Tumor in-situ vaccine based on mesenchymal stem cells as well as preparation method and application of tumor in-situ vaccine

Abstract

The invention discloses a tumor in-situ vaccine based on mesenchymal stem cells as well as a preparation method and application of the tumor in-situ vaccine. The tumor in-situ vaccine disclosed by the invention is ingeniously combined with the loading capacity of liposome nanoparticles, and aims to realize precise in-situ targeted therapy on solid tumors and metastatic tumors. The tumor in-situ vaccine based on the mesenchymal stem cells disclosed by the invention shows a remarkable effect in tumor treatment. Specifically, the vaccine fully utilizes the tumor tropism of the high-penetrability mesenchymal stem cells, so that the high-penetrability mesenchymal stem cells can be efficiently gathered in tumor tissues in a short time. By precisely delivering the liposome nanoparticles to a tumor site, the vaccine can induce tumor cells to generate immunogenic death, thereby releasing an injury-related molecular pattern. These DAMPs can activate pattern recognition receptors (PRRs) on the surface of dendritic cells (DC cells), thereby initiating a series of immune responses.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a tumor in-situ vaccine based on mesenchymal stem cells, its preparation method and application. Background Art

[0002] With the booming development of the field of tumor immunotherapy, tumor vaccines, as a revolutionary treatment method, are increasingly attracting the deep attention of researchers and clinicians. Such vaccines can achieve significant therapeutic effects by stimulating the specific immune response of the body, inducing the immune system to accurately identify and eliminate tumor cells. The core mechanism lies in providing tumor-associated antigens (TAAs) to the body, effectively activating key immune cells such as T cells and B cells, enabling them to accurately target and attack tumor cells.

[0003] However, despite the great therapeutic potential shown by tumor vaccines, various limitations of traditional vaccine preparation methods have seriously hindered their wide application. Traditional methods usually involve cumbersome in vitro cell culture, complex antigen extraction and other steps, which are not only costly, but also have many problems such as complex production processes and unstable curative effects, resulting in uneven effects of existing tumor vaccines in clinical applications. More intractably, due to the high heterogeneity of tumors, their surfaces often present diverse antigens, and traditional vaccines are difficult to comprehensively cover all tumor antigens, which undoubtedly further limits the therapeutic effect.

[0004] In addition, the significant defects of traditional tumor vaccines in terms of safety have greatly restricted their wide application in actual clinical practice. Specifically, tumor-associated antigens (TAAs), as non-mutated self-antigens, often fail to stimulate strong T cell responses in clinical immunotherapy, but may instead induce an immune tolerance state, making the vaccine unable to effectively activate the expected immune response. More seriously, after vaccination, the immune response against TAAs may get out of control and potentially develop into an attack on self-tissues, that is, an autoimmune response, which poses an additional threat to the health of patients.

[0005] The commonly used adjuvant components in traditional vaccines, although can enhance the immunogenicity of vaccines to a certain extent, may also non-specifically activate the innate immune system and induce chronic inflammatory responses. This chronic inflammation may not only exacerbate cell-mediated immune responses, but also trigger systemic immunopathological changes, further increasing the uncertainty and risk during the treatment process. Therefore, solving the safety problems of traditional tumor vaccines is crucial for promoting the progress of the field of tumor immunotherapy.

[0006] In this context, it is particularly urgent and important to develop new methods for preparing tumor vaccines, especially vaccines that can activate immune responses in situ in the body while ensuring safety. In recent years, tumor immunotherapy strategies based on mesenchymal stem cells (MSCs) have emerged and attracted extensive attention in the research community. As a type of pluripotent stem cells with self-renewal, strong differentiation potential, and immunomodulatory functions, MSCs have shown unique advantages in tumor immunotherapy. They can accurately migrate to the tumor site by virtue of the "tumor homing" property and become ideal tumor-targeted delivery carriers. At the same time, MSCs can also secrete various cytokines (such as interferons, interleukins, etc.), skillfully regulate the local immune microenvironment, and significantly enhance the immune system's response to tumors.

[0007] In recent years, mesenchymal stem cells have been widely used in the treatment research of tumors due to their unique tumor tropism and safety in vivo injection. Currently, more than 50 clinical trials have been registered. Intravenous injection of MSCs is relatively safe in clinical applications. Within 24 hours, the main acute adverse reaction related to MSC treatment is fever, which is the only adverse event significantly related to MSC treatment. In most cases, this fever is mild and does not require special treatment, and patients can recover naturally. Long-term observation has not found an increase in microbial infections and tumorigenesis caused by MSCs in patients. Randomized controlled clinical trials have shown that MSC treatment does not increase the risk of infection. MSCs have inestimable potential in the preparation and application of tumor vaccines, especially in improving vaccine delivery efficiency, enhancing immunogenicity, and inducing tumor-site-specific immune responses, showing extremely broad prospects.

[0008] Although mesenchymal stem cells (MSCs) have shown great application potential and prospects in the field of tumor vaccines, they still face many challenges that need to be solved urgently in practical applications. For the cell therapy of solid tumors, whether anti-tumor drugs and immune cells can effectively chemotax and enter the deep part of the primary tumor and metastatic tumors is the key factor determining the success of treatment. However, the vascular structure of tumor tissues often has functional defects and is rich in extracellular matrix, and these characteristics constitute a solid barrier, greatly hindering the effective infiltration of immune cells.

[0009] Specifically, the high accumulation of substances such as hyaluronic acid in the tumor microenvironment will lead to a significant increase in interstitial pressure, which in turn greatly reduces the perfusion volume of drugs. This not only affects the distribution and efficacy of drugs but also makes it difficult for immune cells to penetrate deep into the tumor tissue and play their due anti-tumor role. Therefore, how to improve the tumor targeting and tumor penetration of MSCs has become the primary problem that needs to be solved urgently.

[0010] In order to overcome this problem, we need to explore effective methods to enhance the accumulation and penetration of MSCs in the tumor microenvironment. By optimizing the homing mechanism of MSCs and improving their migration and penetration properties, we can expect to achieve wider and deeper infiltration of drugs and immune cells in tumor tissues, thereby significantly improving the efficacy of anti-tumor treatment. This not only helps to overcome the resistance of tumor tissues to treatment, but also provides new ideas and directions for the development of more efficient and safe tumor vaccines.

