Multifunctional coordination polymer for activating thermotherapy-induced in-situ vaccine as well as preparation method and application of multifunctional coordination polymer

Abstract

The invention discloses a multifunctional coordination polymer for activating a thermal therapy induced in-situ vaccine as well as a preparation method and application of the multifunctional coordination polymer, a blood vessel disrupting agent, a photosensitizer and a contrast agent Gd3 + are constructed and integrated, the blood vessel disrupting agent is used for opening a tumor blood vessel barrier and increasing permeation of a photosensitive drug, and the photosensitizer and tumor bleeding further improve the photo-thermal effect; according to the present invention, the photothermal anti-tumor immune response is enhanced, the Gd < 3 + >-mediated nuclear magnetic imaging is cooperated to achieve the precise positioning treatment on the tumor, the synergistic interaction effect is achieved, the tumor immunotherapy is enhanced, the solubility of the indissolvable drug can be improved, the toxicity can be reduced, the delivery efficiency of the molecular targeting drug can be improved, and the further combination with the anti-PD-1 immunotherapy can be achieved; therefore, the compound has a promising application prospect in preparation of anti-tumor drugs.

Description

Technical Field

[0001] The invention relates to a multifunctional coordination polymer, in particular to a multifunctional coordination polymer for activating an in-situ vaccine induced by hyperthermia, and also to a preparation method and application of the multifunctional coordination polymer. Background Art

[0002] Cancer is one of the most important medical challenges worldwide. Its high morbidity and mortality make it a major obstacle to extending human life. In recent years, cancer immunotherapy has shown great promise in the field of treatment. Among them, in situ tumor vaccines are extracted from dead tumor cells during treatment, avoiding the complex steps of antigen screening and containing all patient-specific tumor antigens, which may reduce immune escape. Photothermal therapy (PTT), as a new strategy for non-invasive tumor treatment, uses photothermal converters (PTAs) to convert light energy into heat energy under near-infrared light (NIR) irradiation to kill tumor cells, showing high selectivity and low invasiveness. In addition, PTT can also induce immunogenic cell death (ICD) of tumor cells, further enhancing the immune recognition ability of in situ tumor vaccines. Therefore, PTT is a promising therapy for initiating in situ tumor vaccines and activating anti-tumor immune responses in vivo.

[0003] Although PTT has shown good prospects in clinical applications, it still faces major challenges in treating deep or large solid tumors. For example, due to the low permeability of blood vessels and high interstitial pressure inside tumor tissue, it is difficult for photosensitizers to penetrate evenly into the deep part of the tumor in nanodelivery systems. In addition, the risk of tumor recurrence and metastasis during treatment, as well as potential thermal damage to normal tissues around the tumor, limit the therapeutic depth and effect of PTT. The key problem is that high-temperature PTT may not effectively induce immunogenic cell death (ICD) and easily cause damage to normal tissues. Although low-temperature PTT can reduce adverse reactions and induce ICD, thereby releasing in situ tumor vaccines, it often cannot induce sufficient immune response to completely eliminate the tumor. First, the enrichment of photosensitizers in tumor tissues is insufficient. This is because nanomaterials rely on high permeability and retention (EPR) effects to enrich in the tumor site after intravenous injection, and penetrate into the tumor tissue and are internalized by tumor cells. Second, when the PTT intensity is insufficient, the residual tumor cells after treatment may proliferate and continue to induce an immunosuppressive tumor microenvironment (TME), inhibiting the effectiveness of the released in situ vaccine. For example, residual tumor cells can recruit immunosuppressive cells such as tumor-associated macrophages (TAMs) and regulatory T cells (Tregs), which can hinder the uptake and antigen presentation of in situ vaccines by dendritic cells (DCs) by secreting cytokines such as interleukin-10. Therefore, if the PTT-induced in situ tumor vaccine fails to activate a sufficient immune response, it may lead to tumor recurrence and metastasis. In addition, in order to avoid thermal damage to normal tissues, traditional photothermal therapy usually chooses incomplete irradiation, which may result in tiny lesions or residual tumor cells that are invisible to the naked eye at the treatment site, thereby increasing the risk of tumor recurrence and metastasis.

[0004] In addition, the lack of reliable imaging systems to guide photothermal ablation and monitor the therapeutic effect during photothermal therapy further exacerbates the recurrence and metastasis problems associated with PTT. Therefore, new strategies are urgently needed to enhance the therapeutic effect of PTT to effectively initiate in situ cancer vaccines and improve clinical efficacy. Summary of the invention

[0005] Purpose of the invention: The present invention aims to solve the problems of insufficient tumor accumulation, tumor recurrence and metastasis during treatment, and thermal damage to normal tissues around the tumor, which lead to poor response to photosensitizer treatment. 3+ The invention discloses a multifunctional coordination polymer capable of activating an in-situ vaccine induced by hyperthermia, which is prepared by the self-assembly principle. The invention also provides a preparation method of the multifunctional coordination polymer and its application in anti-tumor treatment.

[0006] Technical solution: The present invention discloses a multifunctional coordination polymer for activating an in situ vaccine induced by hyperthermia. The multifunctional coordination polymer is prepared from a vascular disruptor, a photosensitizer, a nuclear magnetic contrast agent, 5'NMP and human serum albumin through the principle of chemical coordination self-assembly. 3+ The polymer is formed by interacting with 5'NMP through coordination bonds, and the polymer encapsulates the vascular disrupting agent and the photosensitizer to obtain a multifunctional coordination polymer;

[0007] Among them, the vascular disrupting agent is a tubulin binding agent or a flavonoid compound with anti-vascular function; the photosensitizer is a precious metal nanoparticle, a metal and non-metal compound or a near-infrared dye; the nuclear magnetic contrast agent is a paramagnetic contrast agent (gadolinium agent), a superparamagnetic contrast agent (superparamagnetic iron oxide), a ferromagnetic contrast agent or a diamagnetic contrast agent; the 5'NMP is a nucleoside monophosphate, including GMP, AMP, CMP or TMP, preferably 5'-GMP.

[0008] Among them, the microtubule protein binder is colchicine, paclitaxel or docetaxel, the flavonoid compound is flavone-8-acetic acid (Flavone-8-acetic acid) or 2,5-hexanthenoxiline (DMXAA); the precious metal nanoparticles are Au, Ag or Pt; the metal and non-metal compound is CuS; the near-infrared dye is indocyanine green (ICG) or IR780.

