Preparation and application of a multifunctional nanoformulation for activating an immune response from an in situ tumor vaccine.

Abstract

This invention provides a nanoformulation capable of enhancing the immune response to in situ tumor vaccines. The nanoformulation comprises zoledronic acid, all-trans retinoic acid, and metal ions. Preferably, the metal ion is a lanthanide metal, such as Gd. 3+ Preferred, zoledronic acid: Gd 3+ The mass ratio of all-trans retinoic acid is 4:1:5. The nano-formulation consists of zoledronic acid and Gd... 3+ Zol / Gd-NPs nanocoagulation polymers were formed by mixing in water. All-trans retinoic acid was dissolved in anhydrous ethanol to prepare albumin-encapsulated nanoparticles (A-NPs), which were then mixed with the Zol / Gd-NPs nanocoagulation polymers and sonicated to obtain the final product. These nanoparticles, on the one hand, can utilize zoledronic acid to eliminate macrophages, thereby reversing the inhibition of intratumoral dendritic cells (DCs) by the tumor immunosuppressive microenvironment; on the other hand, all-trans retinoic acid can significantly increase the expression of MHC-I and tumor antigens in tumor cells, thus ensuring the activation of CD8. + T cells can effectively recognize and kill tumors, generating a systemic anti-tumor immune response.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation and application of a multifunctional nano-formulation that activates an in situ tumor vaccine to generate an immune response. Background Technology

[0002] Immunotherapy has revolutionized clinical cancer treatment. As a key component of immunotherapy, cancer vaccines aim to utilize the host's immune system to eliminate tumors. Among these, neoantigen vaccines utilizing tumor mutations have shown promising results in small-scale clinical trials. However, these vaccines face challenges, including the high cost of long-term (2-4 months) neoantigen identification and screening, the spatiotemporal heterogeneity of tumors, and the low immunogenicity of neoantigens within tumors, which severely hinder clinical translation.

[0003] In contrast, in situ tumor vaccines utilize antigens derived from the entire tumor cell during treatment. They do not require recognition or screening and contain all tumor-derived antigens to minimize immune escape. Therefore, in situ tumor vaccines based on whole-tumor antigens represent a promising direction for tumor vaccine research.

[0004] Radiation therapy is a crucial treatment for clinical cancer, affecting approximately 50-60% of cancer patients. Clinical data demonstrate that radiation therapy effectively induces the release of tumor antigens in the irradiated area, leading to in situ vaccine formation and activation of a systemic immune response (distant effects). However, the frequency of radiation-induced distant effects is significantly lower than 1%. Statistical data shows that only 46 cases of distant effects were reported between 1969 and 2014. Even when combined with immune checkpoint inhibitors, the response rate remains unsatisfactory. Therefore, there is an urgent need to develop innovative treatment strategies to enhance radiation-induced distant effects.

[0005] To effectively enhance the distal effect, antigens need to be efficiently presented to dendritic cells (DCs). First, cancer antigens must be taken up and presented by dendritic cells to effectively activate CD8+. + T cells. Secondly, the antigen must be presented directly on the MHC-I receptor of tumor cells in order to activate CD8 cells. + T cells recognize and kill tumors. However, both of these processes are often blocked by the tumor and its immunosuppressive microenvironment. On the one hand, immunosuppressive TAMs can inhibit dendritic cell infiltration and function by secreting a series of cytokines. On the other hand, the low immunogenicity of tumor cells, including the lack of tumor antigens and MHC-I expression, leads to CD8+ inhibition. + T cells cannot be activated to recognize and kill tumors.

[0006] Currently, most research focuses on developing tumor neoantigen vaccines, using several or even dozens of neoantigens as vaccines to activate the body's immune response, hoping to induce tumor-specific T cells that can inhibit tumor growth and thus achieve precise immunotherapy. However, more than 90% of the tumor mutation antigens discovered so far are single-base mutations (SNVs), differing from unmutated proteins in the body by only one amino acid. This high similarity often results in low immunogenicity. Furthermore, clinical antigen prediction is based on biopsy samples from single tumor lesions, and the prediction results cannot encompass all mutation-derived neoantigens from the entire heterogeneous tumor. The tumor immunosuppressive microenvironment (TIME) is also a significant obstacle limiting neoantigen vaccine treatment of solid tumors. For these reasons, the anti-tumor immune response induced by neoantigen vaccines is relatively weak and difficult to effectively inhibit the malignant progression of the entire tumor.

[0007] In contrast, in situ tumor vaccines derived from whole cancer cell lysates eliminate the need for neoantigen recognition and screening, can generate endogenous tumor antigens, and ensure that the generated antigens can be effectively absorbed and processed by sufficient dendritic cells (DCs), providing a promising approach to enhance anti-tumor immunotherapy.

[0008] However, the tumor immunosuppressive microenvironment significantly inhibits the activation of tumor immune responses by in situ tumor vaccines by suppressing the infiltration and function of dendritic cells (DCs). In particular, tumor-associated macrophages (MAMs), a crucial immunosuppressive cell population in the immunosuppressive microenvironment (comprising approximately 30-50% of tumor cell volume, and even exceeding 50% in breast cancer), inhibit DC tumor infiltration and activation through various mechanisms. For example, VEGF, IL-10, and TGF-β secreted by macrophages can inhibit DC migration and function, respectively, preventing them from activating radiotherapy-induced anti-tumor immune responses. More importantly, numerous studies have shown that radiotherapy further promotes TAM infiltration, a significant factor contributing to the inability of DCs to effectively activate anti-tumor immune effects. Furthermore, tumor cells themselves can reduce their autoimmunity by decreasing tumor-specific antigens or MHC-I, leading to immune escape. In this situation, even if tumor-specific CD8+ is produced... + T cells are also unable to recognize and kill tumors.