[0011] Secondly, how to enhance the immune stimulation effect of MSCs to ensure that they can effectively activate the immune system and induce a strong anti-tumor immune response is also an important issue facing current research. In addition, how to combine MSCs with tumor in situ vaccines and achieve accurate generation of in situ vaccines at the tumor site to improve the treatment effect and reduce systemic side effects is also a technical problem that needs to be solved urgently.

[0012] Therefore, the preparation method and application research of tumor in situ vaccines based on mesenchymal stem cells have become a major research hotspot in the field of tumor immunotherapy. By cleverly combining the tumor targeting of MSCs with the advantages of in situ vaccine generation, we are expected to achieve efficient and accurate immune response induction in the tumor microenvironment, thereby significantly improving the treatment effect, reducing side effects, and overcoming many difficulties in traditional tumor vaccine treatment. For example, in the tumor microenvironment, MSCs can serve as excellent carriers, carrying tumor antigens or immune activation factors, and accurately triggering in situ immune responses at the tumor site. This can not only greatly improve the immunogenicity of the vaccine, but also significantly enhance the persistence and specificity of the anti-tumor immune response, providing cancer patients with a safer and more effective new treatment option. Summary of the invention

[0013] Purpose of the invention: In view of the numerous challenges faced by solid tumors and their metastases in immunotherapy, the technical problem to be solved by the present invention is to provide an in situ tumor vaccine based on highly penetrating mesenchymal stem cells for solid tumors and their metastases.

[0014] The technical problem that the present invention needs to solve is to provide a method for preparing a tumor in situ vaccine based on mesenchymal stem cells.

[0015] The final technical problem to be solved by the present invention is to provide an application of a tumor in situ vaccine based on mesenchymal stem cells in the preparation of drugs for treating tumors.

[0016] Technical solution: To solve the above technical problems, the present invention provides a tumor in-situ vaccine based on mesenchymal stem cells. The tumor in-situ vaccine comprises mesenchymal stem cells and drug-loaded liposome nanoparticles loaded on the surface of the mesenchymal stem cells. The drug-loaded liposome nanoparticles are prepared from a tumor-targeting penetrating peptide (iRGD), hyaluronidase (HAase), a chemotherapeutic drug, and an immune adjuvant.

[0017] The innovative strategy of loading and modifying liposome nanoparticles with the tumor-penetrating peptide iRGD and hyaluronidase HAase on the cell surface of MSCs (mesenchymal stem cells) of the present invention aims to significantly enhance the tumor penetrability and targeting of MSCs, thereby improving the efficacy of anti-tumor treatment.

[0018] c-iRGD is a cyclic polypeptide with the sequence CRGDKGPDC, which can specifically recognize and bind to the αvβ3 and αvβ5 integrin receptors on the surface of tumor neovessels. This precise targeting enables MSCs to directly act on tumor cells, significantly reducing the toxicity and damage to surrounding healthy tissues. In addition, c-iRGD also has the function of a transmembrane peptide, which can better guide drugs into the cell interior using the NRP-1 receptor. In the tumor microenvironment, proteases can cleave the c-iRGD part, exposing the second receptor-binding motif (CendR sequence), which binds to NRP-1 on the surface of tumor cells, thereby promoting enhanced tumor vascular permeability and improving drug permeability.

[0019] Hyaluronic acid HA is an important component in the tumor microenvironment. Its high accumulation can lead to an increase in interstitial pressure, affecting drug perfusion and the infiltration of immune cells. As a hyaluronic acid-degrading enzyme, HAase can effectively degrade hyaluronic acid, thereby reducing interstitial pressure and improving vascular perfusion and drug distribution in tumor tissues. By modifying MSCs with liposome nanoparticles loaded with HAase, the penetrability and drug delivery efficiency of MSCs in tumor tissues can be further enhanced.

[0020] Among them, the mesenchymal stem cells are derived from human bone marrow, umbilical cord, adipose tissue, cord blood, amnion, placenta, thymus, synovium, liver, or dental pulp.

[0021] Among them, the preparation method of the drug-loaded liposome nanoparticles is as follows:

[0022] (1) Mix iRGD and DSPE-PEG-Mal in a molar ratio of 0.25:1 to 2:1 in a HEPEs solution and react at room temperature to obtain a DSPE-PEG-iRGD solution;

[0023] (2) Mix HAase and DSPE-PEG-NHS in a molar ratio of 0.25:1 to 2:1 in an HEPEs solution, and react at room temperature to obtain a DSPE-PEG-HAase solution;

[0024] (3) Mix the chemotherapeutic drug and the immune adjuvant with DSPE-PEG-NHS in a molar ratio of 0.5:1 to 2:1 in an HEPEs solution respectively to obtain Product 1 and Product 2;

[0025] (4) Take DSPE-PEG-iRGD, DSPE-PEG-HAase, Product 1, and Product 2 and mix them, and react at room temperature to obtain drug-loaded liposome nanoparticles.

[0026] Among them, the chemotherapeutic drug includes one or more of 5-fluorouracil, oxaliplatin, irinotecan, anthracycline compounds, mitoxantrone, idarubicin, bleomycin, or bortezomib.

[0027] Among them, the immune adjuvant includes one or more of TLR agonists, cytokines, interferons, interleukins, GM-CSF, immune checkpoint inhibitors, or natural immune activators.

[0028] The present invention also includes the preparation method of the mesenchymal stem cell-based in-situ tumor vaccine, comprising the following steps:

[0029] (1) Mix iRGD and DSPE-PEG-Mal in a molar ratio of 0.25:1 to 2:1 in an HEPEs solution, and react at room temperature to obtain a DSPE-PEG-iRGD solution;

[0030] (2) Mix HAase and DSPE-PEG-NHS in a molar ratio of 0.25:1 to 2:1 in an HEPEs solution, and react at room temperature to obtain a DSPE-PEG-HAase solution;

[0031] (3) Mix the chemotherapeutic drug and the immune adjuvant with DSPE-PEG-NHS in a molar ratio of 0.5:1 to 2:1 in an HEPEs solution respectively to obtain Product 1 and Product 2;

[0032] (4) Take DSPE-PEG-iRGD, DSPE-PEG-HAase, Product 1, and Product 2 and mix them, and react at room temperature to obtain drug-loaded liposome nanoparticles;

[0033] (5) Take mesenchymal stem cells and resuspend them to obtain a cell suspension, and add the drug-loaded liposome nanoparticles to the cell suspension to obtain a mesenchymal stem cell-based in-situ tumor vaccine.