[0009] Among them, the multifunctional coordination polymer is a cylindrical structure nanoparticle with a particle size of about 60nm.

[0010] The method for preparing the multifunctional coordination polymer comprises the following steps:

[0011] (1) mixing an aqueous solution of 5'-NMP, a vascular damaging agent and a photosensitizer, and stirring to obtain a mixed solution;

[0012] (2) slowly adding the aqueous solution of the nuclear magnetic resonance contrast agent to the mixed solution prepared in step (1), stirring, and centrifuging to collect the precipitate produced by stirring;

[0013] (3) The precipitate obtained in step (2) is washed with water and collected by centrifugation, and a nanoscale multifunctional coordination polymer for activating the in situ vaccine induced by hyperthermia is obtained under the action of human serum albumin and ultrasound.

[0014] The molar ratio of 5'-NMP, vascular disrupting agent, photosensitizer, nuclear magnetic resonance contrast agent and human serum albumin is 50-300:10-50:1-10:20-200:1-6.

[0015] The method comprises dropping a nuclear magnetic resonance contrast agent into a solution containing 5'-NMP, a vascular disrupting agent and a photosensitizer to form a compatible polymer, and further preparing a nano-scale coordinated polymer under the action of ultrasound at 0°C.

[0016] The multifunctional coordination polymer can also be used in the preparation of anti-tumor drugs.

[0017] Wherein, the tumor is a solid tumor or a solid tumor with metastasis, and the solid tumor is melanoma, colorectal cancer, breast cancer, liver cancer, glioma, bile duct cancer or pancreatic cancer.

[0018] Among them, in the application, the administration method is intravenous injection or intratumoral injection of nano-preparations; light source irradiation can treat tumors in the body through interventional technologies such as optical fiber and endoscope.

[0019] The application may also be a combination of multifunctional coordination polymer-mediated photothermal therapy and PD-1 antibody.

[0020] Specifically, the coordination polymer accumulates in tumor tissues, and the photosensitizer converts light energy into heat energy, using the thermal effect to kill tumor cells, inducing more powerful tumor immunogenic cell death, triggering the release of tumor self-antigens and damage-related molecular patterns and activating antigen-presenting cells; the activated antigen-presenting cells migrate to the draining lymph nodes to start and activate naive T cells. The number of toxic T cells is increased, and inflammatory chemokines are released to recruit immune cells to the tumor microenvironment and enhance the anti-tumor immune response.

[0021] Principle of the invention: The present invention utilizes drug self-assembly to form coordination polymers to exert photothermal sensitization, wherein a vascular disruptor is used to break the vascular barrier and promote the accumulation of coordination polymers to penetrate tumor tissues; a photosensitizer is used to convert light energy into heat energy, thereby generating a high temperature effect, leading to the destruction of tumor cells and killing tumor cells with thermal effects; nuclear magnetic contrast agents are used for imaging to enhance the physiological and anatomical imaging details of tumor tissues in vivo, so that the tumor position can be accurately located throughout the treatment process, achieving precise photothermal treatment. The coordination polymer is obtained by self-assembling a vascular disruptor, a photosensitizer, and a nuclear magnetic contrast agent, and the above components are combined to achieve a synergistic effect, a vascular disruptor is used to open the tumor vascular barrier and increase the penetration of photosensitizers, and tumor bleeding (hematoporphyrin photosensitizer) is used to further enhance the photothermal effect, thereby enhancing the photothermal anti-tumor immune response, and Gd 3+ Mediated nuclear magnetic resonance imaging enables precise treatment of tumors.

[0022] Solve the problem of insufficient accumulation of nano drugs in tumor sites: Vadimezan (ASA404, DMXAA) is a flavonoid derivative and is considered to be one of the most promising anti-tumor vascular drugs. Studies in recent years have shown that Vadimezan can specifically destroy vascular endothelial cells in tumor tissues, further amplify the EPR effect, and thus enhance drug accumulation in tumors. Therefore, the present invention uses Vadimezan to enhance the EPR effect, further promote the intratumoral accumulation effect based on coordination polymers, and thus enhance the immunogenic death of tumor cells induced by photothermal therapy.

[0023] Solve the problem of tumor recurrence and metastasis during photothermal therapy: Cancer growth and metastasis are usually mediated by immunosuppression and immune escape. Compared with other treatments, immunotherapy can activate the body's own immune system to eradicate cancer cells and has a lasting anti-tumor effect, so it is more conducive to inhibiting cancer metastasis and recurrence. Photothermal therapy can induce immunogenic death of tumor cells, stimulate tumor cells to release tumor-associated antigens and damage-associated molecular patterns, promote dendritic cells to present antigens to activate cytotoxic T cells, initiate systemic anti-tumor immune responses, and inhibit tumor recurrence and metastasis.

[0024] Solve the problem of tumor residue or damage to normal tissue in photothermal therapy: Medical imaging technology can achieve non-destructive, real-time imaging and long-term tracking at the in vivo level, monitor the accumulation of nanomedicines in the tumor site, and provide guidance for photothermal therapy. The resolution of magnetic resonance imaging cannot meet clinical needs in some cases, so magnetic resonance contrast agents are needed in some magnetic resonance examinations to achieve the purpose of enhanced contrast. According to literature reports, about 30% of magnetic resonance examinations require the use of contrast agents. Gadolinium chelates are a commonly used magnetic resonance contrast agent in clinical practice. Traditional gadolinium contrast agents lack targeting, have a short half-life, low relaxation efficiency, and large-scale use can lead to toxic side effects. The present invention prepares the gadolinium contrast agent into a coordination polymer to make it targeted, prolong the retention time in the blood, improve the relaxation efficiency, reduce the total dose, and thus reduce toxic side effects. Therefore, combining tumor imaging diagnostic methods with new treatment methods to achieve imaging-guided tumor treatment is an important method to solve tumor residues and normal tissue damage.