[0009] Furthermore, the tumor immunosuppressive microenvironment is composed of numerous and complex inhibitory cells and inhibitory factors. It is difficult for a single cytokine or checkpoint inhibitor to reverse the inhibition of dendritic cells (DCs) by the tumor immunosuppressive microenvironment, and it may also be accompanied by fatal toxic side effects.

[0010] Therefore, there is an urgent need to develop a synergistic therapeutic strategy to eliminate macrophages to reverse the suppression of intratumoral dendritic cells (DCs) by the tumor immunosuppressive microenvironment, while simultaneously significantly increasing MHC-I expression in tumor cells to ensure the activation of CD8.+ T cells can effectively recognize and kill tumors to improve systemic immune responses. Summary of the Invention

[0011] This invention constructs a multifunctional nanoformulation that activates an in situ tumor vaccine to generate an immune response. On one hand, zoledronic acid can overcome the problem of the tumor immunosuppressive microenvironment by eliminating macrophages. On the other hand, ATRA significantly enhances the expression of MHC-I and tumor antigens in tumor cells, addressing the issue of low immunogenicity of tumor cells. This strategy activates cytotoxic T lymphocytes by triggering a stronger antigen presentation, thereby effectively recognizing and killing tumors and inducing a systemic anti-tumor immune response.

[0012] The technical solution of this invention is as follows:

[0013] The first objective of this invention is to provide a nanoformulation capable of enhancing the immune response to an in situ tumor vaccine, the nanoformulation comprising: zoledronic acid, all-trans retinoic acid, and metal ions.

[0014] In the nano-formulation, zoledronic acid forms a coordination polymer with metal ions, and the coordination polymer encapsulates all-trans retinoic acid.

[0015] Furthermore, the metal ion is a lanthanide metal, and preferably, the framework structure is Gd. 3+ .

[0016] Furthermore, the nano-formulation contains zoledronic acid: Gd 3+ The mass ratio of all-trans retinoic acid is 2-6:0.5-2:3-7. Preferably, the nano-formulation contains zoledronic acid:Gd 3+ The mass ratio of all-trans retinoic acid is 4:1:5.

[0017] In this invention, albumin in the nano-formulation acts as a carrier to encapsulate the all-trans retinoic acid system. There are no special restrictions on the amount of albumin used; it is sufficient to complete the encapsulation and form an emulsion-like ATRA colostrum (A-NPs).

[0018] In a particular embodiment, the nanoformulation contains zoledronic acid: Gd 3+ The mass ratio of all-trans retinoic acid is 2-6:0.5-2:3-7. Preferably, the nano-formulation contains zoledronic acid:Gd 3+ The mass ratio of all-trans retinoic acid is 4:1:5.

[0019] Furthermore, the nano-formulation is zoledronic acid, Gd 3+Zol / Gd-NPs nano-coordination polymers were formed by mixing in water; all-trans retinoic acid was dissolved in anhydrous ethanol to prepare albumin-encapsulated A-NPs formulations; then, Zol / Gd-NPs and A-NPs nanoparticles were thoroughly mixed to obtain the coordination polymer AZ-NPs.

[0020] Preferably, the concentration of all-trans retinoic acid in anhydrous ethanol in the nano-formulation is 0.5%.

[0021] A second objective of this invention is to provide a method for preparing the aforementioned nano-formulation, wherein the method comprises:

[0022] (1) Mix zoledronic acid aqueous solution with GdCl3·6H2O aqueous solution thoroughly, stir, and centrifuge to collect the precipitate; wash the obtained precipitate with pure water, and centrifuge to collect Zol / Gd-NPs nano-coordination polymer;

[0023] (2) Weigh all-trans retinoic acid and dissolve it in anhydrous ethanol, then mix it thoroughly with PBS containing albumin to obtain a mixed solution; sonicate the mixed solution to obtain ATRA colostrum (A-NPs);

[0024] (3) The ATRA colostrum obtained in step (2) is used to resuspend the Zol / Gd-NRs nanoscale coordination polymer obtained in step (1), and ultrasonic treatment is performed to obtain the nano-formulation.

[0025] In step (1), the bisphosphonic acid group in the Zol structure acts as a bidentate ligand, reacting with Gd in the GdCl3·6H2O aqueous solution. 3 + Metal coordination bonds are formed, and Zol / Gd-NRs nanoscale coordination polymers (Z-NPs) are synthesized and constructed through a coordinated self-assembly method.

[0026] In step (2), ATRA nanoparticles coated with albumin (HSA) are used to form ATRA colostrum (A-NPs);

[0027] In step (3), the carboxyl groups of albumin can react with the Gd in the Z-NPs nanoparticles prepared in step (1). 3+ The coordination between ions allows Z-NPs to exhibit a significant adaptive encapsulation ability for anionic nanomaterials and proteins. Based on this, Gd in HSA carboxyl groups and Z-NPs... 3+ Driven by ion interactions, ATRA@HSA was encapsulated into Z-NPs to prepare AZ-NPs.

[0028] Preferably, the concentration of all-trans retinoic acid in anhydrous ethanol in step (2) is 0.5%;

[0029] A third objective of this invention is to provide the use of zoledronic acid and all-trans retinoic acid in combination in the preparation of a medicament capable of enhancing the immune response to an in situ tumor vaccine.