[0034] The present invention also includes the application of the mesenchymal stem cell-based in-situ tumor vaccine in the preparation of drugs for treating tumors.

[0035] Among them, the tumor includes but is not limited to breast cancer.

[0036] Among them, the application includes treating a tumor-bearing subject by subcutaneous, intratumoral, peritumoral or intraperitoneal injection.

[0037] The core innovation of the present invention is reflected in two aspects: First, the present invention proposes a unique modification method that can enhance the in-situ tumor penetrability and targeting of mesenchymal stem cells; Second, a drug-loaded in-situ vaccine based on highly penetrable mesenchymal stem cells is developed. By modifying liposome nanoparticles loaded with the tumor-penetrating peptide iRGD and hyaluronidase HAase on the surface of MSCs cells, the present invention aims to solve the problem that immune cells are difficult to effectively infiltrate tumor tissues in tumor treatment. This innovative strategy combines the tumor targeting and transmembrane ability of iRGD, and the degradation effect of HAase on hyaluronic acid, to jointly promote the accumulation, penetration and drug delivery of MSCs in tumor tissues. Liposome nanoparticles (LNP) as a drug carrier have the advantages of protecting drugs from external media damage and reducing side effects. In the present invention, LNP is used to load the tumor-penetrating peptide iRGD and hyaluronidase HAase, and is anchored on the cell surface of MSCs through specific modification techniques. Combining the tumor targeting and transmembrane ability of iRGD, and the degradation effect of HAase on hyaluronic acid, the present invention is expected to significantly improve the accumulation, penetration and drug delivery efficiency of MSCs in tumor tissues. This not only helps to overcome the resistance of tumor tissues to treatment, but also provides new ideas and directions for the development of more efficient and safe tumor vaccines. This innovative strategy has broad application prospects in the field of tumor treatment, especially in precision medicine and personalized treatment. By optimizing the homing mechanism of MSCs and improving their migration and penetration characteristics, it is expected to achieve a wider and deeper infiltration of drugs and immune cells in tumor tissues, thus significantly improving the efficacy of anti-tumor treatment.

[0038] In addition, the present invention further introduces an immune adjuvant, which activates the immune system through multiple signaling pathways, significantly enhances the proliferation and activity of CD8+ T cells, improves the efficiency of anti-tumor immune response, promotes the maturation of DC cells, and strengthens the interaction between NK cells and DC cells, thereby reversing the immunosuppressive state in the tumor microenvironment. The vaccine strategy of the present invention also has a synergistic effect with chemotherapeutic drugs, further enhancing the anti-tumor effect. At the same time, the vaccine can establish long-term immune memory and effectively prevent the recurrence and metastasis of tumors. By activating innate and adaptive immune responses and precisely regulating immune cells in the tumor microenvironment, the present invention provides a new and efficient immune activation pathway, opening up new prospects for tumor treatment.

[0039] In a specific embodiment, the in-situ tumor vaccine of the present invention can be used in a treatment method for a subject with a tumor. The method includes injecting a therapeutically effective amount of modified mesenchymal stem cells into the patient's blood through intravenous injection. These modified mesenchymal stem cells can carry and deliver liposome nanoparticles to the tumor site, thereby exerting the above-mentioned immunotherapeutic effect.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The vaccine ingeniously combines the loading capacity of liposome nanoparticles (LNPs) to achieve precise in-situ targeted therapy for solid tumors and metastatic tumors. The in-situ tumor vaccine based on mesenchymal stem cells described in the present invention has shown remarkable effects in tumor treatment. Specifically, the vaccine makes full use of the tumor tropism of highly penetrative mesenchymal stem cells, enabling them to efficiently aggregate inside the tumor tissue within a short time (within 48 hours). By precisely delivering liposome nanoparticles to the tumor site, the vaccine can induce immunogenic cell death (ICD) of tumor cells, thereby releasing damage-associated molecular patterns (DAMPs). These DAMPs can activate pattern recognition receptors (PRRs) on the surface of dendritic cells (DC cells), thereby initiating a series of immune responses. Description of the Drawings

[0041] Figure 1 To evaluate the particle size and charge characterization of liposome nanoparticles. The particle size and charge characterization of DSPE-PEG2000-NHS liposome nanoparticles, highly penetrative liposome nanoparticles encapsulating iRGD and hyaluronidase HAase, and highly penetrative drug-loaded liposome nanoparticles encapsulating the chemotherapeutic drug doxorubicin, immune adjuvant R848, iRGD, and hyaluronidase HAase.

[0042] Figure 2 To evaluate the change in cell viability after modification of mesenchymal stem cells.

[0043] Figure 3 To verify the loading of liposome nanoparticles on mesenchymal stem cells, flow cytometry was used for inspection. When preparing the nanoparticles, we labeled FAM fluorescence for iRGD and CY5 fluorescence for HAase. The results showed that the liposome nanoparticles encapsulating DOX, R848, iRGD, and HAase were successfully attached to the cell surface.

[0044] Figure 4To evaluate the in vivo distribution within 48 h after intravenous injection of a tumor vaccine based on highly penetrative mesenchymal stem cells. DiR fluorescent dye was used to take near-infrared fluorescence images of mice at 4, 6, 24, and 48 hours after intravenous injection to detect the drug distribution at each time period. (A) Representative mice near-infrared imaging of each group at different time periods; (B) Fluorescence intensity of MSCs in tumor tissues detected at different time points.

[0045] Figure 5 To verify the therapeutic potential of a tumor in-situ vaccine based on mesenchymal stem cells against CT26 subcutaneous transplanted tumors, six groups were randomly divided for treatment according to different therapeutic drugs: NS, MSC, DR / M, DR / Mi, DR / MH, DR / MiH. (A) Tumor diameter development; (B) Tumor size; (C) Tumor weight.