[0025] Furthermore, gadolinium was selected as a nuclear magnetic resonance contrast agent, and at 0.05 mM [Gd 3+] concentration, so the MRI sensitivity of VI@Gd-NPs is better than that of Magnevist, and the maximum signal of VI@Gd-NPs can last until 24 hours after injection. Due to the enhanced accumulation of tumors, this extends the diagnostic window of MRI imaging. Therefore, non-destructive, real-time imaging and long-term tracking at the in vivo level can be achieved to monitor the location of tumors in the body and achieve precise treatment. Vadimezan was selected as a vascular destructive agent, which has high safety in clinical trials and can specifically destroy tumor blood vessels. In tumors treated with VI@Gd-NPs, the ICG signal was significantly higher than that of the I@Gd-NPs group, which effectively increased the intratumoral delivery of the photosensitizer ICG, promoted the penetration and accumulation of nanoparticles in tumor tissues, improved drug efficacy, and reduced side effects.

[0026] The principle of the above coordination polymer is as follows: (1) Multifunctional coordination polymer is prepared by using the principle of drug self-assembly; (2) Photothermal therapy is performed 6 hours after tail vein injection; (3) Through vascular disruptors, coordination polymers are targeted and accumulated in tumor tissues, and photosensitizers convert light energy into heat energy, killing tumor cells by thermal effect, inducing more powerful tumor immunogenic cell death, triggering the release of tumor autoantigens and damage-associated molecular patterns (DAMPs) and activating antigen-presenting cells; (4) Activated antigen-presenting cells migrate to draining lymph nodes to start and activate naive T cells. The number of toxic T cells is increased, and inflammatory chemokines are released to recruit immune cells to the tumor microenvironment, thereby generating a systemic anti-tumor immune response.

[0027] The present invention can kill tumor cells, release tumor-associated antigens, and activate specific memory T cells in two cold solid tumors, 4T1 breast cancer (low immunogenicity, high malignancy, and easy spontaneous metastasis) and CT26 (immune cold tumor), producing a significant difference in the treatment of solid tumors. In the application of coordination polymers, laser therapy can also be combined with anti-PD-1 immune checkpoints to significantly increase the CD8 + It reduces the infiltration of T cells and helper T cells, and increases the number of effector memory T cells and central memory T cells in the spleen, effectively activating the systemic anti-tumor immune response and further significantly improving the therapeutic effect on tumors.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The multifunctional coordination polymer of the present invention has significant therapeutic effects in the treatment of solid tumors. It can kill tumor cells, release tumor-associated antigens, and activate specific memory T cells in two cold solid tumors, 4T1 breast cancer (low immunogenicity, high malignancy, and easy spontaneous metastasis) and CT26 (immune cold tumor), and produce a significant difference in the treatment effect of solid tumors;

[0029] (2) The coordination polymer of the present invention significantly inhibited tumor growth in the mouse MC38 colorectal cancer model. The proportion of macrophages in the primary tumor was significantly reduced by half compared with the saline control group, and Treg cell infiltration was also reduced by nearly 10 times. Not only that, the treatment can also significantly reduce the infiltration of immunosuppressive macrophages and Tregs in distal tumors; the systemic immune response of VI@Gd-NPs+laser treatment successfully reversed the tumor's immunosuppressive microenvironment;

[0030] (3) In the mouse CT26 colorectal cancer model, the coordination polymer of the present invention significantly inhibited the growth of tumors in the treatment group. In the CT26 bilateral tumor model group, the combination with PD-1 antibody can further promote systemic anti-tumor immune response, inhibit the growth of distal tumors, and reduce the expression of tumor-infiltrating CD8 + T cells and CD4 + T cells increased by at least 7-fold and 3-fold, respectively; when the CT26 tumor was challenged three times, the cured mice were 100% suppressed, thus achieving long-term anti-tumor immunotherapy effects and preventing tumor recurrence;

[0031] (4) In the mouse 4T1 breast cancer model, the coordination polymer of the present invention significantly inhibited the tumor growth in the treatment group, with a complete response rate (CR) of 58.3%. After the tumor was removed, the survival of the mice was monitored, and the survival of the mice in the treatment group was significantly prolonged, and the metastatic lesion load in the lung tissue of the VI@Gd-NPs+Laser group was significantly reduced.

[0032] (5) The coordination polymer of the present invention can improve the solubility of poorly soluble drugs and reduce their toxicity, thereby enhancing the efficiency of molecular targeted drug delivery. For some tumors that are difficult to surgically remove and prone to recurrence, the coordination polymer of the present invention can be used in the preparation of anti-tumor drugs to mediate photothermal therapy, which can effectively treat and reduce the risk of recurrence, and can be further combined with anti-PD-1 immunotherapy to enhance the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the assembly of the multifunctional coordination polymer VI@Gd-NPs of the present invention and a schematic diagram of the research idea;

[0034] Figure 2 This is a TEM electron microscope photo of the multifunctional coordination polymer VI@Gd-NPs of the present invention;

[0035] Figure 3 is the particle size distribution of the multifunctional coordination polymer VI@Gd-NPs of the present invention;

[0036] Figure 4 It is the Zeta potential diagram of the multifunctional coordination polymer VI@Gd-NPs of the present invention;

[0037] Figure 5 The stability changes of the multifunctional coordination polymer of the present invention within 96 hours, including: (a) relative change of particle size within 96 hours; (b) change of Zeta potential within 96 hours; (c) change of PDI within 96 hours;

[0038] Figure 6 It is the UV-visible absorption spectrum of the multifunctional coordination polymer VI@Gd-NPs of the present invention;

[0039] Figure 7 Characterization of the temperature rise characteristics of the multifunctional coordination polymer VI@Gd-NPs of the present invention;

[0040] Figure 8 The temperature change of the multifunctional coordination polymer VI@Gd-NPs temperature increase cycle of the present invention;

[0041] Fig. 9 The uptake of the multifunctional coordination polymer VI@Gd-NPs of the present invention in CT26 cells;

[0042] Fig.10 This is the situation of inducing cell apoptosis after the multifunctional coordination polymer VI@Gd-NPs of the present invention is combined with laser irradiation;

[0043] Fig.11 The multifunctional coordination polymer VI@Gd-NPs of the present invention induces immunogenic death after combined laser irradiation, wherein: (a) immunofluorescence light confocal microscope photo, DAPI (blue), CRT (green); (b) quantitative result of CRT on the cell membrane; (c) immunofluorescence confocal photo, DAPI (blue), HMGB1 (red); (d) quantitative result of HMGB1 released into the cell; (e) quantitative result of ATP released into the cell;

