Effect

[0144] B16-OVA cells were dissolved in sterile PBS (4 × 10⁶ cells / mL), and a 1:1 volume ratio of matrix gel was added. Two × 10⁵ cells were subcutaneously inoculated into C57BL / 6 mice. On day 5 after inoculation, tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to eliminate lymphocytes. The tumor-bearing mice were randomly divided into 6 groups, with each group receiving the following drug doses: 5 × 10⁶ OT-1CD8+ T cells activated for 5 days, aPD1 (30 μg), iRGD (200 μg), CXCL9 (15 μg), and CMP (DSPE-PEG2000-CXCL9 linked to SMP). On day 6 after tumor cell inoculation, the mice were given the first dose of the drug according to the following groupings. Four days after drug administration, each mouse in group ① received 200 μL of sterile PBS via tail vein, while each mouse in the other groups received 5 × 10⁶ OT-1CD8+ T cells activated for 5 days via tail vein.

[0145] ① Tail vein injection of CD8+T+aPD1

[0146] ② Tail vein injection of CD8+T+aPD1+iRGD

[0147] ③ Tail vein injection of CD8+T+aPD1 + subcutaneous injection of free CXCL9

[0148] ④ Tail vein injection of CD8+T+aPD1+iRGD + subcutaneous injection of free CXCL9

[0149] ⑤ Inject CD8+T+aPD1 via tail vein + inject CMP@Gel (loaded with 15μg CXCL9) subcutaneously.

[0150] ⑥ Tail vein injection of CD8+T+aPD1+iRGD + subcutaneous injection of CMP@Gel (loaded with 15μg CXCL9)

[0151] The mice were observed every two days to assess their survival, and changes in body weight and tumor size were recorded for each group during the treatment period. A tumor volume exceeding 1000 mm³ was considered the experimental termination point for each mouse.

[0152] 1.17 In vivo tumor-suppressing effect study of thermosensitive gel co-loaded with CXCL9 and aPD1

[0153] B16-OVA cells were dissolved in sterile PBS (1×10⁷ cells / mL), and a 4:1 volume ratio of matrix gel was added. 8×10⁵ cells were subcutaneously inoculated into C57BL / 6 mice. On day 6 post-inoculation, tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to eliminate lymphocytes. The tumor-bearing mice were randomly divided into 6 groups, with each group receiving the following drug doses: 5×10⁶ OT-1CD8+ T cells activated for 5 days, aPD1 (30 μg), iRGD (200 μg), and CXCL9 (15 μg). On day 7 after tumor cell inoculation, the mice were given the first dose of the drug according to the following groupings. Four days after drug administration, each mouse in group ① received 200 μL of sterile PBS via tail vein, while each mouse in the other groups received 5×10⁶ OT-1CD8+ T cells activated for 5 days via tail vein.

[0154] ① Tail vein injection of PBS

[0155] ② Tail vein injection of CD8+T

[0156] ③ Tail vein injection of CD8+T+iRGD + subcutaneous injection of CMP@Gel (containing 15μg CXCL9)

[0157] ④ Tail vein injection of CD8+T+iRGD+aPD1 + subcutaneous injection of CMP@Gel (loaded with 15μg CXCL9)

[0158] ⑤ Tail vein injection of CD8+T+iRGD + subcutaneous injection of CMP / aPD1@Gel (containing 15μg CXCL9 + 30μg aPD1)

[0159] ⑥ Tail vein injection of CD8+T+ subcutaneous injection of CMP / aPD1 / iRGD@Gel (loaded with 15μg CXCL9+30μg aPD1+200μg iRGD)

[0160] The mice were observed every two days to assess their survival, and changes in body weight and tumor size were recorded for each group during the treatment period. A tumor volume exceeding 1000 mm³ was considered the experimental termination point for each mouse.