[0030] Furthermore, the mass ratio of zoledronic acid to all-trans retinoic acid is 2-6:3-7; preferably, the mass ratio of zoledronic acid to all-trans retinoic acid is 4:5.

[0031] A fourth objective of this invention is to provide the use of the aforementioned nanoformulations in the preparation of medicaments capable of enhancing the immune response to tumor in situ vaccines.

[0032] Furthermore, intravenous infusion of the drug, combined with radiotherapy, can enhance the immune response generated by the tumor in situ vaccine.

[0033] In a specific embodiment, the cancer treatment protocol of this invention involves a first tail vein infusion of a multifunctional nano-formulation, followed by synergistic radiotherapy at a dose of 6 Gy 24 hours after administration. Two administrations and two radiotherapy sessions constitute one complete treatment course.

[0034] Furthermore, the tumor is a solid tumor or a solid tumor with metastases.

[0035] In the nano-formulation system of this invention:

[0036] a. The zoledronic acid can eliminate macrophages, reverse the inhibition of intratumoral dendritic cells by the tumor immunosuppressive microenvironment, and solve the problem of the tumor immunosuppressive microenvironment;

[0037] b. The all-trans retinoic acid can significantly increase MHC-I expression in tumor cells, thereby ensuring the activation of CD8. + T cells can effectively recognize and kill tumors, and can also induce tumor differentiation, thereby enhancing the immunogenicity of tumor cells;

[0038] c. By synergistically combining zoledronic acid and all-trans retinoic acid, cytotoxic T lymphocytes are activated through a stronger antigen presentation effect, thereby effectively recognizing and killing tumors and triggering a systemic anti-tumor immune response.

[0039] d. Nanoparticles, as targeted therapy carriers, significantly enhanced CD8. + The proportion of T cells infiltrating tumors on the radiotherapy side and in distant tumors can be increased, and the percentage of effector memory T cells in the spleen can be increased. This can improve the solubility of poorly soluble drugs and reduce toxicity, improve the delivery efficiency of molecularly targeted drugs, and enhance the therapeutic effect.

[0040] e. Radiotherapy can induce tumor cell death and generate endogenous tumor antigens; the above-mentioned nano-preparations combined with radiotherapy can enhance drug efficacy while reducing toxic side effects, making the treatment process safer and more effective;

[0041] f. This invention significantly inhibits primary, distal, and metastatic tumor lesions in various immunocold tumor models such as CT26 and 4T1. More importantly, the systemic antitumor immune response induced by RT+AZ-NPs treatment can effectively prevent multiple tumor recurrences over a long period (109 days).

[0042] The radiotherapy method in this invention is one or more of the following: α, β, γ rays produced by radioactive isotopes, X-rays produced by various X-ray therapy machines or accelerators, charged particles such as electrons, protons and heavy ions;

[0043] The combination therapy of the present invention is used to enhance tumor immunotherapy, including any one of solid tumors or solid tumors with metastases such as melanoma, colorectal cancer, breast cancer, liver cancer, glioma, bile duct cancer, and pancreatic cancer.

[0044] The beneficial effects of this invention are as follows:

[0045] The multifunctional nanoformulation constructed in this study can, on the one hand, utilize zoledronic acid to eliminate macrophages, thereby reversing the inhibition of intratumoral dendritic cells by the tumor immunosuppressive microenvironment; on the other hand, all-trans retinoic acid can significantly increase the expression of MHC-I and tumor antigens in tumor cells, thus ensuring the activation of CD8. + T cells can effectively recognize and kill tumors, generating a systemic anti-tumor immune response.

[0046] The technical solution of this invention is based on drug delivery technology, a comprehensive system for regulating drug distribution within the body, which plays an important role in many diseases such as tumors and inflammation. Compared with traditional drug delivery methods, nanomedicines can utilize the high permeability and long retention effect (EPR effect) of solid tumors to accumulate in tumor tissue, endowing the system with different functions such as targeting, thereby improving drug efficacy while reducing toxic side effects, making the treatment process safer and more effective.

[0047] The AZ-NPs constructed in this invention are readily available and the preparation method is simple. The Z-NPs used in this invention are a functionalized nanoparticle drug delivery platform capable of flow cytometry analysis and cell imaging by encapsulating fluorescent dyes FITC and Cy5.5; they can also be further loaded with chemotherapeutic drugs (doxorubicin, mitoxantrone, etc.), photosensitizers (alcium blue, photoporphyrin), and photothermal agents (ICG, etc.) to achieve combined therapy. It is expected that this nanoparticle drug delivery and therapeutic platform, combined with other therapies, will be used in the future for anti-tumor immunotherapy. Attached Figure Description

[0048] Figure 1 Characterization of AZ-NPs nanoscale coordination polymers; Figure 1 a is a transmission electron microscope (TEM) image of AZ-NPs (scale bar 60 nm). Figure 1 b is the particle size distribution diagram of AZ-NPs;

[0049] Figure 2 The potential of AZ-NPs nanoscale coordination polymers is given, where A-NPs and Z-NPs are control groups;

[0050] Figure 3 To quantify the encapsulation efficiency of AZ-NPs nanoscale coordination polymers using ultraviolet spectrophotometry; Figure 3 'a' is the Zol standard curve. Figure 3 b is the ATRA standard track. Figure 3 c represents the Zol and ATRA content results in AZ-NPs prepared quantitatively using a UV spectrophotometer;

[0051] Figure 4 To assess the stability of AZ-NPs nanoscale coordination polymers, the particle size and dispersion index (PDI) of AZ-NPs were determined over a 7-day period.