[0044] Fig.12 Characterization of the ability of the multifunctional coordination polymer VI@Gd-NPs of the present invention to destroy tumor blood vessels, wherein: (a) Representative photos of tumors in CT26 tumor mice at different time points (0h, 2h, 6h, 12h, 24h, 48h) after intravenous injection of normal saline or VI@Gd-NPs (Vadimezan, 15mg / kg; ICG, 8mg / kg); (b) Representative images of H&E-stained tumor sections 6 hours after intravenous injection of normal saline or VI@Gd-NPs;

[0045] Fig.13 is the tumor accumulation capacity of the multifunctional coordination polymer VI@Gd-NPs of the present invention; wherein a is the average fluorescence intensity of ICG in the tumor after administration of the preparation; b is a representative fluorescence photo of ICG; c is the average fluorescence intensity of ICG in the tumor, heart, liver, spleen, lung and kidney;

[0046] Fig.14 In vivo photothermal imaging of tumors using the multifunctional coordination polymer VI@Gd-NPs of the present invention;

[0047] Fig.15 The magnetic resonance imaging ability of the multifunctional coordination polymer VI@Gd-NPs of the present invention is compared with Magnevist, wherein: (a) T1-weighted magnetic resonance (MR) images of VI@Gd-NPs and Magnevist in PBS; (b) longitudinal relaxation rate (r1) measurement of VI@Gd-NPs and Magnevist; (cd) magnetic resonance (MR) images of VI@Gd-NPs (Gd concentration: 30 mg / kg) and Magnevist in tumors, kidneys and livers at 0 hours, 6 hours, 24 hours and 48 hours after injection; (eg) relative MRI signal intensity of VI@Gd-NPs and Magnevist in tumors (e), kidneys (f) and livers (g) at 0 hours, 6 hours, 24 hours and 48 hours after injection;

[0048] Fig.16 This is a study on the treatment of MC38 colon cancer mice by laser treatment of the multifunctional coordination polymer VI@Gd-NPs of the present invention, including: (a) tumor growth curves of mice receiving various treatment regimens (n=10); (b) weight changes of mice; (c) individual tumor growth trajectories of mice, and the complete remission rate was calculated;

[0049] Fig.17 This is a study on the treatment of CT26 colon cancer mice by laser therapy of the multifunctional coordination polymer VI@Gd-NPs combined with anti-PD-1 immune checkpoints of the present invention, wherein (a) a schematic diagram shows the scheme of VI@Gd-NPs + laser combined with PD-1 antibody to treat mice carrying CT26 tumors; (b) primary and distant tumor growth curves of mice receiving various treatment schemes (n=10); (c) individual distant tumor growth curves of mice, and the partial response rate was calculated;

[0050] Fig.18 The present invention is an immunological evaluation of the multifunctional coordination polymer VI@Gd-NPs laser therapy combined with anti-PD-1 immune checkpoints on CT26 colon cancer mice, wherein (ac) quantitative analysis of cytotoxic (CD3+CD8+) and helper (CD3+CD4+) T cells in the tumor determined by flow cytometry; (df) quantitative analysis of effector memory T cells (CD44+CD62L-) and central memory T cells (CD44+CD62L+) determined by flow cytometry;

[0051] Fig.19The tumor growth curve of mice cured after laser treatment with the multifunctional coordination polymer VI@Gd-NPs of the present invention was re-challenged three times;

[0052] Fig. 20 This is an investigation of the inhibition of 4T1 breast cancer tumor metastasis by laser treatment of the multifunctional coordination polymer VI@Gd-NPs of the present invention, including: (a) 4T1 tumor growth curve over time; (b) survival rate of treated mice; (c) quantitative analysis of lung metastasis; (d) lung metastasis images of mice treated with different methods; (e) representative H&E staining of lung metastasis in each group;

[0053] Fig.21 This is the analysis of serum biochemical and blood indexes in the CT26 tumor-bearing mouse model treated with laser by the multifunctional coordination polymer VI@Gd-NPs of the present invention; wherein ac is a liver function index; d is a kidney function index; eh is a blood-related examination index;

[0054] Fig. 22 H&E staining of major organ tissues in the CT26 tumor-bearing mouse model treated with laser of the multifunctional coordination polymer VI@Gd-NPs of the present invention. DETAILED DESCRIPTION

[0055] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0056] Example 1

[0057] The multifunctional coordination polymer for activating the in situ vaccine induced by hyperthermia of the present invention comprises: a vascular destructive agent is Vadimezan (VDA), a photosensitizer is ICG, a nuclear magnetic contrast agent is a paramagnetic contrast agent (gadolinium agent) Gd chloride, 5'NMP is 5'-GMP disodium salt and 1-5% human serum albumin, and the preparation method is:

[0058] (1) 30 mL of an aqueous solution containing 10 mM 5'-GMP disodium salt was mixed with 20 mL of VDA (Vadimezan 15 mg) or ICG (ICG 8 mg) or VDA & ICG (Vadimezan 15 mg, ICG 8 mg) solution, respectively, and stirred for 30 minutes;

[0059] (2) 20 mL of an aqueous solution containing 10 mM Gd chloride was slowly added to the mixed solution and stirred at room temperature for 1 hour. A precipitate was formed during the stirring process and the precipitate was collected by centrifugation (4000 x g, 20 minutes).

[0060] (3) The collected precipitate was washed three times with pure water and collected by centrifugation (4000 x g, 20 minutes). Subsequently, under the action of human serum albumin and ultrasound, the products were obtained and labeled as V@Gd-NPs, I@Gd-NPs and the multifunctional coordination polymer VI@Gd-NPs for activating the in situ vaccine induced by hyperthermia of the present invention, respectively. The preparation process is as follows Figure 1 shown.

[0061] Characterization of multifunctional coordination polymer VI@Gd-NPs:

[0062] The particle morphology and size of VI@Gd-NPs were observed by transmission electron microscope (TEM) to characterize their appearance. An appropriate amount of prepared VI@Gd-NPs was ultrasonically dispersed before observation, 1-2 drops were dripped onto the copper mesh with a dropper, and the excess liquid was absorbed with filter paper. After standing and drying, it was placed under TEM for observation and photography. The results are shown in Figure 1. Figure 2 As shown, the coordination polymer now presents a cylindrical structure with a particle size of about 60 nm.

[0063] The particle size (Diameter) and polymer dispersity index (PDI) of nanoparticles are important indicators for evaluating nanoparticles. Take 2mL of the prepared nanoparticle solution and test it with a particle size analyzer. The results are as follows Figure 3 As shown, VI@Gd-NPs have a smaller average diameter of 56.89 nm, which is consistent with the TEM results.