[0161] 1.18 In vivo immunological evaluation of thermosensitive gel co-loaded with CXCL9 and aPD1

[0162] B16-OVA cells were dissolved in sterile PBS (1.25 × 10⁷ cells / mL), and a 4:1 volume ratio of matrix gel was added. 1 × 10⁶ cells were subcutaneously inoculated into C57BL / 6 mice. On day 7 post-inoculation, tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to eliminate lymphocytes. The tumor-bearing mice were randomly divided into four groups, with each group receiving the following drug doses: 5 × 10⁶ Pmel-1 CD8+ T cells activated for 5 days, aPD1 (30 μg), iRGD (200 μg), and CXCL9 (15 μg). On day 8 after tumor cell inoculation, the mice were given the first dose of the drug according to the following groupings. Four days after drug administration, each mouse in group ① received 200 μL of sterile PBS via tail vein, while each mouse in the other groups received 5 × 10⁶ Pmel-1 CD8+ T cells activated for 5 days via tail vein.

[0163] ① Tail vein injection of PBS

[0164] ② Tail vein injection of CD8+T

[0165] ③ Tail vein injection of CD8+T+iRGD+aPD1 + subcutaneous injection of free CXCL9

[0166] ④ Tail vein injection of CD8+T+iRGD + subcutaneous injection of CMP / aPD1@Gel (containing 15μg CXCL9 + 30μg aPD1)

[0167] Changes in body weight and tumor volume were recorded in each group of mice every two days. Two days after the second administration, mice were euthanized, and tumors, spleens, inguinal lymph nodes, and peripheral blood were collected. Peripheral blood, spleen cells, and lymph node cells after erythropoiesis were stained with antibodies by flow cytometry to detect the expression levels of Thy1.1, CD8a, CD4, and CD45. Cells obtained from tumor grinding were incubated in eBioscience™ Cell Stimulation Cocktail (Invitrogen) at 37°C for 6 hours, and the expression levels of Thy1.1, CD8a, CD25, IFN-γ, and Granzyme B were detected by flow cytometry.

[0168] 1.19 Distal tumor inhibition effect of thermosensitive gel co-loaded with CXCL9 and aPD1

[0169] B16-OVA cells were dissolved in sterile PBS (1×10⁷ cells / mL), and mixed with matrix gel at a 4:1 volume ratio. The cells were then subcutaneously seeded at both ends of C57BL / 6 mice, with a seeding density of 8×10⁵ cells. On day 6 after seeding, tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg / kg to eliminate lymphocytes. The tumor-bearing mice were randomly divided into four groups, with each group receiving the following drug doses: 5×10⁶ OT-1CD8+ T cells activated for 5 days, aPD1 (30 μg), iRGD (200 μg), and CXCL9 (15 μg). On day 7 after tumor cell seeding, the mice were given the first dose of the drug according to the following groupings. Four days after drug administration, each mouse in group ① received 200 μL of sterile PBS via tail vein, while each mouse in the other groups received 5×10⁶ OT-1CD8+ T cells activated for 5 days via tail vein.

[0170] ① Tail vein injection of PBS

[0171] ② Tail vein injection of CD8+T

[0172] ③ Tail vein injection of CD8+T+iRGD+aPD1 + subcutaneous injection of free CXCL9

[0173] ④ Tail vein injection of CD8+T+iRGD + subcutaneous injection of CMP / aPD1@Gel (containing 15μg CXCL9 + 30μg aPD1)

[0174] The mice were observed every two days to assess their survival, and changes in body weight and tumor size were recorded for each group during the treatment period. A tumor volume exceeding 1000 mm³ was considered the experimental termination point for each mouse.

[0175] 1.20 Rechallenge Experiment to Cure Tumors in Mice

[0176] B16-OVA cells were dissolved in sterile PBS (1.25 × 10⁷ cells / mL), added to a 4:1 volume ratio of matrix gel, and subcutaneously inoculated into C57BL / 6 mice at a density of 1 × 10⁶ cells. A Rechallenge tumor-bearing model was established by co-constructing cured C57BL / 6 mice with age-matched healthy C57BL / 6 mice. Free aPD1 (30 μg) was injected via the tail vein on days 11, 14, 18, 21, 24, and 27 after tumor inoculation. Starting on day 8 after tumor inoculation, the survival status of the mice was observed every two days, and changes in body weight and tumor size were recorded for each group during treatment. A tumor volume exceeding 1000 mm³ was considered the experimental termination point for each mouse.