[0052] Figure 5 To enhance the expression of tumor antigens and MHC-I molecules in AZ-NPs; Figure 5 a. Relative expression levels of different genes in the AZ-NPs-treated group and the Saline-treated group; Figure 5 b shows the expression of the neoantigen in the Saline and AZ-NPs treatment groups; Figure 5 c represents the expression of damage-associated molecular patterns (DAMPs) in the Saline and AZ-NPs treatment groups; Figure 5 de represents the gating strategy and quantitative results of flow cytometry detection of MHC-I;

[0053] Figure 6 The metabolic distribution of IZ-NPs in mice; Figure 6 Image a is a representative image of tumor, heart, liver, spleen, lung and kidney imaging obtained from dissection of a mouse 48 hours after intravenous injection; Figure 6 b represents the quantitative results of fluorescence intensity in the organ (n=5); Figure 6 c is a representative image of mouse fluorescence imaging at different time points;

[0054] Figure 7 To initiate DC-mediated in situ tumor antigen presentation for AZ-NPs+RT; Figure 7 a) RNA sequencing treatment regimen: Tumor tissues from patients treated with RT and AZ-NPs+RT were collected 3 days after treatment and RNA-seq gene analysis was performed. Figure 7 b. Volcano plot of differentially expressed genes: Orange and blue areas represent genes that were significantly upregulated or downregulated by AZ-NPs+RT treatment compared to tumors treated with RT (n=3); Figure 7c.GO enrichment analysis revealed the three gene pathways with the highest enrichment levels;

[0055] Figure 8 The changes in tumor volume in the B16F10 subcutaneous tumor model;

[0056] Figure 9 To enhance the immunotherapeutic effect of AZ-NPs+RT in CT26 tumor-bearing mice, Figure 9 a. Treatment regimen for CT26 tumor-bearing mice Figure 9 bc represents the average tumor growth curves for the primary tumor and the distal tumor, respectively. Figure 9 d represents CD8 in the distal tumor + T cells and CD4 + Representative flow cytometry image of T cells Figure 9 ef represents CD8 in the primary tumor and the distal tumor, respectively. + T cell ratio Figure 9 gh represents the quantitative analysis of CD8+ immunofluorescence staining in distal tumors. + T cells and CD4 + T cell results, Figure 9 i represents immunofluorescence staining of the distal tumor;

[0057] Figure 10 AZ-NPs+RT enhances DC cell activation. Figure 10 a represents the gating strategy for flow cytometry analysis of DCs. Figure 10 bc represents flow cytometry analysis of CD11c in tumor-draining lymph nodes. + Percentage of DCs and their respective flow cytometry plots (n=5). Figure 10 de is the tumor draining lymph node CD80 + CD86 + Representative flow cytometry plots and quantitative plots of cell percentage (n=5). Figure 11 The proportion of macrophages in CT26 tumors after AZ-NPs+RT treatment. Figure 11 a is a representative image from flow cytometry. Figure 11 b represents quantitative analysis (n=6);

[0058] Figure 12 The expression of MHC-I in CT26 tumor cells after AZ-NPs+RT treatment. Figure 12 'a' represents the circle-gate strategy. Figure 12 b represents the quantitative analysis results (n=6);

[0059] Figure 13 The treatment endpoint after AZ-NPs+RT therapy is the level of spleen effector memory T cells (TEM). Figure 13 'a' represents the circle-gate strategy. Figure 13 b represents the quantitative analysis results (n=5);

[0060] Figure 14 It induces long-term and tumor-specific immunosuppression in AZ-NPs+RT. Figure 14 a represents the treatment regimen used in the CT26-cured mouse immune memory assay. Figure 14 b. Percentage of mice without tumors after the first tumor challenge. Figure 14 c represents the average tumor growth curve during the three CT26 tumor re-challenge periods. Figure 14 d represents the tumor growth curve of CT26. Figure 14 e represents the 4T1 tumor growth curve. Figure 14 f shows microscopic images of CT26 tumor cells co-incubated with different concentrations of spleen cells from cured mice (red arrows indicate cell aggregates formed by spleen cells and tumor cells). Figure 14 g represents the cell viability of CT26 or 4T1 tumor cells and spleen cells from CT26-cured mice after 24 hours of incubation (x-axis represents the ratio of spleen cells to tumor cells (n=5)).

[0061] Figure 15 To assess the efficacy of AZ-NPs+RT in enhancing immunotherapy for 4T1 tumors. Figure 15 a represents the in situ tumor volume measurement results. Figure 15 b represents the distal tumor volume measurement results (n=8);

[0062] Figure 16 This describes the lung metastasis status in the 4T1 drug efficacy model. Figure 16 Image a is a panoramic scan of a H&E section of lung tissue (the arrow points to the metastatic lesion; scale bar is 100 μm). Figure 16 b shows images of lung tissue from each group. Figure 16 c represents the quantitative results of lung metastases;

[0063] Figure 17 For the safety evaluation of AZ-NPs+RT treatment, Figure 17 ac represents serum biochemical indicators in CT26 tumor-bearing mice 14 days after different treatments, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN). Figure 17 dh represents the complete blood count (CBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet count (PLT) of CT26 tumor-bearing mice 14 days after different treatments. Figure 17 Image i shows hematoxylin and eosin (H&E) staining of the heart, liver, spleen, lungs, and kidneys.

[0064] Figure 18 A schematic diagram illustrating how AZ-NPs+RT treatment enhances the efficacy of immunotherapy. Detailed Implementation

[0065] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0066] As described in the embodiments:

[0067] AZ-NPs refer to nano-formulations formed by encapsulating a polymer of Zol and Gd in colostrum formed by ATRA and HSA.