[0064] Zeta potential is an indicator of the electrical stability of nanoparticles and their interaction with ions in the surrounding medium. Take 2 mL of the prepared nanoparticle solution and use the phase analysis light scattering (PALS) program in the particle size analyzer to detect it. The results are shown in Figure 2. Figure 4 As shown, the average Zeta potentials of V@Gd-NPs, I@Gd-NPs, and VI@Gd-NPs are −15.67 mV, −14.85 mV, and −10.27 mV, respectively.

[0065] Stability of VI@Gd-NPs:

[0066] The particle size, Zeta potential and PDI of the prepared nanoscale coordination polymer were tested for 96 hours by DLS and PALS. Figure 5 It shows that V@Gd-NPs, I@Gd-NPs and VI@Gd-NPs have good stability, which provides important guarantee for subsequent in vivo experimental applications.

[0067] Example 2

[0068] Spectral analysis of VI@Gd-NPs

[0069] The spectral absorption of the diluted free Vadimezan solution, free ICG solution, unloaded Gd-NPs solution and VI@Gd-NPs solution was scanned at an excitation light wavelength of 300nm-1000nm using a UV-visible spectrophotometer.

[0070] The results are as follows Figure 6 As shown, VI@Gd-NPs showed the same ultraviolet (UV) absorption peaks as free Vadimezan and free ICG at 353nm and 782nm, respectively, confirming that Vadimezan and ICG were successfully coordinated and polymerized with Gd-NPs.

[0071] Example 3

[0072] Verification of the photothermal effect of VI@Gd-NPs in vitro

[0073] The photothermal effect in vitro was used to verify that the ICG carried in the nanopolymer can be converted to light and heat. 1 mL of V@Gd-NPs solution, 1 mL of I@Gd-NPs solution, or 1 mL of VI@Gd-NPs solution was placed in a 1.5 mL EP tube and irradiated with an 808 nm near-infrared laser at a power of 2 W / cm2 for 90 seconds, while a thermometer was used to measure every 15 seconds. After 90 seconds, the solution temperature naturally dropped to room temperature before the next cycle.

[0074] The results are as follows Figure 7 , 8 As shown in the figure, under the laser irradiation with a wavelength of 808nm and an excitation energy of 2W / cm2, I@Gd-NPs and VI@Gd-NPs showed obvious temperature rise characteristics, indicating that the photothermal conversion ability of ICG was not affected by polymer coordination. More importantly, the photothermal conversion ability of ICG was not affected by continuous temperature rise cycles, indicating that VI@Gd-NPs can be used in repeated laser treatments.

[0075] Example 4

[0076] Preparation of VH@Gd-NPs and study of their cellular uptake ability

[0077] The preparation method of VH@Gd-NPs is the same as that of Example 1, the vascular disrupting agent is Vadimezan (VDA), the photosensitizer is hematoporphyrin, the nuclear magnetic contrast agent is the paramagnetic contrast agent (gadolinium agent) Gd chloride (GdCl3), 5'NMP is 5'-GMP disodium salt and 1-5% human serum albumin.

[0078] (1) Mix 30 mL of an aqueous solution containing 10 mM 5'-GMP disodium salt with 20 mL of an aqueous solution containing 1 mM Hemin and 15 mg Vadimezan, and stir for 30 minutes;

[0079] (2) 20 mL of an aqueous solution containing 10 mM Gd chloride was slowly added to the mixed solution and stirred at room temperature for 1 hour. A precipitate was formed during the stirring process and the precipitate was collected by centrifugation (4000 x g, 5 minutes);

[0080] (3) The collected precipitate was washed three times with pure water and collected by centrifugation (4000× g, 5 min). Subsequently, under the action of human serum albumin and ultrasound, nanoscale multifunctional coordination polymer VH@Gd-NPs for activation of hyperthermia-induced in situ vaccine were obtained.

[0081] CT26 cells were cultured at 3 × 10 cells / mL in 1640 medium containing 10% FBS and 1% penicillin / streptomycin in an atmosphere of 5% CO2 and 37°C. 4 The density of cells / well was seeded in a 24-well plate covered with a glass disk. After cell attachment, VH@Gd-NPs were incubated for 4 h. Lysosomes and nuclei were labeled using Lysotracker and DAPI, respectively. Subsequently, tumor cells were washed three times with PBS, and images were acquired and analyzed by confocal laser scanning microscopy (CLSM).

[0082] The results are as follows Fig. 9 As shown, confocal laser scanning microscopy (CLSM) imaging shows the uptake of VH@Gd-NPs by CT26 cells. The nucleus, lysosomes and VH@Gd-NPs are stained with DAPI (blue), Lysotracker (green) and heme (red), respectively. The yellow area shows the localization of heme@Gd-NPs in lysosomes. It can be seen that VH@Gd-NPs can enter CT26 tumor cells through endocytosis and are mainly located in the cell lysosomes.

[0083] Example 5

[0084] Study on apoptosis and immunogenic death induced by VI@Gd-NPs combined with laser therapy

[0085] CT26 tumor cells were cultured at 1x 10 4 Cells were seeded in 96-well plates at a density of 1.5 × 10 cells / well and incubated with PBS, V@Gd-NPs, I@Gd-NPs, or VI@Gd-NPs for 4 h. 2) irradiation for 30 seconds, and then continue to incubate for 12 hours. Cell activity is measured by using CCK-8 kit according to the manufacturer's instructions. In order to further visually observe live / dead cells, treated CT26 cells are stained with Calcein-AM / PI cell activity / cytotoxicity detection kit for 30 minutes and imaged using a fluorescent microscope. Cell apoptosis is measured by Annexin V / PI staining and flow cytometry.

[0086] The results are as follows Fig.10 The cells were treated with PBS, V@Gd-NPs, I@Gd-NPs, and VI@Gd-NPs for 24 hours, with or without 2.0 W / cm 2 Laser irradiation for 20 seconds. CT26 cells stained with Annexin V-FITC / PI were then quantitatively analyzed by flow cytometry. It can be seen that I@Gd-NPs and VI@Gd-NPs significantly increased CT26 cell apoptosis after 808nm laser irradiation, and the cell apoptosis level was increased three times compared with the single photothermal group.