[0177] 2. Experimental Results

[0178] 2.1 Determination of BSA NP / SMP particle size, zeta potential, and drug loading

[0179] The n-hydroxysuccinimide (NHS) functional groups at both ends of 3,3'-dithiobis(sulfosuccinimide propionate) (DTSSP) can undergo an amide reaction with the amino groups in proteins, crosslinking monomeric serum albumin (BSA) to form carrier particles. Adjusting the molar reaction ratio of BSA to DTSSP yields nanoparticles (NP) and submicron particles (SMP) of different sizes, with uniform particle size distribution and a particle size distribution (PDI) less than 0.3. Figure 2 A, Table 1). When the molar ratio of BSA:DTSSP is 1:200, the resulting NP particle size is ~340nm ( Figure 2 B), this ratio was chosen for subsequent NP experiments. We tried three different methods to prepare SMP ( Figure 2 C), the SMP particles obtained by method 3 have the largest size, approximately 891 nm, and are uniformly distributed. Figure 2 DE, Table 2). The potentials of NP and SMP are -2.2±0.8mV and -1.5±0.9mV, respectively. Figure 2 F). TEM observation of the morphology of NP / SMP revealed that NP / SMP exhibits a spherical structure. TEM images showed that the NP particle size was approximately 270 nm and the SMP particle size was approximately 700 nm, consistent with the trend of hydrated particle size measured by a particle size analyzer. Figure 2 GH).

[0180] After co-incubating DSPE-PEG (DP-FITC) linked to FITC fluorescent molecules with BSA SMP, DP-FITC can be linked to the BSA vector via DSPE. Figure 2 I). Keeping the total NP / SMP amount constant (1 mg), the amount of DP-FITC that can bind to NP / SMP is dose-dependent. When the amount of DP-FITC added exceeds 40 μg, the binding of DP-FITC to BSA tends to saturate. Each mg of NP can bind a maximum of approximately 36 μg of DP-FITC, and each mg of SMP can bind a maximum of approximately 34 μg of DP-FITC. Figure 2 J).

[0181] Cytochrome C (CC) consists of 108 amino acid residues, with a molecular weight of 12.3 kDa, an isoelectric point of 8.7, and 16 lysine residues. CXCL9 consists of 105 amino acid residues, with a molecular weight of 12.2 kDa, an isoelectric point of 9.8, and 21 lysine residues. Therefore, CC is suitable as a model protein for CXCL9 to study its physicochemical properties. Reacting CC with the fluorescent molecule Cy5 with DP yields CC-Cy5-DP. CC-Cy5-DP@NP represents the binding of CC-Cy5-DP to NP, and CC-Cy5@SMP represents the binding of CC-Cy5 to SMP. CC-Cy5-DP specifically binds to NP / SMP via DSPE. NP can load 32 μg CC-Cy5 / mg NP, and SMP can load 39 μg CC-Cy5 / mg SMP, which is more than three times the amount of CC loaded via electrostatic adsorption (CC-Cy5@SMP). Figure 2 K).

[0182] 2.2 Preparation and Characterization of Thermosensitive Hydrogels

[0183] An aqueous solution of PLGA-PEG-PLGA polymer undergoes a phase transition upon heating, forming a non-flowing colloid. Further heating causes the precipitation of a white substance. The phase transition is reversible upon cooling. Figure 3 A). PLGA-PEG-PLGA polymer aqueous solutions gelled at 37°C at concentrations ranging from 14% (m / v) to 22% (m / v), with higher w / v ratios resulting in lower gelation temperatures. To ensure gelation at room temperature but rapid gelation at body temperature, hydrogels with a w / v ratio of 16% were used in subsequent experiments. Figure 3 B). Thermosensitive hydrogels can encapsulate up to ~0.35 mg CC / mg PLGA-PEG-PLGA ( Figure 3 C). The encapsulation efficiency of the hydrogel for 15 μg of free CC-Cy5 on both BSANP and SMP was close to 100%, indicating that the BSA carrier does not affect the loading of chemokines. The loading efficiency of the hydrogel for aPD1 was also close to 100%, indicating that the molecular weight and type of protein drug have no significant effect on the drug loading efficiency of the hydrogel. Figure 3 D).

[0184] 2.3 In vitro degradation and drug release kinetics of thermosensitive gel

[0185] A thermosensitive copolymer solution containing 10% Ponceau Red was plated in a CAR-T pattern on the surface of a petri dish and gelled at 37°C. PBS was then added to submerge the hydrogel, and the dish was placed on a shaker (100 rpm, 37°C) and observed daily. During the 8-day observation period, no significant changes were observed in the thermosensitive gel for the first 4 days. After 4 days, the hydrogel gradually began to degrade over time. Figure 4 A).