[0046] Figure 6 To evaluate the anti-tumor efficacy of a tumor vaccine based on highly penetrative mesenchymal stem cells against breast cancer in-situ solid tumors. Three groups were randomly divided for treatment according to different therapeutic drugs: NS, drug-loaded liposome nanoparticles encapsulating DOX and R848, and MSCs modified with highly penetrative drug-loaded liposome nanoparticles encapsulating DOX, R848, iRGD, and HAase. (A) Tumor diameter development; (B) Tumor size; (C) Tumor weight.

[0047] Figure 7 Representative images of evaluating the anti-tumor efficacy of a tumor vaccine based on highly penetrative mesenchymal stem cells against breast cancer lung metastatic tumors. Four groups were randomly divided for treatment according to different therapeutic drugs: NS, DR (DOX and R848), DR / M (MSCs loaded with drug-loaded liposome nanoparticles encapsulating DOX and R848), DR / MiH (MSCs modified with highly penetrative drug-loaded liposome nanoparticles encapsulating DOX, R848, iRGD, and HAase). The results showed that compared with the control group, the fluorescence in the lungs of the DR / MiH vaccine group was significantly lower than that of other control groups, indicating good anti-metastatic tumor efficacy.

[0048] Figure 8 To evaluate the effect of a tumor vaccine based on highly penetrative mesenchymal stem cells on the tumor microenvironment. Tumor tissues were collected from CT26 model mice with colon cancer 10 days after the last treatment for immune response analysis. The results showed that compared with the control group, cytotoxic T lymphocytes (CTLs) in the DR / MiH vaccine group effectively infiltrated into the tumor site, and the proportion of functional dendritic cells (DCs, CD11c + CD80 + CD86 + DC) was significantly up-regulated.

[0049] Figure 9To evaluate the effect of a tumor vaccine based on highly penetrative mesenchymal stem cells on the tumor microenvironment, tumor tissues were collected 10 days after the last treatment of 4T1 model mice with breast cancer for immune response analysis. The results showed that, compared with the control group, helper T cells (CD4 + T) in the DR / MiH vaccine group effectively infiltrated into the tumor site, and the proportion of functional dendritic cells (CD11c + CD80 + CD86 + DC) was significantly upregulated, and the polarization of M2 macrophages to M1 macrophages increased. Detailed implementation manners

[0050] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0051] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0052] In one embodiment, the chemotherapeutic drugs capable of inducing immunogenic death of tumors include, but are not limited to, the following: 5-fluorouracil (5-FU), oxaliplatin, irinotecan, anthracyclines including doxorubicin and epirubicin, mitoxantrone, idarubicin, bleomycin, bortezomib.

[0053] In one embodiment, the immunoadjuvants include TLR (Toll-like receptor) agonists: TLR7 / 8 agonists, TLR9 agonists, Poly I:C; cytokines; interferons (IFN); interleukins (IL); granulocyte-macrophage colony-stimulating factor (GM-CSF); immune checkpoint inhibitors;

[0054] PD-1 / PD-L1 inhibitors; CTLA-4 inhibitors; natural immune activators; cyclophosphamide; silymarin; tumor-associated antigens (TAA); carcinoembryonic antigen (CEA); MAGE class antigens; tumor immune cell therapy adjuvants; IL-2 or IL-15;

[0055] CD40 agonists; nanomaterials and delivery carriers; liposomes, nanoparticles; dendrimers; antibody-based immunopotentiators; anti-CD40 antibodies; anti-OX40 antibodies; immunomodulatory drugs; thalidomide; vitamin D and vitamin A derivatives, etc.

[0056] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the following will specifically describe the technical solution of the present invention in combination with specific examples and comparative examples. However, it should be understood that providing these examples is only for specific illustration and does not limit the scope of the present invention in any way.

[0057] Example 1

[0058] 1. Cell lines and reagent materials

[0059] 1) Murine CT26 colon cancer and 4T1 breast cancer cells, 4T1-luc breast cancer cells (i.e., 4T1 breast cancer cells labeled with firefly luciferase) were preserved by the Research and Experiment Center of the Department of Oncology, Nanjing Drum Tower Hospital, School of Medicine, Nanjing University. Human umbilical cord blood mesenchymal stem cells MSC were provided and preserved by the Stem Cell and Regenerative Medicine Research Center of Nanjing Drum Tower Hospital, School of Medicine, Nanjing University. CT26 cells and 4T1 cells were cultured in PRMI 1640 medium (Corning, USA) containing 10% fetal bovine serum, and the incubation conditions were 37°C and 5% CO2. MSC cells were cultured in low-glucose DMEM medium (Gibco, USA) containing 10% fetal bovine serum, and the incubation conditions were 37°C and 5% CO2.

[0060] 2) Specific antibodies and reagents used in flow cytometry: PerCP / Cyanine5.5 anti-mouse CD3 (BD Pharmingen, USA); Brilliant Violet510 anti-mouse CD4 (BD Pharmingen, USA); FITC anti-mouse CD8 (BD Pharmingen, USA); PE anti-mouse CD11c (BD Pharmingen, USA); APC anti-mouse CD80 (BD Pharmingen, USA); PerCP / Cyanine7 anti-mouse CD86 (BD Pharmingen, USA); PerCP / Cyanine5.5 anti-mouse CD11b (BD Pharmingen, USA); APC anti-mouse F4 / 80 (BD Pharmingen, USA); PE anti-mouse CD206 (BD Pharmingen, USA); Permeabilization and fixation solution (BD Pharmingen, USA).

[0061] 2. Mice

[0062] In this experiment, specific pathogen-free (SPF)-level female BALB / c mice, 4 - 6 weeks old and weighing 18 - 20 g, were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. All mice were housed in the SPF-level experimental animal center of Nanjing Drum Tower Hospital Cancer Institute. The environmental temperature was 22 - 24°C, with 12 h of light and 12 h of darkness each day, and the relative humidity was 50 - 60%. The use and operation of experimental animals strictly followed the regulations and guidelines of the Animal Experiment Management Committee of Nanjing Drum Tower Hospital.