[0087] CT26 cells were cultured at 1x 10 5 The cells were seeded at a density of 100 cells / well in a 12-well plate containing a confocal slide and incubated with PBS, V@Gd-NPs, I@Gd-NPs, or VI@Gd-NPs for 4 h. 2 ) for 30 seconds and then continued to incubate for 12 hours. To confirm the cell surface translocation of CRT and the cellular release of HMGB1, the treated CT26 cells were incubated with anti-CRT antibody or anti-HMGB1 antibody for 1 hour, followed by incubation with ALEXA FLUOR 488-conjugated secondary antibody for 30 minutes, washed three times with PBS, and then stained with DAPI nuclei and photographed using an activated confocal microscope. To detect ATP release, CT26 cells were incubated with 5x 10 3 The cells were seeded at a density of 100 cells / well in a 96-well plate and incubated with PBS, V@Gd-NPs, I@Gd-NPs, or VI@Gd-NPs for 4 h. 2 ) for 30 seconds and then continued to incubate for 24 hours. The culture medium was collected and the release of ATP was detected by ATP detection kit according to the manufacturer's instructions.

[0088] The results are as follows Fig.11 As shown, VI@Gd-NPs-mediated photothermal therapy can effectively amplify ICD and enhance the exposure or release of DAMPs (HMGB-1, ATP, and CRT).

[0089] Example 6

[0090] VI@Gd-NPs induce tumor bleeding and enhance drug accumulation

[0091] First, in order to study the damage effect of tumor blood vessels, CT26 tumor-bearing mice (150-200 mm 3 )VI@Gd-NPs (Vadimezan 15 mg / kg, ICG 8 mg / kg) were injected intravenously. Six hours after injection, the mice were sacrificed and tumor tissues were collected to study bleeding by H&E staining.

[0092] In order to evaluate the accumulation ability of VI@Gd-NPs in tumor tissue, CT26 tumor-bearing mice (150-200 mm 3 ) were treated with VI@Gd-NPs tail vein injection. The mice were anesthetized with isoflurane and fixed in an animal tank. Fluorescence imaging was performed using a small animal living bioluminescence imager. Whole-body fluorescence images of the mice were taken 6 hours after administration. The mice were killed 6 hours later and fluorescence images of their main organs were taken. Fluorescence quantitative analysis was performed using Living Image Version4.5 Software.

[0093] The results are as follows Fig.12 As shown, VI@Gd-NPs coordinated with Vadimezan does not affect its ability to destroy tumor blood vessels and can effectively increase the infiltration of red blood cells into tumors. Fig.13 The distribution of drugs in mice 6 hours after intravenous injection of saline or VI@Gd-NPs. Compared with other organs, VI@Gd-NPs accumulated significantly in tumors and were significantly different from the control group I@Gd-NPs. The above results show that Vadimezan not only destroys tumor blood vessels, but also destroys the barrier that prevents nanoparticles from infiltrating into tumors, thereby enhancing permeability. Therefore, VI@Gd-NPs have the ability to enhance drug accumulation and infiltration in tumors.

[0094] Example 7

[0095] Photothermal effect of VI@Gd-NPs in vivo

[0096] CT26 tumor-bearing mice (150-200 mm3) were intravenously injected with saline, V@Gd-NPs (Vadimezan 15 mg / kg), I@Gd-NPs (8 mg / kg), and VI@Gd-NPs. Six hours after intravenous injection, the mice were anesthetized and their tumor sites were irradiated with 808 nm laser at a power of 2 W / cm 2 Thermographic images were taken every 10 seconds. The temperature of the tumor site was recorded by an infrared thermal imaging camera (Nanjing University, Nanjing).

[0097] The results are as follows Fig.14 As shown in Figure 2, VI@Gd-NPs induce a stronger photothermal effect in vivo. 2 ) In vivo photothermal imaging of tumors treated with saline, V@Gd-NPs, I@Gd-NPs, and VI@Gd-NPs (Vadimezan, 15 mg / kg; ICG, 8 mg / kg) after 90 seconds. The photothermal effect was the strongest in the VI@Gd-NPs group because Vadimezan destroyed tumor blood vessels and increased the infiltration of the drug in the tumor site, more photosensitizer ICG penetrated deep into the tumor, and bleeding in the tumor site increased the infiltration of red blood cells, further enhancing the photothermal effect.

[0098] Example 8

[0099] VI@Gd-NPs in vivo MRI imaging

[0100] The clinical process of photothermal therapy is accompanied by the problem of incomplete tumor elimination. Good tumor contrast effect can accurately guide photothermal therapy to completely eliminate tumor lesions. 3+ ) is commonly used in MRI (magnetic resonance imaging) because its ions are paramagnetic (i.e. contain unpaired electrons).

[0101] In order to evaluate the accumulation of VI@Gd-NPs in tumor tissues and the 3+ ions, VI@Gd-NPs([Gd 3+ ]=30mg / kg) or Magnevist ([Gd 3+ ]=30mg / kg) was intravenously injected into CT26 tumor-bearing mice (150-200mm 3 ). Mice were anesthetized with isoflurane and fixed in an animal trough. An MRI scanner (Biospec 7T / 20USR, Germany) was operated to obtain T1-weighted images. The parameters of T1-weighted imaging were as follows: flip angle = 90, TR = 400ms, TE = 8.90ms, FOV = 8.00 / 4.53cm, matrix = 256x 256, SI = 1.0 / 1.0mm, averages = 3, slices = 12, NEX = 4. MRI images were observed at 0, 6, 24 and 48 hours and analyzed with ImageJ software.

[0102] The results are as follows Fig.15 As shown, Fig.15VI@Gd-NPs have good MRI contrast ability. (a) T1-weighted magnetic resonance (MR) images of VI@Gd-NPs and Magnevist in PBS; (b) Longitudinal relaxation rate (r1) measurement of VI@Gd-NPs and Magnevist; (cd) Magnetic resonance (MR) images of VI@Gd-NPs (Gd concentration: 30 mg / kg) and Magnevist in tumors, kidneys, and livers at 0 h, 6 h, 24 h, and 48 h after injection; (eg) Relative MRI signal intensity of VI@Gd-NPs and Magnevist in tumors (e), kidneys (f), and livers (g) at 0 h, 6 h, 24 h, and 48 h after injection. Compared with Magnevist, T1-weighted images show that VI@Gd-NPs have a better MRI contrast than Magnevist at 0.05 mM [Gd 3+ ] concentration, which showed clear MRI contrast, indicating that the MRI sensitivity of VI@Gd-NPs was superior to that of Magnevist. Intravenous injection of Magnevist and VI@Gd-NPs showed maximum signal intensity in the tumor area 6 hours after injection, while the Magnevist signal began to decrease in the following 48 hours, however, the maximum signal of VI@Gd-NPs could last until 24 hours after injection, which extended the diagnostic window of MR imaging due to the enhanced accumulation of the tumor.