[0186] Next, we investigated the in vitro release kinetics of free CC-Cy5, CC-Cy5 linked to BSA NPs and SMPs (CC-Cy5-DP@NP, CC-Cy5-DP@SMP), and CC-Cy5 loaded in thermosensitive gels (CC-Cy5@Gel, CC-Cy5-DP@NP@Gel, and CC-Cy5-DP@SMP@Gel). All samples were placed in dialysis bags (MW = 300 kDa) with one end sealed tightly, ensuring a liquid volume of 200 μL. The other end was then sealed, and the bags were placed in 50 mL centrifuge tubes containing 20 mL of PBS, maintained at 37 °C. Free CC-Cy5 was released rapidly, almost entirely diffusing outside the dialysis bag within 24 hours. In the CC-Cy5@Gel group, the hydrogel delayed the release of CC-Cy5. However, due to the large pore size of the hydrogel, CC-Cy5 with a molecular weight of only 12 kDa flowed out of the hydrogel, with nearly 80% of CC-Cy5 released within 3 days. Compared with the free group, the CC-Cy5-DP@NP and CC-Cy5-DP@SMP groups delayed the release of CC-Cy5, reaching 80% release at 3 and 4 days, respectively. This is mainly because the NP and SMP carriers themselves cannot pass through the dialysis bag, and CC-Cy5 can only be released after the NP / SMP has degraded. Loading CC-Cy5-DP@NP and CC-Cy5-DP@SMP into hydrogels (CC-Cy5-DP@NP@Gel, CC-Cy5-DP@SMP@Gel) can further slow down the release of CC-Cy5. However, compared to CC-Cy5-DP@NP, CC-Cy5-DP@Gel showed less significant sustained-release effect, mainly due to the smaller particle size of NP, which prevents it from remaining in the hydrogel for an extended period. The sustained-release effect of the CC-Cy5-DP@SMP@Gel group was significantly improved compared to CC-Cy5-DP@SMP, and the release time was extended by nearly 5 times compared to the free CC-Cy5 group. Figure 4 B). The total amount of CC-Cy5 released cumulatively by each carrier was basically the same. The release of free aPD1 in the hydrogel takes approximately 7 days, due to its relatively large molecular weight (150 kDa), thus its release period in the in vitro hydrogel system is longer than that of free CC-Cy5. Figure 4 C).

[0187] 2.4 Chemokine migration in vitro experiment

[0188] ELISA results showed that the activity of CXCL9 decreased slightly with increasing DSPE-PEG-NHS reaction molar ratio. To improve the yield of CXCL9-DP while minimizing the loss of CXCL9 activity, we ultimately determined a reaction molar ratio of 1:5. The resulting CXCL9-DP exhibited 97.6% of the bioactivity of unreacted CXCL9. Figure 4 D). To investigate the ability of chemokine hydrogels to recruit CD8+ T cells in vitro, we designed a Transwell migration assay (…). Figure 4 E). MS1 cells were first plated in the upper chamber. Two days later, equal volumes of free and BSA-loaded CXCL9 were added to the lower chamber, along with an equal volume of T cell culture medium containing cytokines. An equal volume of activated CD8+ T cells and the same T cell culture medium were added to the upper chamber, maintaining the liquid levels in both chambers. The cell count in the lower chamber was counted daily. Compared to the No CXCL9 group, the free CXCL9 group showed a significant increase in CD8+ T cells in the lower chamber, approximately 2.3 times after 8 days, demonstrating that the chemokine CXCL9 can induce CXCR3+ T cell migration. The number of CD8+ T cells recruited by DP-modified CXCL9 (CXCL9-DP) was essentially the same as that recruited by unmodified CXCL9, indicating that DP modification did not affect the chemotactic function of CXCL9. After 8 days, the number of CD8+ T cells recruited to the lower chamber in the CXCL9-DP@NP group and the CXCL9-DP@SMP group was approximately 2.3 times and 3.3 times that of the CXCL9 group, respectively, indicating that the binding of CXCL9-DP to NP / SMP can prolong the release of CXCL9 and extend its duration of action. Both the CXCL9-DP@NP@Gel group and the CXCL9-DP@SMP@Gel group recruited more CD8+ T cells to the lower chamber than the CXCL9-DP@NP group and the CXCL9-DP@SMP group. The number of CD8+ T cells collected in the lower chamber after 8 days in the thermosensitive hydrogel-encapsulated CXCL9-DP@SMP group was nearly 7 times that of the No CXCL9 group and nearly 2 times that of the CXCL9-DP@SMP group. Figure 4 F).