[0063] Construction of CT26 subcutaneous tumor-bearing mouse model: Resuscitate cryopreserved CT26 tumor cells, routinely culture them to the logarithmic growth phase, collect the cells, resuspend them with normal saline and adjust the cell concentration to 1×10 7 / mL, and place them on ice for later use. Prepare healthy female BALB / c mice, depilate and prepare the skin on the abdomen, place them in separate cages and mark them with ear tags. Inject 100 μL of CT26 tumor cell suspension subcutaneously into the left abdomen of each mouse near the groin to construct CT26 subcutaneous tumor-bearing mouse model.

[0064] 3. Preparation of Liposome Nanoparticles (LNP), iRGD-Loaded Mesenchymal Stem Cells (abbreviated as M-i), Hyaluronidase (Shanghai Aladdin Biochemical Technology Co., Ltd.)-Loaded Mesenchymal Stem Cells (abbreviated as M-H), Highly Penetrating Mesenchymal Stem Cells (abbreviated as M-iH), and In Situ Vaccine Loaded with Chemotherapeutic Drugs and Immune Adjuvants that Can Induce Tumor Immunogenic Death (abbreviated as DR / MiH):

[0065] 1) Preparation of DSPE-PEG-iRGD and DSPE-PEG-HAase

[0066] Step 1: Mix C-iRGD-FAM (Shanghai Taopu Biotechnology Co., Ltd.) and DSPE-PEG2000-Mal (LaysanBio, USA) at a molar ratio of 1:1 in a 0.5 mM HEPEs (Shanghai Beyotime Biotechnology Co., Ltd.) solution, adjust the pH to 6.5, protect with nitrogen, and shake at room temperature for 48 hours; add the obtained reaction product into a 3500 Da dialysis bag and dialyze in double-distilled water, changing the dialysis solution every 8 hours for at least 4 times to remove unreacted C-iRGD-FAM and DSPE-PEG2000-Mal; detect the concentration of the dialysis product with a BCA protein concentration assay kit to obtain the product DSPE-PEG-iRGD. Aliquot and store at -80 °C or prepare the obtained product into a powder with a freeze dryer and store at -20 °C.

[0067] Step 2: Add 5 μL of the activated 10 mM CY5 (MedChemExpress, USA) dye working solution to the 10 mg / ml HAase solution, mix well and place in the dark, and shake and incubate at room temperature for 60 minutes to fully mix the two reactants to obtain the product HAase-CY5. Mix HAase-CY5 and DSPE-PEG2000-NHS (Shanghai Aladdin Biochemical Technology Co., Ltd.) at a molar ratio of 1:1 in a 0.5 mM HEPEs solution, adjust the pH to 8, protect with nitrogen, and shake at room temperature for 48 hours; add the obtained reaction product into a 3500 Da dialysis bag and dialyze in double-distilled water, changing the dialysis solution every 8 hours for at least 4 times to remove unreacted HAase, CY5, and DSPE-PEG2000-NHS; detect the concentration of the dialysis product with a BCA protein concentration assay kit to obtain the product DSPE-PEG-HAase. Aliquot and store at -80 °C or prepare the obtained product into a powder with a freeze dryer and store at -20 °C.

[0068] 2) Preparation of LNP, LNP-iRGD, LNP-HAase, and LNP-iRGD-HAase

[0069] Step 1: Dissolve DSPE-PEG2000-NHS, DSPE-PEG-iRGD, and DSPE-PEG-HAase in 10 mL of chloroform at a molar ratio of 4:1 to DSPE-PEG2000-N3 respectively to form 3 homogeneous lipid solutions.

[0070] Step 2: Dissolve DSPE-PEG-iRGD, DSPE-PEG-HAase, and DSPE-PEG2000-N3 in 10 mL of chloroform at a molar ratio of 4:4:1 to form a homogeneous lipid solution.

[0071] Step 3: Collect the 4 lipid solutions from the above two steps and slowly rotary evaporate them at 37 °C using a rotary evaporator to remove chloroform and form a homogeneous lipid film. Add 1×PBS buffer to the lipid film and shake well to fully hydrate the lipid film to form liposomes.

[0072] Step 4: Collect the hydrated liposome solution and transfer it to an ultrasonic instrument for ultrasonic treatment to homogenize the particle size of the liposomes. The ultrasonic conditions are: power 200 W, pulse mode (working for 3 seconds, interval 3 seconds), total time 15 minutes.

[0073] Step 5: Centrifuge and collect the product at 10000 rpm for 60 min; resuspend it with double-distilled water and filter it 20 times through a 100 nm filter using a liposome extruder to obtain LNP, LNP-iRGD, LNP-HAase, and LNP-iRGD-HAase. Aliquot and store at -80 °C or prepare the obtained product into a powder using a freeze dryer and store at -20 °C.

[0074] 3) Preparation of in-situ tumor vaccine DR / MiH based on mesenchymal stem cells:

[0075] In this example, the chemotherapeutic drug doxorubicin DOX and the immune adjuvant R848 are used as examples

[0076] Step 1: Mix DOX with DSPE-Hyd-PEG2000-NHS (Xi'an Ruixi Biotechnology Co., Ltd.) at a molar ratio of 1:1 in a 0.5 mM HEPEs solution (simultaneously, mix R848 with DSPE-PEG2000-NHS at a molar ratio of 1:1 in a 0.5 mM HEPEs solution), adjust the pH to 8.0, protect with nitrogen gas, and shake at 4°C for 48 hours. Add the obtained reaction product to a 3500 Da dialysis bag and dialyze in double-distilled water, changing the dialysis solution every 8 hours for at least 4 times to remove unreacted DOX, R848, DSPE-Hyd-PEG2000-NHS, and DSPE-PEG2000-NHS; obtain the products DSPE-Hyd-PEG-DOX and DSPE-PEG-R848. Aliquot and store at -80°C or prepare the obtained product into a powder using a freeze dryer and store at -20°C.

[0077] Step 2: Dissolve DSPE-Hyd-PEG-DOX, DSPE-PEG-R848, and DSPE-PEG2000-N3 at a molar ratio of 4:4:1 in 10 mL of chloroform to form a homogeneous lipid solution. Dissolve DSPE-PEG-iRGD, DSPE-Hyd-PEG-DOX, DSPE-PEG-R848, and DSPE-PEG2000-N3 at a molar ratio of 4:4:4:1 in 10 mL of chloroform to form a homogeneous lipid solution. Dissolve DSPE-PEG-HAase, DSPE-Hyd-PEG-DOX, DSPE-PEG-R848, and DSPE-PEG2000-N3 at a molar ratio of 4:4:4:1 in 10 mL of chloroform to form a homogeneous lipid solution. Dissolve DSPE-PEG-iRGD, DSPE-PEG-HAase, DSPE-Hyd-PEG-DOX, DSPE-PEG-R848, and DSPE-PEG2000-N3 at a molar ratio of 4:4:4:4:1 in 10 mL of chloroform to form a homogeneous lipid solution.