[0103] In addition, both Magnevist and VI@Gd-NPs are metabolized by the kidneys, and VI@Gd-NPs showed a significantly slower renal metabolism rate compared with Magnevist. The above results show that VI@Gd-NPs can effectively accumulate in tumor tissues, making it an effective means of in vivo MR imaging and capable of accurately guiding anti-tumor photothermal therapy.

[0104] Example 9

[0105] Study on the treatment of MC38 colon cancer mice by VI@Gd-NPs combined with laser therapy

[0106] MC38 colorectal cancer cells were digested, washed with PBS, and resuspended in RPMI-1640 medium to prepare 2x10 5 The suspension of 100 cells / mouse was injected subcutaneously into the left side of C57 female mice. When the MC38 tumor volume reached about 100-150mm 3 At 4 pm, mice were injected with Saline, V@Gd-NPs, I@Gd-NPs, and VI@Gd-NPs via intravenous injection. 6 h after intravenous injection, mice were anesthetized with 2% sodium pentobarbital and illuminated with an 808 nm laser (2.0 W / cm 2 ) irradiated the tumor site. Tumor volume and body weight were measured every other day. When the tumor volume reached about 1500mm3 MC38 tumor cells were cultured at 1x10 6 Cells were injected subcutaneously into the right flank of C57 female mice. Tumor volume and body weight were measured every other day.

[0107] The results are as follows Fig.16 As shown in the figure, the primary tumor growth curve of MC38 tumor model mice (n=10). It can be seen that VI@Gd-NPs synergistic laser treatment significantly improved the immunotherapy effect, slowed down the primary tumor of mice, significantly improved the treatment remission rate, and VI@Gd-NPs synergistic laser treatment had no significant effect on the weight of mice.

[0108] Example 10

[0109] Study on the treatment of CT26 colon cancer mice by VI@Gd-NPs synergistic laser therapy combined with anti-PD-1 immune checkpoint

[0110] CT26 colorectal cancer cells were digested, washed with PBS, and resuspended in RPMI-1640 medium to make 2x10 6 The suspension of 1×10 cells / mouse was injected subcutaneously into the left flank of female BALB / C mice. 5 The cells were injected subcutaneously into the right flank of female BALB / C mice. When the primary CT26 tumor reached a volume of approximately 100-150 mm 3 At 1 ℃, mice were injected with Saline, V@Gd-NPs, I@Gd-NPs, and VI@Gd-NPs via intravenous injection. 6 h after intravenous injection, mice were anesthetized with 2% sodium pentobarbital and illuminated with an 808 nm laser (2 W / cm 2 ) irradiated the tumor site. Anti-PD-1 antibody (10 mg / kg) was administered intraperitoneally on days 1, 3, 5, and 7 after treatment, anti-CD8 antibody was administered intraperitoneally on days 1 (200 μg / mouse), 3 (100 μg / mouse), and 5 (100 μg / mouse) after treatment, and anti-CD20 antibody was administered intraperitoneally on days 1 (200 μg / mouse) and 3 (100 μg / mouse) after treatment. Tumor volume and body weight were measured every other day. On day 25 after tumor implantation, mice were killed by cervical dislocation, and distant tumors and spleens were collected. Distant tumors were weighed and ground to prepare single-cell suspensions, and cells were labeled with anti-CD3, anti-CD45, anti-CD4, and anti-CD8 fluorescent antibodies and analyzed by flow cytometry. Single-cell suspensions were obtained by crushing splenocytes through a 40 μm filter and lysing red blood cells. To study the formation of immune blocking and effector memory T cells, cells were labeled and stained with anti-CD3, anti-CD8, anti-CD19, anti-CD44, and anti-CD62L fluorescent antibodies and analyzed by flow cytometry.

[0111] The results are as follows Fig.17As shown in the figure, VI@Gd-NPs+Laser combined with anti-PD-1 immune checkpoint therapy has the best therapeutic effect. VI@Gd-NPs synergistic laser therapy combined with anti-PD-1 immune checkpoint therapy significantly increased the infiltration of CD8+T cells and helper T cells in distal tumors and increased the number of effector memory T cells and central memory T cells in the spleen of these mice. The results are shown in the figure. Fig.18 The above results show that this combination therapy can effectively activate systemic anti-tumor immune response and greatly improve the therapeutic effect.

[0112] Embodiment 11

[0113] Study on VI@Gd-NPs synergistic with laser therapy to enhance long-term tumor-specific immune memory response

[0114] In the CT26 tumor model, mice were cured after treatment with VI@Gd-NPs and 808nm laser. The control group used untreated blank mice. In the first challenge, CT26 colorectal cancer cells were digested, washed with PBS, and resuspended in RPMI-1640 medium to make 2×10 5 The suspension of 5×10 cells / mouse was injected subcutaneously into the right side of BALB / C female mice. For the second and third challenges, 5×10 5 cells / cell and 1×10 6 The suspension of 10 cells / mouse was injected subcutaneously into the right side of female BALB / C mice.

[0115] The results are as follows Fig.19 Figure 2 shows the tumor growth curves of cured and untreated mice during three re-challenges. The three tumor challenges were on days 130, 161, and 182 after the first treatment (n=5); CT26 tumors in cured mice were 100% inhibited, indicating that VI@Gd-NPs synergistic laser treatment can achieve long-term anti-tumor immunotherapy effects and prevent tumor recurrence.

[0116] Example 12

[0117] Study on the inhibition of 4T1 breast cancer metastasis by VI@Gd-NPs synergistic with laser therapy

[0118] 4T1 metastatic breast cancer cells were digested, washed with PBS, and resuspended in RPMI-1640 medium to prepare 2x 10 5 The suspension of 100 cells / cell was injected subcutaneously into BALB / C female mice. When the tumor volume of 4T1 tumor-bearing mice reached about 150-200mm 3At 4 pm, saline, V@Gd-NPs, I@Gd-NPs, and VI@Gd-NPs were injected intravenously. 6 h after intravenous injection, mice were anesthetized with 2% sodium pentobarbital and laser irradiation was performed at 808 nm (2 W / cm 2 ) irradiated the tumor site for 90 seconds. Tumor volume was measured every other day. To further study 4T1 spontaneous lung metastasis, when the tumor volume reached approximately 1000 mm 3 When the primary tumor was removed, the lungs were harvested and the metastatic tumor nodules in the lungs were counted.