[0068] IZ-NPs refer to nanoparticles formed by labeling Z-NPs with indocyanine green (ICG). The preparation method is as follows: ICG (2 mg) is dissolved in 2.5 mM zoledronic acid solution (6.0 mL). Then, 4 mL of 2.5 mM GdCl3·6H2O aqueous solution is slowly added, and the mixture is incubated overnight. Next, the precipitate at the bottom is collected by centrifugation (12000 × g, 30 min) and washed three times with pure water. Finally, the precipitate is resuspended in PBS containing 3% HSA (10 mL) and sonicated four times (8 min) at 950 W and 40% intensity at 25 °C to obtain IZ-NPs.

[0069] Z-NPs refer to polymers formed by Zol and Gd, prepared as follows: 4.0 mL of a 2.5 mM GdCl3·6H2O aqueous solution is slowly added to 6.0 mL of a 2.5 mM zoledronic acid solution, mixed thoroughly, and stirred overnight. The precipitate is then collected by centrifugation (12000 × g, 30 min) and washed three times with pure water. It is resuspended in 10 mL of PBS containing 3% HSA and sonicated five times (10 min) at 25°C with 950 W and 30% intensity. A-NPs refer to colostrum formed by ATRA and HSA, prepared as follows: 5 mg of ATRA is dissolved in 1.0 mL of anhydrous ethanol, mixed with 9 mL of PBS containing 3% HSA, and sonicated four times (8 min) at 25°C with 950 W and 30% intensity.

[0070] ATRA stands for all-trans retinoic acid.

[0071] Zol refers to zoledronic acid.

[0072] Example 1: Preparation of AZ-NPs

[0073] (1) Mix an aqueous solution of 2.5 mM zoledronic acid (4 mg, 6.0 mL) with an aqueous solution of 2.5 mM GdCl3·6H2O (4.0 mL, Gd... 3+Mix thoroughly (1 mg by mass), stir overnight, and collect the precipitate by centrifugation (12000×g, 30 min). Wash the obtained precipitate three times with pure water (1.0 mL), and collect the Zol / Gd-NRs nanoscale coordination polymer by centrifugation (12000×g, 30 min).

[0074] (2) Weigh 5 mg of all-trans retinoic acid and dissolve it in 1 mL of anhydrous ethanol. Then mix it thoroughly with 1.5 mL of 20% albumin and 7.5 mL of sterile PBS. Sonicate the above mixture (950 W, 30% intensity, 25 °C, 10 min) to obtain ATRA colostrum.

[0075] (3) Finally, the Zol / Gd-NRs nanoscale coordination polymer collected by the last washing and centrifugation was resuspended in ATRA colostrum and subjected to ultrasonic treatment (950W, 30% intensity, 25℃, 8 minutes) to obtain AZ-NPs multifunctional nano-formulation, namely the nano-formulation that can enhance the immune response of tumor in situ vaccines.

[0076] Example 2 Characterization of AZ-NPs

[0077] The particle size and morphology of the AZ-NPs prepared in Example 1 were observed using a transmission electron microscope (TEM). An appropriate amount of ultrasonically dispersed AZ-NPs was taken, dropped onto a copper grid, allowed to stand and dry, and then placed under a transmission electron microscope to observe the field of view and take pictures.

[0078] The AZ-NPs constructed in this invention are multifunctional nano-formulations with rod-like structures approximately 30–150 nm in length and a zeta potential of -5.0–-7.5 mV. The encapsulation efficiency of Zol is 57.01–70.31%, and that of ATRA is 88.67–97.00%. ATRA is effectively loaded, and the particle size and PDI change are small under storage conditions at 4 °C, indicating good stability.

[0079] Typical images such as Figure 1 As shown in figure a, the results show that the nano-formulation has a rod-like structure with a length of approximately 70.40 nm. Figure 1 b is the particle size distribution diagram of AZ-NPs, showing that the particle size of AZ-NPs is around 70nm.

[0080] Freshly prepared AZ-NPs nanoparticles were analyzed using a particle size analyzer. Typical results are as follows: Figure 2 As shown, the results indicate that the AZ-NPs zeta potential is -6.50 mV, and since both Z-NPs and A-NPs are negatively charged, this suggests that they have good stability and low cytotoxicity.

[0081] Zol and ATRA in the prepared AZ-NPs were quantified using a UV spectrophotometer. Typical results are shown below. Figure 3 As shown, the encapsulation efficiency of Zol was 64.65%, while that of ATRA was 92.10%.

[0082] After initial size and polydispersity index (PDI) of the newly prepared AZ-NPs were tested, the samples were stored in a 4°C refrigerator in the dark and monitored for seven consecutive days to characterize the stability of the coordination polymer.

[0083] The results are as follows Figure 4 As shown, AZ-NPs exhibited minimal changes in particle size and PDI under storage conditions of 4℃, indicating good stability.

[0084] Example 3: AZ-NPs enhance the expression of tumor antigens and MHC-I molecules.