[0189] 2.5 In vivo biodegradation and drug release of thermosensitive gel

[0190] A mixed solution of CC-Cy5-DP@SMP and PLGA-PEG-PLGA polymers was subcutaneously injected into normal BALB / c mice and C57BL / 6 mice bearing B16-OVA xenografts, respectively. It was observed that after injection of the mixed solution, in normal BALB / c mice (… Figure 5 A) and tumor-bearing C57BL / 6 mice ( Figure 5B) Both can form hydrogels in vivo. Over time, the hydrogel degrades in vivo, releasing CC-Cy5. The subcutaneous blue color in mice gradually fades and almost disappears after 8 days. We also used a small animal in vivo imaging system to detect the release of the drug loaded in the thermosensitive gel in vivo. Free CC-Cy5 or CC-Cy5-loaded particles and thermosensitive gel were injected subcutaneously into female BALB / c mice, and Cy5 fluorescence at the injection site was detected at different time points. Figure 6 (AB). The fluorescence intensity of both the free CC-Cy5 group and the CC-Cy5@Gel group decreased rapidly in vivo, with 65% of the fluorescence disappearing after 1 day. This may be due to the small molecular weight of CC-Cy5, which easily leaks from the hydrogel, thus the release kinetics of CC-Cy5@Gel are similar to those of free CC-Cy5. The subcutaneous fluorescence intensity of the CC-Cy5-DP@NP and CC-Cy5-DP@NP@Gel groups remained at approximately 50% after 2 days, about twice that of the free CC-Cy5 group. This is likely due to the retention of BSA nanoparticles under the skin; however, because the NP particle size is not large enough, it easily leaks from the gel, resulting in little difference between the CC-Cy5-DP@NP@Gel and CC-Cy5-DP@NP groups. The release curves of the CC-Cy5-DP@SMP group were similar to those of the CC-Cy5-DP@SMP group, but the hydrogel loaded with CC-Cy5-DP@SMP significantly improved the drug retention effect. The CC-Cy5-DP@SMP@Gel group maintained 80% fluorescence signal after 2 days and still had 20% signal after 8 days, which is consistent with the in vitro release kinetics results.

[0191] 2.6 OT-1 mouse T cell activation expression of CD8 and CXCR3

[0192] CD8+ T cells obtained from OT-1 transgenic mice can specifically recognize the MHC class I molecule-OVA257-264 antigen peptide complex, thus recognizing and killing B16-OVA tumor cells. To investigate the proportion of CXCR3+ and CD8+ T cells in spleen cells during in vitro activation and expansion, and to optimize T cell activation conditions, OT-1 mouse spleens were crushed, split open, and co-incubated with OVA257-264 antigen peptide and cytokines. After 3 days of activation and culture, lymphocytes were purified with Ficoll and then cultured and proliferated. After splitting open, only 25% of the OT-1 mouse spleen cells were CD8+ T cells. After 3 days of activation and Ficoll purification, the proportion of CD8+ T cells reached 70%. Continuing culture until day 5, the proportion of CD8+ T cells reached 98%. Figure 7AB). Initially, only 7.21% of the spleen cells from OT-1 mice expressed the CXCL9 receptor CXCR3. After 3 days of incubation, the CXCR3-expressing cells were only 8.8%, but by day 5, the percentage of CXCR3+ cells reached 59%. Figure 7 Therefore, spleen cells from OT-1 mice cultured for 5 days using this activation method were selected as T cells to be adopted into the mice.