[0078] Collect the above 4 lipid solutions and slowly rotary evaporate them at 37°C using a rotary evaporator to remove chloroform and form a homogeneous lipid film. Add 1×PBS buffer to the lipid film and shake well to fully hydrate the lipid film to form 4 liposomes.

[0079] Step 3: Collect the 4 hydrated liposome solutions and transfer them to an ultrasonic instrument for ultrasonic treatment to homogenize the particle size of the liposomes. The ultrasonic conditions are: power 200 W, pulse mode (work for 3 seconds, interval for 3 seconds), total time 15 minutes.

[0080] Step 4: Collect the four products by centrifugation at 10000rpm for 60min; resuspend with double distilled water, filter 20 times with a liposome extruder 100nm filter membrane to obtain the products DR / LNP, DR / LNP-iRGD, DR / LNP-HAase, and DR / LNP-iH. Store the products at -80℃ or prepare them into powder with a freeze dryer and store at -20℃.

[0081] Step 5: Select the stable MCS digestion and centrifuge, wash twice with 1× PBS, and resuspend to 1×10 6 / mL, calculated as 1×10 6 cells: 10 μg of NHS-PEG-DBCO (purchased from Xi'an Qiyue Biotechnology Co., Ltd., Chinese name: active ester polyethylene glycol diphenyl cyclooctyne, molecular weight 1000) was added, incubated at 37°C with shaking for 30 min, washed twice with 1×PBS, and the supernatant was discarded to obtain NHS-PEG-DBCO-modified MCS.

[0082] Step 6: NHS-PEG-DBCO modified MCS was added at 1×10 6 cells: 5 μg were added to DR / LNP, DR / LNP-iRGD, DR / LNP-HAase, and DR / LNP-iH liposome nanoparticles. Incubated with shaking at 37°C overnight, DR / M mesenchymal stem cells loaded with DOX and R848, DR / Mi mesenchymal stem cells modified with iRGD, DR / MH mesenchymal stem cells modified with HAase, and DR / MiH mesenchymal stem cell-based tumor in situ vaccine were obtained.

[0083] 4. Characterization of LNP, LNP-iRGD-HAase and DR / LNP-iH

[0084] The particle size and charge of LNP, LNP-iRGD-HAase and DR / LNP-iH nanoparticles were characterized by DLS. The results are shown in Figure 1 The particle size of blank liposome nanoparticles LNP was about 150nm, and the charge was negative; the particle size of nanoparticles LNP-iRGD-HAase loaded with iRDG and HAase increased slightly to about 200nm, and the charge was negative; the particle size of nanoparticles DR / LNP-iH loaded with iRDG, HAase, DOX, and R848 increased slightly to about 200nm, and the charge was negative.

[0085] 5. Construction of high-penetrability mesenchymal stem cells and cell viability detection

[0086] Construction of high permeability mesenchymal stem cells: NHS-PEG-DBCO modified MCS was added at a rate of 1×10 6Cells: 5 μg of LNP-iRGD, LNP-HAase, and LNP-iRGD-HAase liposome nanoparticles were added respectively. After overnight shaking incubation at 37 °C, mesenchymal stem cells M-i, M-H, and M-iH modified with iRGD, HAase, and iRGD-HAase were obtained respectively.

[0087] Cell viability detection: Penetrating liposome nanoparticles encapsulating iRGD, penetrating liposome nanoparticles encapsulating HAase, and highly penetrating liposome nanoparticles encapsulating iRGD and HAase with the same mass were co-incubated with mesenchymal stem cells for 24 hours and 48 hours respectively. Using 1×PBS buffer with the same mass as the blank control, the MTT cell proliferation and cytotoxicity detection kit (Shanghai Beyotime Biotechnology Co., Ltd.) was used to detect the changes in cell viability of mesenchymal stem cells after co-incubation. The results show that Figure 2 it can be seen that the modification with iRGD and HAase has no obvious effect on cell viability.

[0088] 6. Flow cytometry detection of in-situ tumor vaccine based on mesenchymal stem cells

[0089] Unmodified mesenchymal stem cells MSC, penetrating drug-loaded mesenchymal stem cells DR / Mi modified with iRGD, and penetrating drug-loaded mesenchymal stem cells DR / MH modified with HAase with the same number were collected as the control group, and highly penetrating drug-loaded mesenchymal stem cells DR / MiH modified with iRGD and HAase were used as the experimental group. They were placed in a 15 mL centrifuge tube, 3 mL of PBS was added, centrifuged at 1200 rpm for 5 min, the supernatant was discarded, washed twice with 1×PBS buffer and then placed in a flow cytometry tube, and flow cytometry was performed to verify the modification efficiency of liposome nanoparticles on mesenchymal stem cells MSC. The iRGD modification is shown in the FITC channel, and the HAase modification is shown in the APC channel. The results of flow cytometry assessment are shown in Figure 3 After overnight co-incubation of liposome nanoparticles with mesenchymal stem cells, they were efficiently loaded on the cell surface.

[0090] 7. In-vivo verification of highly penetrating mesenchymal stem cells

[0091] The drug distribution at each time period was recorded by a small animal in-vivo imaging system. Mice with CT26 subcutaneous xenograft tumor models were intravenously injected with DiR (China Bridge Bio)-labeled in-situ tumor vaccine based on mesenchymal stem cells (DR / MiH) (5×10 6 / mouse), and DR / M, DR / MH, and DR / Mi labeled with DiR were used as controls respectively. Near-infrared fluorescence images were detected at 2, 4, 24, and 48 hours after injection. Subsequently, the tumors and important organs of the mice were excised for imaging, and the fluorescence measurement results were quantitatively analyzed. The representative results of each group are shown in Figure 4After intravenous injection, MSCs enter the blood circulation, first passing through the lungs and then reaching the liver. The abundant blood sinus structure and high-density endothelial cells in the liver provide a large number of "landing points" for MSCs, making it easy for them to be intercepted and aggregated. 24 hours after intravenous injection, MSCs reach the tumor tissue through the blood circulation. The high penetrability modification enables MSCs to accumulate more in the tumor site. 48 hours after intravenous injection, the DR / MiH group shows stronger tumor tropism and homing ability compared with other penetrant mesenchymal stem cell groups, demonstrating the synergistic effect of the combined modification of iRGD and HAase on mesenchymal stem cells.