[0085] RNA sequencing and a CT26 tumor neoantigen library were used to detect the expression of CT26 neoantigen after AZ-NPs treatment. CT26 tumor cells were cultured at a density of 2.5 × 10⁶ cells per well. 5 Tumor cells were seeded at a density of 10 μM in 6-well plates and incubated for 24 hours. They were then treated with AZ-NPs ([ATRA] = 10 μM) for 72 hours. After incubation, tumor cells were collected for RNA sequencing analysis (n = 3). Cells with a p-value less than 0.05 were selected for neoantigen analysis. Finally, CT26 neoantigens were evaluated based on CT26 tumor mutations and the neoantigen library. Figure 5 As shown in figure a, multiple tumor mutational antigens significantly increased after AZ-NPs treatment. These antigens may be recognized as exogenous proteins, thereby triggering an immune response. Figure 5 As shown in b, four tumor neoantigens were also significantly increased; these tumor neoantigens have been shown to activate tumor-specific immune responses. Figure 5 As shown in c, AZ-NPs treatment also significantly increased the expression of damage-associated molecular pattern proteins (DAMPs), which may act as an endogenous immune adjuvant to stimulate DCs.

[0086] The cells were collected and analyzed using flow cytometry. For example... Figure 5 As shown in de, AZ-NPs treatment can increase the expression level of MHC-I in tumor cells.

[0087] In summary, AZ-NPs treatment can increase the expression of tumor antigens, DAMPs, and MHC-I, thereby enhancing the anti-tumor immune response.

[0088] Example 4: Study on the tumor targeting and accumulation of AZ-NPs

[0089] Indocyanine green (ICG) fluorescent dye was dissolved in zoledronic acid aqueous solution, then thoroughly mixed with GdCl3·6H2O aqueous solution, stirred overnight, centrifuged, washed, and centrifuged again to obtain Zol / Gd-NRs nanoprecipitates encapsulating ICG. After resuspension and sonication, IZ-NPs multifunctional nanoformulation was obtained, which is to add ICG to the zoledronic acid aqueous solution described in step (1) of Example 1, with the remaining operations the same as in Example 1. CT26 tumor-bearing mice were treated with IZ-NPs via tail vein injection. Fluorescence imaging was performed using a small animal in vivo bioluminescence imaging system. Whole-body fluorescence images of mice were taken at 0h, 12h, 24h, 38h, and 48h after administration.

[0090] The results are as follows Figure 6 As shown, IZ-NPs accumulated in in situ tumor tissue will return to the lungs via systemic venous blood and be metabolized and excreted from the body. IZ-NPs are almost undetectable in other normal organs, indicating that AZ-NPs have good tumor targeting and retention effects.

[0091] Example 5

[0092] Three days after treatment with saline + RT and AZ-NPs + RT (NPs×2, RT×2), tumors were collected according to sequencing requirements. RNA quality was assessed using an Agilent 2100 Expert bioanalyzer, with library preparation and sequencing performed by Biotech Corporation (Shanghai, China) on an Illumina Nova Seq 6000 platform. The raw data were processed, and differentially expressed genes between Saline + RT and AZ-NPs + RT tumor tissues were compared through gene pathway enrichment analysis. Genes significantly upregulated or downregulated by AZ-NPs + RT treatment compared to RT-treated tumors were identified. Figure 7 b).

[0093] The results are as follows Figure 7 As shown in c, a pathway analysis based on GO enrichment was performed on differentially expressed genes. Compared with RT, the first three pathways in the AZ-NPs+RT group were significantly enriched in terms of exogenous antigens, peptide antigens, antigen processing and presentation, indicating that it can reverse the inhibition of intratumoral dendritic cells by the tumor immunosuppressive microenvironment.

[0094] Example 6: Treatment of B16-OVA tumor-bearing mice with AZ-NPs

[0095] BALB / c female mice were subcutaneously injected with 5×10 5 One melanoma B16-OVA cell was used, and the mouse tumor was allowed to grow to 120 mm. 3They were divided into 5 groups according to Saline, Saline+RT, Z-NPs+RT, A-NPs+RT, and AZ-NPs+RT.

[0096] Among them, the Saline group received 200 μL of normal saline via tail vein injection, the Saline+RT group received 200 μL of normal saline via tail vein injection and radiotherapy, the Z-NPs+RT group received 200 μL of Z-NPs via tail vein injection and radiotherapy, the A-NPs+RT group received 200 μL of A-NPs via tail vein injection and radiotherapy, and the AZ-NPs+RT group received 200 μL of AZ-NPs via tail vein injection and radiotherapy.

[0097] The mice were treated on days 7 and 12 after tumor inoculation. The nanomedicine was injected via the tail vein, followed by radiation therapy at a dose of 6 Gy × 2 at the tumor site 24 hours later. Radiation therapy was administered every 5 days for a total of 2 sessions. Tumors were collected on day 4 after the second radiation therapy. During this period, tumor volume and body weight were measured and recorded daily.

[0098] The results are as follows Figure 8 As shown, the Saline group had the largest tumor volume, the Saline+RT group showed a certain anti-tumor effect, and although the Z-NPs+RT and A-NPs+RT groups had stronger tumor-suppressing effects than the Saline+RT group, there was no significant difference. The AZ-NPs+RT group had the smallest tumor volume and its anti-tumor effect was significantly stronger than other groups. The tumor volume growth curve of the B16-OVA unilateral melanoma-bearing mouse model showed that AZ-NPs could synergistically inhibit the growth of mouse melanoma with radiotherapy and produce the efficacy of in situ vaccine with radiotherapy, which was significantly better than treatment with zoledronic acid or all-trans retinoic acid alone.