[0193] 2.7 In vivo tumor-suppressing effect of chemokine biogels

[0194] To investigate the in vivo efficacy of CXCL9-DP@SMP@Gel (CMP@Gel), we subcutaneously constructed a B16-OVA tumor model in female C57BL / 6 mice and began treatment when the tumor volume was approximately 35 mm3. The following groups were selected for tail vein injection: CD8+T+aPD1 (T+aPD1), CD8+T+aPD1+iRGD (T+aPD1+iRGD), CD8+T+aPD1+subcutaneous free CXCL9 (T+aPD1+CXCL9), CD8+T+aPD1+iRGD+subcutaneous free CXCL9 (T+aPD1+CXCL9+iRGD), CD8+T+aPD1+subcutaneous CMP@Gel (T+aPD1+CMP@Gel), and CD8+T+aPD1+iRGD+subcutaneous CMP@Gel (T+aPD1+CMP@Gel+iRGD). Figure 8 A).

[0195] The T+aPD1+CXCL9 group showed better anti-tumor effects than T+aPD1, demonstrating that CXCL9's chemotactic recruitment ability to CD8+ T cells contributes to enhancing the tumor-suppressive effect of adoptive T cells. The results of comparing the T+aPD1+iRGD group with the T+aPD1 group, and the T+aPD1+CMP@Gel+iRGD group with the T+aPD1+CMP@Gel group, demonstrate that the tumor vascular penetration performance of the iRGD peptide improves the efficacy of adoptive T cells against solid tumors. The results of the T+aPD1+CXCL9+iRGD group, compared with T+aPD1+CXCL9 and T+aPD1+iRGD, demonstrate that the combination of iRGD and CXCL9 further enhances the therapeutic effect compared to using either alone. The results of the T+aPD1+CMP@Gel+iRGD group, compared with T+aPD1+CXCL9+iRGD, demonstrate that the sustained release of hydrogel can promote the long-term peritumoral release of CXCL9, thereby continuously recruiting adoptive T cells to the tumor site and significantly improving the therapeutic effect. Figure 8B). During the treatment, the body weight of mice in each group did not decrease significantly, indicating that there was no significant toxicity in any group, thus verifying the in vivo safety of this dosage of gel and drug. Figure 8 C). Survival curves show that the T+aPD1+iRGD group, T+aPD1+CXCL9 group, T+aPD1+CXCL9+iRGD group, T+aPD1+CMP@Gel group, and T+aPD1+CMP@Gel+iRGD group all inhibited tumor growth to some extent compared to the T+aPD1 group. However, the T+aPD1+CMP@Gel+iRGD group showed the best efficacy, with 80% (4 / 5 mice) of tumors being cleared. Figure 8 D) significantly prolonged the survival of mice.

[0196] 2.8 In vivo tumor-suppressing effect of thermosensitive gels co-loaded with multiple drugs

[0197] The above in vivo tumor suppression experiments have demonstrated that the use of iRGD peptide and the loading of chemokine CXCL9 onto a thermosensitive gel can significantly improve the efficacy of adoptive T cells against solid tumors. Figure 8 Next, we further verified whether aPD1 is necessary in treatment, and whether aPD1 and iRGD peptide can be co-loaded with CXCL9 in a hydrogel to further enhance efficacy. Because... Figure 8 The T+aPD1+CMP@Gel+iRGD group showed significant therapeutic effects. To demonstrate the improved efficacy of the optimized biogel, treatment was initiated only when the tumor volume reached 60 mm³. The following groups were included: PBS group, tail vein injection CD8+T group (T only), tail vein injection CD8+T+iRGD+subcutaneous injection CMP@Gel group (T+iRGD+CMP@Gel), tail vein injection CD8+T+iRGD+aPD1+subcutaneous injection CMP@Gel group (T+iRGD+aPD1+CMP@Gel), tail vein injection CD8+T+subcutaneous injection of a thermosensitive gel co-loaded with CMP, aPD1, and iRGD group (T+CMP / aPD1 / iRGD@Gel), and tail vein injection CD8+T+iRGD+subcutaneous injection of a thermosensitive gel co-loaded with CMP and aPD1 group (T+iRGD+CMP / aPD1@Gel). Figure 9A). Results from the T+iRGD+aPD1+CMP@Gel group and the T+iRGD+CMP@Gel group demonstrated that immune checkpoint inhibition of aPD1 is necessary to improve the efficacy of solid tumor treatment. Results from the T+iRGD+CMP / aPD1@Gel group and the T+iRGD+aPD1+CMP@Gel group demonstrated that aPD1 loaded in hydrogel was more beneficial in enhancing the therapeutic effect of combination therapy compared to tail vein injection of free aPD1. Results from the T+CMP / aPD1 / iRGD@Gel group and the T+iRGD+CMP / aPD1@Gel group demonstrated that there was no statistically significant difference in the tumor inhibition curves between free iRGD peptide and iRGD loaded in thermosensitive gel at the statistical time points. Figure 9 B), but in the T+CMP / aPD1 / iRGD@Gel group, only 16.7% of mice had their tumors completely cleared, while in the T+iRGD+CMP / aPD1@Gel group, 66.7% of mice had their tumors disappear (B). Figure 9 D). During the treatment, the body weight of mice in each group did not decrease significantly, indicating the drug's safety in vivo. Figure 9 C).