[0092] 8. Verification of the tumor-suppressing effect of the in-situ vaccine based on highly penetrant mesenchymal stem cells

[0093] 1) Verification of the tumor-suppressing effect in the subcutaneous CT26 tumor model

[0094] Step 1: Resuscitate the cryopreserved CT26 tumor cells, routinely culture them to the logarithmic growth phase, collect the cells, resuspend them with normal saline, and adjust the cell concentration to 1×10 7 / mL, and place them on ice for later use.

[0095] Step 2: Prepare 36 healthy BALB / c female mice, depilate and prepare the skin on the abdomen, place them in separate cages and mark them with ear tags. Subcutaneously inject 100 μL of the tumor cell suspension into the left abdomen of each mouse near the inguinal position to construct a subcutaneous transplanted tumor model, denoted as Day 0. Observe the tumor growth of the mice daily, and use a vernier caliper to measure the long diameter D L (mm) and the short diameter D S (mm) of the tumor, and estimate the tumor volume according to the following formula:

[0096] V(mm 3 ) = 0.5×D L ×D S 2

[0097] Step 3: When the tumor volume grows to about 80 - 100 mm 3 on Day 7, randomly divide the 36 mice into 6 groups, with 6 mice in each group. Intravenously inject 200 μL of NS (normal saline); MSC (1×10 6 ); DR / M (1×10 6 , 5 μg / mouse), DR / Mi (1×10 6 , 5 μg / mouse), DR / MH (1×10 6 , 5 μg / mouse); DR / MiH (1×10 6 , 5 μg / mouse) on the 8th, 10th, and 12th days respectively. Press with a sterile cotton swab for 30 s after injection.

[0098] Step 4: Subsequently, the changes in tumor volume and body weight were detected every other day. When the tumor volume reached 1500 mm 3 or the maximum tumor diameter reached 20 mm, the mice were immediately euthanized, which was regarded as reaching the survival endpoint. As Figure 5 shown: The tumors of the mice in the NS group grew rapidly; the tumors of the mice injected with MSCs alone grew faster than those in the NS group in the later stage. Our countermeasure might be that MSCs had a tumor-promoting effect on this tumor model; the tumor growth was slightly inhibited after loading drugs on MSCs alone; the tumor growth was significantly inhibited by loading drugs on MSCs modified with HAase or iRGD alone, but the tumor growth could not be completely inhibited; while the tumor growth was significantly inhibited by loading drugs on MSCs combined with HAase and iRGD, that is, the mesenchymal stem cell-based in-situ tumor vaccine. It was proved that DR / MiH had a significant tumor-inhibiting effect in subcutaneous transplanted tumors.

[0099] 2) Verification of the tumor-inhibiting effect of the in-situ 4T1 tumor model

[0100] Step 1: Prepare 18 healthy BALB / c female mice and 4T1 cells. Each mouse was injected with 100 μL of tumor cell suspension (concentration of 1×10 7 / mL) into the second pair of fat pads to construct an in-situ transplanted tumor model.

[0101] Step 2: On Day 7, the tumor volume grew to approximately 80 - 100 mm 3 , and then all the mice were randomly divided into 3 groups, with 6 mice in each group. On the 8th, 10th, and 12th days, 200 μL of NS; DR / LNP (5 μg / mouse); DR / MiH (1×10 6 , 5 μg / mouse) were injected intravenously. After injection, the injection site was pressed with a sterile cotton swab for 30 s.

[0102] Step 3: Subsequently, the changes in tumor volume and body weight were detected every other day. When the tumor volume reached 1500 mm 3 or the maximum tumor diameter reached 20 mm, the mice were immediately euthanized, which was regarded as reaching the survival endpoint. As Figure 6 shown: Compared with the NS group, the treatment with drug-loaded liposome nanoparticles alone had a slight inhibitory effect on tumor growth, while the DR / MiH group had a significant inhibitory effect on tumor growth. It was proved that DR / MiH also had a significant tumor-inhibiting effect in in-situ tumors.

[0103] 3) Verification of the tumor-inhibiting effect of the lung metastasis 4T1 tumor model

[0104] Step 1: Prepare 16 healthy BALB / c female mice and 4T1-Luc cells. Each mouse was injected with 200 μL of 4T1-Luc tumor cell suspension (concentration of 5×10 5( / mL) to establish a lung metastasis model by intravenous injection of 4T1 cells.

[0105] Step 2: Inject D-Luciferin potassium (purchased from MedChemExpress, USA) intraperitoneally, and take pictures of the fluorescence in the lungs of mice with a small animal imager. Stable lung fluorescence can be photographed on Day 7. Randomly divide all mice into 4 groups, with 4 mice in each group. Place them in separate cages and mark them with ear tags. Intravenous injection of 200 μL NS is performed on days 7, 9, and 11 respectively; DOX-R848 drug (1.25 μg DOX, 1.25 μg R848 / mouse); DR / M (1×10 6 , 5 μg / mouse); DR / MiH (1×10 6 , 5 μg / mouse). After injection, press with a sterile cotton swab for 30 s.

[0106] Step 3: Subsequently, detect the changes in fluorescence images every 2 - 3 days, record the lung fluorescence and the body weight of mice. After 21 days of inoculating tumor cells, euthanize the mice, which is regarded as reaching the survival endpoint, record and draw the survival curve. Subsequently, excise the important organs of the mice for imaging and quantitatively analyze the fluorescence measurement results. Representative results of each group are shown in Figure 7 : After treatment with DR / MiH, the lung metastasis of mouse breast cancer tumors was significantly inhibited.