[0099] Example 7: Treatment of CT26 tumor-bearing mice with AZ-NPs

[0100] BALB / c female mice were subcutaneously injected with 5×10 5 CT26 cells were collected, and mouse tumors were allowed to grow to 120 mm. 3 The mice were divided into five groups according to Saline, Saline+RT, Z-NPs+RT, A-NPs+RT, and AZ-NPs+RT, with the treatment method for each group being the same as in Example 5. The mice were treated on days 10 and 17 after tumor inoculation. The nanomedicine was injected via the tail vein, followed by radiotherapy at a dose of 6 Gy × 2 at the tumor site 24 hours later. Radiotherapy was administered once a week for a total of two sessions. On the day following the second radiotherapy session, the mice were subcutaneously inoculated with 1 × 10⁻⁶ N·m² of the nanomedicine. 5CT26 cells were used to form distal tumors. Primary tumors were collected from mice on day 5 following the second radiotherapy. On day 14 after distal tumor inoculation, mice received a tail vein injection of AZ-NPs, and distal tumors were collected on day 7 post-treatment. Tumor volume and body weight were measured and recorded daily during this period.

[0101] The results are as follows Figure 9-12 As shown, where, Figure 9 The immunotherapeutic effect of AZ-NPs+RT in CT26 tumor-bearing mice was enhanced, showing that AZ-NPs can synergistically increase CD8 levels with radiotherapy. + T cells and CD4 + The infiltration ratio of T cells in in situ and distant tumor tissues can inhibit tumor growth and exert a systemic anti-tumor effect.

[0102] Figure 10 To demonstrate the synergistic effect of AZ-NPs on the activation of dendritic cells (DCs), synergistic radiotherapy showed that AZ-NPs can promote the release of tumor-associated antigens from tumor tissue, activate the maturation of DCs, and produce the efficacy of an in situ vaccine. The activation effect was significantly better than that of zoledronic acid or all-trans retinoic acid alone.

[0103] Figure 11 The proportion of macrophages in CT26 tumors after AZ-NPs+RT treatment showed that, compared with the Saline+RT group and the A-NPs group, AZ-NPs synergistic radiotherapy could significantly inhibit the infiltration of macrophages in the tumor and reverse the tumor immunosuppressive microenvironment.

[0104] Figure 12 The study compared AZ-NPs + RT therapy to MHC-I expression in CT26 tumor cells, showing that compared to the Saline + RT group and the Z-NPs group, AZ-NPs combined with radiotherapy significantly promoted MHC-I expression in the tumor and increased the expression of CD8+ cells. + The opportunity for T cells to recognize and kill.

[0105] In summary, the growth curves of the primary tumor and distal tumor volume in mice, as well as the size of the primary and distal tumors and the corresponding tumor weight quantification maps after treatment, indicate that AZ-NPs combined with radiotherapy stimulated systemic immunity in mice, effectively inhibited the growth of distal tumors of CT26 colorectal cancer, and promoted the production of distal radiotherapy effects. This suggests that AZ-NPs synergistic radiotherapy can slow down the growth of the primary tumor in mice and significantly inhibit the growth of distal tumors.

[0106] Example 8

[0107] Mice euthanized by cervical dislocation were dissected, and subcutaneous tumors and spleens were removed. After pressing, grinding, filtering, centrifugation, and resuspending, spleen cells were obtained. PE-CD3, FITC-CD8a, APC-CD44, PerCP / Cy5.5-CD62L, and a mixed antibody were added to the spleen cells. The proportions of effector memory T cells (TEM) and central memory T cells (TCM) in the mouse spleen were analyzed by flow cytometry. The results are as follows: Figure 13 As shown, the results indicate that compared to the Saline, Saline+RT, and Z-NPs groups, AZ-NPs synergistic radiotherapy can significantly promote the development of CD8+ T cells into memory T cells in the spleen, thereby preventing tumor recurrence.

[0108] like Figure 14 As shown in a, for cured mice, a cycle of 1×10⁻⁶ was performed every 28 days. 5 CT26 cells were injected subcutaneously into cured mice to challenge the tumor, with untreated mice serving as controls. Mice injected subcutaneously with tumor cells received no further treatment.

[0109] During this period, the tumor volume of the mice was measured and recorded daily, and the results were as follows: Figure 14 As shown in the results, the cured mice developed specific immune memory against CT26 tumors, but had no significant inhibitory effect on 4T1 tumors.

[0110] CT26 and 4T1 cells were seeded in 96-well plates. Mouse spleens were harvested using the same method as above to obtain spleen cells. After counting the spleen cells, the densities of CT26 and 4T1 cells were adjusted. The concentration of mouse spleen effector T cells was set, and they were mixed with target cells (tumor cells) at ratios of 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, and co-cultured at 37°C and 5% CO2 for 24 hours. After 24 hours, the supernatant was collected, and CCK-8 was added to each well to detect cell viability.

[0111] After incubation for a suitable period, cell morphology was observed using an optical microscope and absorbance was measured at a wavelength of 450 nm. The results are as follows: Figure 14 As shown in fg, the results indicated that after treatment, spleen cells and CT26 cells from the tumor formed significant aggregates. More importantly, the viability of CT26 cells was significantly reduced, while the viability of 4T1 cells remained unchanged after 24 hours of co-incubation with spleen cells. This suggests that the immune memory generated by AZ-NPs+RT treatment is tumor-specific and durable.

[0112] In summary, the results indicate that AZ-NPs combined with radiotherapy significantly improve the immunotherapy effect of CT26 tumors after radiotherapy and establish a specific anti-tumor immune memory effect.