[0198] 2.9 In vivo immunological evaluation of thermosensitive gel co-loaded with CXCL9 and aPD1

[0199] To investigate the in vivo immunological effects of a thermosensitive gel co-loaded with CMP and aPD1 combined with iRGD, we subcutaneously constructed a B16-OVA tumor model in female C57BL / 6 mice. Spleen cells from Pmel-1 mice were extracted and activated with gp100 peptide. After the tumor volume reached 85 mm³, activated Pmel-1 Thy1.1+CD8+ T cells were injected for adoptive T cell therapy. The following groups were included: a blank control group (PBS), a tail vein injection CD8+T group (T only), a tail vein injection CD8+T+iRGD+aPD1+subcutaneous injection of free CXCL9 group (T+iRGD+aPD1+CXCL9), and a tail vein injection CD8+T+iRGD+subcutaneous injection CMP / aPD1@Gel group (T+iRGD+CMP / aPD1@Gel). The biogel group co-loaded with CXCL9 and aPD1 significantly reduced tumor size compared to the statistically significant drug injection. Figure 10 AC). Six days after initial treatment, cells from mouse tumors, spleen, lymph nodes, and peripheral blood were analyzed. In the tumors, there was no statistically significant difference in the proportion of CD8+ T cells among lymphocytes across the groups (AC). Figure 11 AB). The co-loaded drug gel group (T+iRGD+CMP / aPD1@Gel group) significantly increased the number of tumor-infiltrating adoptive T cells (ACTs) compared to the same dose of free drug. The proportion of ACTs in lymphocytes and CD8+ T cells was 4.1 and 3.4 times that of the free drug group, respectively. Figure 11More importantly, the number of adoptive T cells per mg of solid tumor in the T+iRGD+CMP / aPD1@Gel group was nearly 21 times higher than that in the free drug T+iRGD+aPD1+CXCL9 group. Figure 11 F). Furthermore, the drug-co-loaded immunogel also significantly increased the number of endogenous CD8+ T cells per mg of solid tumor, with the T+iRGD+CMP / aPD1@Gel group showing a 6-fold increase compared to the free drug T+iRGD+aPD1+CXCL9 group. Figure 11 G). The gel group not only increased the number of adoptive T cells compared to the free drug group, but also increased the IFN-γ (G) content of adoptive T cells in the T+iRGD+CMP / aPD1@Gel group. Figure 12 AB), Granzyme B ( Figure 12 The expression level of CD8+ T cells was 1.5 to 3.5 times that of endogenous CD8+ T cells in the T+iRGD+CMP / aPD1@Gel group, indicating stronger tumor cell killing ability. Thermosensitive gel also increased the expression level of inguinal lymph nodes compared to free drugs. Figure 13 AB), Spleen ( Figure 13 CD) and blood ( Figure 13 The percentage of adoptive T cells in the EF group was 2.4, 2.6, and 1.8 times that in the free drug group, respectively.