[0107] 9. Detection of changes in immune cells in the tumor microenvironment by flow cytometry

[0108] For the CT26 tumor model mice and control mice and the orthotopic 4T1 tumor model mice and control mice treated in step 8, the mice were sacrificed one week after the last administration, and the tumor tissues were removed and photographed. Single cell suspensions of spleen and lymph nodes were prepared by mechanical grinding method, washed 3 times with PBS and reserved. At the same time, the tumor tissues were cut into small pieces and digested with collagenase type IV (Gibco, USA, catalog number, 9001 - 12 - 1) at 37 °C with gentle stirring for 2 h. The tumor suspension was passed through a 70 μm cell sieve to remove large fragments or undigested tumor tissue blocks. Subsequently, all cell samples were resuspended in pre-cooled PBS, stained with the corresponding specific antibodies in the dark at 4 °C for 30 min, washed and then analyzed. The data was collected with a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using software FlowJo (Tree Star). The results are shown in Figure 8 、 Figure 9 : In the CT26 model, compared with the control group, CD80 in the DR / MiH group + CD86 +The proportion of DCs (52.7%) was significantly upregulated, almost three times that of the NS group (17.4%); the expression of CD3 + CD8 + T cells in the tumors of the DR / MiH group was significantly higher than that of all other treatment groups, indicating a strong cytotoxic T cell response was induced in the tumors and lymph nodes. In the 4T1 model, compared with the control group, the proportion of CD80 + CD86 + DCs in the tumors of the DR / MiH group was significantly upregulated, almost twice that of the NS group (36.7%); the expression of CD3 + CD4 + T cells in the tumors of the DR / MiH group was significantly higher than that of all other treatment groups. The proportion of M1 macrophages (CD11b + F4 / 80 + CD86 + ) in the DR / MiH group was about 1.5 times that of the NS group. And the proportion of M2 macrophages (CD11b + F4 / 80 + CD206 + ) in the DR / MiH group decreased significantly, about 0.5 times that of the NS group. It indicates that the in-situ vaccine based on mesenchymal stem cells successfully reversed the polarization of tumor-associated macrophages.

[0109] 10. Safety analysis

[0110] Establish various tumor models in step 8 by the above method and treat the mice. The mice were sacrificed 10 days after the last treatment. The heart, liver, spleen, lungs and kidneys were fixed in 4% paraformaldehyde, routinely dehydrated, paraffin-embedded, sectioned, and then stained with HE sections. Photographs were taken under an optical microscope to observe whether there were lesions in the organs of each group, and the safety of this treatment mode was evaluated. No tissue damage was seen in the images.

Claims

1. A tumor in-situ vaccine based on mesenchymal stem cells, characterized in that, The in-situ tumor vaccine comprises mesenchymal stem cells and drug-loaded liposome nanoparticles loaded on the surface of the mesenchymal stem cells, and the drug-loaded liposome nanoparticles are prepared from a tumor-targeting penetrating peptide, hyaluronidase, a chemotherapeutic drug, and an immune adjuvant.

2. The tumor in-situ vaccine based on mesenchymal stem cells according to claim 1, characterized in that The mesenchymal stem cells are derived from human bone marrow, umbilical cord, adipose tissue, cord blood, amnion, placenta, thymus, synovium, liver, or dental pulp.

3. The mesenchymal stem cell-based in-situ tumor vaccine according to claim 1, wherein The preparation method of the drug-loaded liposome nanoparticles comprises the following steps: (1) Mix iRGD and DSPE-PEG-Mal at a molar ratio of 0.25:1 to 2:1 in an HEPEs solution, and react at room temperature to obtain a DSPE-PEG-iRGD solution; (2) Mix HAase and DSPE-PEG-NHS at a molar ratio of 0.25:1 to 2:1 in an HEPEs solution, and react at room temperature to obtain a DSPE-PEG-HAase solution; (3) Mix the chemotherapeutic drug and the immune adjuvant with DSPE-PEG-NHS at a molar ratio of 0.5:1 to 2:1 in an HEPEs solution respectively to obtain product 1 and product 2; (4) Take DSPE-PEG-iRGD, DSPE-PEG-HAase, product 1, and product 2 and mix them, and react at room temperature to obtain the drug-loaded liposome nanoparticles.

4. The mesenchymal stem cell-based in-situ tumor vaccine according to claim 3, wherein the chemotherapeutic drug comprises one or more of 5-fluorouracil, oxaliplatin, irinotecan, anthracyclines, mitoxantrone, idarubicin, bleomycin, or bortezomib.

5. The mesenchymal stem cell-based in-situ tumor vaccine according to claim 3, wherein the immune adjuvant comprises one or more of TLR agonists, cytokines, interferons, interleukins, GM-CSF, immune checkpoint inhibitors, or natural immune activators.

6. The preparation method of the mesenchymal stem cell-based in-situ tumor vaccine according to any one of claims 3 to 5, characterized in that, Comprises the following steps: (1) Mix iRGD and DSPE-PEG-Mal at a molar ratio of 0.25:1 to 2:1 in an HEPEs solution, and react at room temperature to obtain a DSPE-PEG-iRGD solution; (2) Mix HAase and DSPE-PEG-NHS at a molar ratio of 0.25:1 to 2:1 in an HEPEs solution, and react at room temperature to obtain a DSPE-PEG-HAase solution; (3) Mix the chemotherapeutic drug and the immune adjuvant with DSPE-PEG-NHS at a molar ratio of 0.5:1 to 2:1 in an HEPEs solution respectively to obtain product 1 and product 2; (4) Take DSPE-PEG-iRGD, DSPE-PEG-HAase, product 1, and product 2 and mix them, and react at room temperature to obtain the drug-loaded liposome nanoparticles; (5) Take mesenchymal stem cells and resuspend them to obtain a cell suspension, and add the drug-loaded liposome nanoparticles to the cell suspension to obtain the mesenchymal stem cell-based in-situ tumor vaccine.

7. Use of the mesenchymal stem cell-based in-situ tumor vaccine according to any one of claims 3 to 5 in the preparation of a drug for treating tumors.

8. The application according to claim 7, wherein The tumor includes breast cancer.

9. The application according to claim 7, wherein The application includes injecting into a tumor-bearing subject for treatment by subcutaneous, intratumoral, peritumoral or intraperitoneal injection.

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