[0113] Example 9: Treatment of 4T1 tumor-bearing mice with AZ-NPs

[0114] BALB / c female mice were subcutaneously injected with 5×10 5 4 T1 cells were collected and mouse tumors were allowed to grow to 120 mm. 3 The mice were divided into three groups according to their dosage: Saline, Saline+RT, and AZ-NPs+RT. Treatment was administered on days 6 and 11 post-tumor inoculation. The nanomedicine was injected via the tail vein, followed by radiotherapy at a dose of 6 Gy × 2 radiated to the tumor site 24 hours later. Radiotherapy was repeated every 5 days for a total of two sessions. On the day following the second radiotherapy session, mice were subcutaneously inoculated with 1 × 10⁻⁶ N·m³ of nanoparticles. 5 Four T1 cells were injected to form a distal tumor. On day 5 post-tumor inoculation, mice were treated with a single tail vein injection of AZ-NPs. Thirty-two days later, the primary lesion, distal tumor, and surrounding skin tissue were surgically removed. Postoperatively, mouse body weight and survival were monitored, and whole lungs were collected one week post-surgery. Throughout this period, tumor volume and body weight were measured and recorded daily.

[0115] The results are as follows Figure 15-16 As shown, compared with Saline and Saline+RT, the AZ-NPs+RT group significantly inhibited the growth of both in situ and distant tumors. Figure 15 ), and can inhibit 4T1 tumor metastasis ( Figure 16 ).

[0116] In summary, AZ-NPs combined with radiotherapy can extend the in situ therapeutic effect of radiotherapy to distant metastatic lesions and effectively inhibit lung metastasis in 4T1 breast cancer mice.

[0117] Example 10: In vivo safety study of AZ-NPs

[0118] CT26 tumor-bearing mice that underwent complete treatment (two sessions of nanomedicine + two sessions of radiotherapy) had their orbital blood collected for complete blood count and serum biochemical analysis after treatment. The biosafety of AZ-NPs was evaluated using these blood count and serum biochemical analyses.

[0119] The results are as follows Figure 17 As shown in ac, there were no significant differences in serum biochemical indicators between mice injected with AZ-NPs via the tail vein and the control group, and the liver and kidney function indicators of the mice were basically similar to those of the control group.

[0120] The results are as follows Figure 17 As shown in dh, there were no significant differences in the blood routine indicators of mice treated with AZ-NPs combined with radiotherapy compared with the control group, and all were within the normal reference range.

[0121] H&E staining analysis was performed on the heart, liver, spleen, lung, kidney, and tumor tissue of the treated mice. The results are as follows: Figure 17 As shown in i, the H&E staining results of the major organs and tissues of mice in each experimental group were not significantly different from those in the control group, indicating that tail vein injection of AZ-NPs does not cause toxic side effects.

[0122] Both free Zol and ATRA have certain toxicity to the body. Common adverse reactions of Zol include gastrointestinal reactions and kidney damage. Kidney function should be checked before Zol injection. It is not recommended for patients with severe renal failure. Administration should be at least 15 minutes after injection, and the dose should not exceed 5 mg. Common adverse reactions of ATRA include dry lips and skin with desquamation, and gastrointestinal reactions. It carries certain teratogenic risks and potential safety hazards.

[0123] The above-mentioned blood routine and serum biochemical indicators were used to evaluate the in vivo safety of AZ-NPs of the present invention. The normal blood routine and serum biochemical indicators proved that AZ-NPs have excellent blood safety in vivo. The biosafety was evaluated by H&E staining of the major organs of mice in each group. The normal H&E staining of the major organs of mice in each group proved that AZ-NPs have good biosafety, specifically: AZ-NPs have good blood safety and no hepatotoxicity or nephrotoxicity.

[0124] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanoformulation capable of enhancing the immune response to an in situ tumor vaccine, characterized in that, The nano-formulation comprises: zoledronic acid, all-trans retinoic acid, and metal ions; wherein, zoledronic acid forms a coordination polymer with the metal ions, and the coordination polymer encapsulates all-trans retinoic acid; the metal ion is Gd. 3+ The nano-formulation is zoledronic acid and Gd. 3+ Zol / Gd-NPs nano-coordination polymers were formed by mixing in water; all-trans retinoic acid was dissolved in anhydrous ethanol to prepare albumin-encapsulated A-NPs formulations; then, Zol / Gd-NPs and A-NPs nanoparticles were thoroughly mixed to obtain the coordination polymer AZ-NPs. Zoledronic acid in the nano-formulation: Gd 3+ The mass ratio of all-trans retinoic acid is 4:1:

5.

2. The nano-formulation according to claim 1, characterized in that, The concentration of all-trans retinoic acid in anhydrous ethanol is 0.5%.

3. The method for preparing the nano-formulation according to any one of claims 1 to 2, characterized in that, The method is as follows: (1) Mix zoledronic acid aqueous solution with GdCl3•6H2O aqueous solution thoroughly, stir, and collect the precipitate by centrifugation; wash the obtained precipitate with pure water, and collect it by centrifugation to obtain Zol / Gd-NPs nano-coordination polymer; (2) Weigh all-trans retinoic acid and dissolve it in anhydrous ethanol, then mix it thoroughly with PBS containing albumin to obtain a mixed solution; sonicate the mixed solution to obtain ATRA colostrum, i.e., A-NPs preparation; (3) The ATRA colostrum obtained in step (2) is used to resuspend the Zol / Gd-NRs nanoscale coordination polymer obtained in step (1), and ultrasonic treatment is performed to obtain the nano-formulation.

4. The use of the nano-formulation of claim 1 in the preparation of a drug that can enhance the immune response of an in situ tumor vaccine, wherein the drug, when administered intravenously and combined with radiotherapy, can enhance the immune response of the in situ tumor vaccine, wherein the tumor is melanoma, colorectal cancer, or breast cancer.

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