[0119] The results are as follows Fig. 20 As shown in the figure, VI@Gd-NPs-mediated photothermal therapy enhanced the immunotherapy effect against 4T1 breast cancer and inhibited metastasis. The tumor volume was significantly suppressed, and the metastatic load of lung tissue was significantly reduced in the VI@Gd-NPs+Laser group. VI@Gd-NPs synergized with laser therapy to activate a strong immune response and effectively inhibited primary and metastatic tumors of 4T1 breast cancer.

[0120] Embodiment 13

[0121] Study on the safety of VI@Gd-NPs synergistic laser therapy

[0122] After the CT26 colorectal cancer-bearing mice were treated with complete therapy (coordination polymer + laser therapy), orbital blood was collected for routine blood analysis and serum biochemical analysis, and all mice were killed by cervical dislocation, and the hearts, livers, spleens, lungs, and kidneys of the mice were collected for H&E analysis.

[0123] The results are as follows Fig. 20 As shown in the figure, the results of blood routine and serum biochemical tests after the mouse tail vein injection of VI@Gd-NPs photothermal therapy showed that the serum biochemical indicators related to liver and kidney function, including ALT, AST, ALP and BUN, did not change significantly; the blood routine indicators such as white blood cell, red blood cell, hemoglobin concentration and hematocrit were all within the normal reference range. Fig.21 , 22 As shown, the H&E staining results of the mouse heart, liver, spleen, lung, and kidney showed that there was no difference between the H&E staining results of the main organ tissues of the mice in each treatment group and those in the control group, indicating that VI@Gd-NPs has good safety and biological tolerance.

[0124] In summary, the multifunctional coordination polymer for activating the in situ vaccine induced by hyperthermia of the present invention is constructed by integrating the vascular disrupting agent, the photosensitizer and the contrast agent Gd 3+, using vascular disruptors to open the tumor vascular barrier, increase the penetration of photosensitizers, and further enhance the photothermal effect by photosensitizers and tumor bleeding (hematoporphyrin photosensitizers), thereby enhancing the photothermal anti-tumor immune response and synergizing with Gd 3+ Mediated nuclear magnetic resonance imaging can achieve precise positioning treatment of tumors, achieve a synergistic effect, enhance tumor immunotherapy, improve the solubility of poorly soluble drugs and reduce toxicity, enhance the efficiency of molecular targeted drug delivery, and can be further combined with laser therapy and / or anti-PD-1 immunotherapy to enhance the treatment effect.

Claims

1. A multifunctional coordination polymer for activating hyperthermia-induced in situ vaccines, characterized in that: The multifunctional coordination polymer is prepared from a vascular disruptor, a photosensitizer, a nuclear magnetic contrast agent, 5'NMP and human serum albumin by chemical coordination self-assembly principle, wherein metal ions in the nuclear magnetic contrast agent and 5'NMP interact with each other through coordination bonds to form a polymer, and the polymer wraps the vascular disruptor and the photosensitizer to obtain the multifunctional coordination polymer.

2. The multifunctional coordination polymer according to claim 1, characterized in that The vascular disruptor is a tubulin binder or a flavonoid compound with anti-vascular function; the photosensitizer is a noble metal nanoparticle, a metal and non-metal compound or a near-infrared dye; the nuclear magnetic contrast agent is a paramagnetic contrast agent, a superparamagnetic contrast agent, a ferromagnetic contrast agent or a diamagnetic contrast agent; the 5'NMP is a nucleoside monophosphate, including GMP, AMP, CMP or TMP.

3. The multifunctional coordination polymer according to claim 2, characterized in that The tubulin binder is colchicine, paclitaxel or docetaxel; the flavonoid compound is flavone-8-acetic acid or 2,5-hexanone theobromine; the noble metal nanoparticles are Au, Ag or Pt; the metal and non-metal compound is CuS; the near-infrared dye is indocyanine green or IR780.

4. A method for preparing the multifunctional coordination polymer according to claim 1, characterized in that: The following steps are involved: (1) mixing an aqueous solution of 5'-NMP, a vascular damaging agent and a photosensitizer, and stirring to obtain a mixed solution; (2) slowly adding the aqueous solution of the nuclear magnetic resonance contrast agent to the mixed solution prepared in step (1), stirring, and centrifuging to collect the precipitate produced by stirring; (3) The precipitate obtained in step (2) is washed with water and collected by centrifugation, and a nanoscale multifunctional coordination polymer for activating the in situ vaccine induced by hyperthermia is obtained under the action of human serum albumin and ultrasound.

5. The method according to claim 4, characterized in that: The molar ratio of 5'-NMP, vascular disrupting agent, photosensitizer, nuclear magnetic contrast agent and human serum albumin is 50-300:10-50:1-10:20-200:1-6, the concentration of human serum albumin is 1-5%, and the temperature condition of ultrasound is 0°C.

6. Use of the multifunctional coordination polymer according to claim 1 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The tumor is a solid tumor or a solid tumor with metastasis, and the solid tumor is melanoma, colorectal cancer, breast cancer, liver cancer, glioma, bile duct cancer or pancreatic cancer.

8. The use according to claim 6, characterized in that: The application is to administer the nano preparation by intravenous injection or intratumoral injection.

9. The use according to claim 6, characterized in that: The application is the combined use of multifunctional coordination polymer-mediated photothermal therapy and PD-1 antibody.

10. The use according to claim 9, characterized in that: Specifically, the multifunctional coordination polymer accumulates in tumor tissues, the photosensitizer converts light energy into heat energy, kills tumor cells by thermal effect, induces more powerful tumor immunogenic cell death, triggers the release of tumor self-antigens and damage-related molecular patterns, and activates antigen-presenting cells; the activated antigen-presenting cells migrate to the draining lymph nodes to start and activate naive T cells. The number of toxic T cells is increased, and inflammatory chemokines are released to recruit immune cells to the tumor microenvironment and enhance anti-tumor immune response.

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