[0200] 2.10 The therapeutic effect of immunomodulatory thermosensitive gel on distal tumors

[0201] To investigate whether co-loaded thermosensitive gel therapy can stimulate a systemic anti-cancer immune response, thereby treating tumors not near the gel and achieving systemic and metastatic therapeutic effects, we designed a distal tumor treatment experiment. B16-OVA was injected into the right back of C57BL / 6 mice to create a "proximal tumor," and a "distal tumor" was injected into the left back. Treatment began when the tumor volume was approximately 55 mm³, and an immunogel was injected near the "proximal tumor." The following groups were established: a blank control group (PBS), a tail vein injection group (CD8+T only), a tail vein injection group (CD8+T+iRGD+aPD1+subcutaneous injection of free CXCL9) (T+iRGD+aPD1+CXCL9), and a tail vein injection group (CD8+T+iRGD+CMP / aPD1@Gel) (T+iRGD+CMP / aPD1@Gel). Figure 14 A). Following treatment, right proximal tumors were successfully eliminated in 87.5% of mice. Figure 14 B). More significantly, the thermosensitive gel also cleared distal tumors in 62.5% of mice, and the average tumor volume in the gel group was only 4% of that in the free drug group ( Figure 14 C). After 60 days of treatment, the survival rate of mice in the T+iRGD+CMP / aPD1@Gel group was 62.5% (5 / 8 mice).Figure 14 E). The above results demonstrate that thermosensitive gel can trigger a systemic immune response, treating systemic or metastatic tumors. Figure 14 It is directed at tumors, rather than being limited to those near the gel injection site.

[0202] 2.11 Immunomodulatory thermosensitive gel promotes the generation of anti-tumor immune memory.

[0203] C57BL / 6 mice that had been cured by the gel group in the previous experiment were subcutaneously inoculated with B16-OVA tumor cells again (90 days later) for a tumor re-challenge experiment. Healthy C57BL / 6 mice of the same age were subcutaneously inoculated with tumor cells as a control group. aPD1 was injected via the tail vein on days 11, 14, 18, 21, 24, and 27 after tumor inoculation. Figure 15 A). 12.5% ​​of the cured mice did not develop tumors after tumor re-challenge, and another 50% of the cured mice had tumors that were completely eliminated by aPD1 after they grew, while every control mouse had a tumor. Ultimately, the average tumor volume in the cured mice was only 17% of that in the control group. Figure 15 (BC). These data indicate that early adoptive T-cell therapy and immunogel treatment can significantly delay tumor recurrence and enhance the response of mice to aPD1 treatment. This demonstrates that immunothermosensitive gel is beneficial for the generation of anti-cancer immune memory, can prevent tumor recurrence, and can significantly improve the therapeutic effect of immune checkpoint inhibitors on recurrent tumors.

[0204] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing chemokine particles, characterized in that: Includes the following steps: 1) The chemokine CXCL9 and the amphiphilic polymer DSPE-PEG-NHS were mixed and reacted to obtain the chemokine-modified form; 2) Mix BSA solution and DTSSP solution to react, and centrifuge the reacted sample solution. Resuspend the solution in PBS to prepare SMP. 3) The chemokine modifier is added to the albumin carrier SMP for binding, thereby obtaining chemokine particles; In step 2), the SMP preparation method can be a two-step or a three-step method. The two-step method includes first adding a portion of BSA solution and DTSSP solution for mixing and reaction, and then adding BSA solution and DTSSP solution to the reaction system to continue the reaction. The three-step method includes first adding a portion of BSA solution and DTSSP solution for mixing and reaction, then adding BSA solution to the reaction system to continue the reaction, and finally adding DTSSP solution to the reaction.

2. The preparation method according to claim 1, characterized in that: The albumin carrier SMP has a particle size of 700~1400nm.

3. A chemokine particle, characterized in that: The chemokine particles are prepared using the preparation method described in any one of claims 1-2.

4. A thermosensitive gel composition, characterized in that: It includes the chemokine particles, PD-1 monoclonal antibody, and thermosensitive hydrogel as described in claim 3.

5. A thermosensitive gel composition, characterized in that: It includes the chemokine particles, PD-1 monoclonal antibody, iRGD peptide, and thermosensitive hydrogel as described in claim 3.

6. Combined immunosuppressive drugs, characterized by: The solution includes the thermosensitive gel composition of claim 4 or 5 applied at the tumor site and the iRGD peptide for intravenous administration.

7. The use of the chemokine particles of claim 3, the thermosensitive gel composition of any one of claims 4-5, and the combined immunomodulatory drug of claim 6 in the preparation of a drug for adoptive cell therapy; wherein, The adoptive cells were CAR-T cells.

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