Composition for realizing double benefits of treatment and immune memory and application thereof

Abstract

The invention discloses a composition capable of achieving double benefits of treatment and immune memory and application of the composition, and belongs to the technical field of biological medicine, and the pharmaceutical composition is composed of 5-carboxyl-8-hydroxyquinoline or pharmaceutically acceptable salt thereof and an anti-tumor drug nano preparation. Preferably, the doxorubicin liposome is composed of 5-carboxyl-8-hydroxyquinoline or a pharmaceutically acceptable salt thereof and a sialic acid functional group modified doxorubicin liposome. The composition can promote antigen presenting cell functions, enhance T cell activity, strengthen T cell memory formation, induce deep remodeling of an immunosuppression microenvironment and training immunity of a congenital immune system, generate strong immune memory, realize comprehensive killing of homologous or heterologous tumors, and has a good application prospect. The recurrence of primary tumors, the generation of secondary primary cancers and the attack of other newborn cancers are prevented.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a composition that achieves dual benefits of treatment and immune memory and its application. Background Art

[0002] Tumors are formed by abnormal proliferation of cells in the human body, usually caused by gene mutations, environmental factors (such as radiation, chemicals), viral infections or bad lifestyle habits (such as smoking).

[0003] According to the different roles played by immune cells in the tumor microenvironment, immune cells can be divided into functional tumor suppressor immune cells and tumor-associated immune cells. NK cells, M1 cells, N1 cells, dendritic cells 1 (DC1 cells), helper T cells 1 (Th1 cells) and CD8 + T cells and other cells are functional tumor-suppressing immune cells, while MDSCs, M2 cells, N2 cells, DC2 cells, Th2 cells, and regulatory T cells (Treg cells) are tumor-associated immune cells. In addition to promoting tumor growth, invasion, and metastasis, tumor-associated immune cells also inhibit functional tumor-suppressing immune cells.

[0004] Chemotherapy is a commonly used anti-tumor treatment, which mainly inhibits tumor growth by directly destroying the DNA of tumor cells or interfering with their growth and metabolic processes. However, due to its lack of targeting, it can damage actively proliferating cells in normal tissues (such as bone marrow precursor cells, functional T cells, antigen-presenting cells, etc.), and directly kill and inhibit the function of functional tumor-suppressing immune cells and tumor-associated immune cells indiscriminately, leading to bone marrow suppression, damage to hematopoietic stem cells / progenitor cells, decrease in lymphocytes, weakening of the immune system's regenerative capacity, and impaired antigen presentation function. Therefore, non-targeted chemotherapy not only has poor anti-tumor efficacy, but also leads to the obstruction of antigen presentation and T cell activation, weakening of immune memory function, and lack of protection when patients face pathogen attacks again, which may lead to the recurrence of primary cancer tumors, the development of secondary primary cancers, and other new cancer attacks.

[0005] Tumor recurrence is the main event leading to death.

[0006] After a patient's first cancer is controlled, new cancers may develop elsewhere in the body that are not directly related to the original cancer. These new cancers are called secondary primary cancers. They are not recurrences or metastases of the original cancer, but rather independent new cancers. According to the NCI, the main causes of secondary primary cancers are: common carcinogenic factors (lifestyle, environment, and medical history), treatment methods (radiotherapy, chemotherapy, immunosuppressants), and genetic susceptibility.

[0007] Immunotherapy, such as antibody therapy (immune checkpoint inhibitors, antibody drugs) and cell therapy (CAR-T, etc.), activates the patient's own immune system to enable it to recognize and attack tumor cells. Unlike chemotherapy, immunotherapy aims to enhance the body's anti-tumor ability rather than directly destroy tumor cells. In recent years, the field of tumor treatment has made significant progress driven by technologies such as molecular targeting and immunotherapy. However, immunotherapy has certain limitations. Immunotherapy may have problems such as uneven efficacy, immune-related adverse reactions, and drug resistance. At the same time, the production and delivery costs, storage stability, and immunogenicity associated with immunotherapy are also issues that should be considered.

[0008] 5-Carboxy-8-hydroxyquinoline (IOX1) is a broad-spectrum inhibitor of 2-OG oxygenase. IOX1 does not require derivatization and can directly inhibit JmjC domain-containing lysine demethylases (JmjC KDMs) in cells. By regulating the epigenetic changes of tumor cells, it inhibits multiple intracellular signaling pathways involved in tumor cell growth and suppresses cellular drug resistance. Patent CN 112915087 B discloses a 5-carboxy-8-hydroxyquinoline-based anti-tumor drug sensitizer and its applications, demonstrating that IOX1 reduces PD-L1 expression in tumor cells. Patent CN 112516141 A discloses a CD47 expression inhibitor and its combined use with immunotherapy drugs, demonstrating that IOX1 reduces CD47 expression in tumor cells, enhancing the efficacy of immunotherapy and providing a safer and more effective alternative to PD-L1 or CD47 inhibitors.

[0009] As of May 2025, there are nearly 15,278 combined tumor trials registered on Clinicaltrials.gov. This shows that combination anti-tumor drugs have gradually gained attention in clinical practice and gradually developed into a mainstream treatment option. Summary of the Invention

[0010] The purpose of the present invention is to provide a composition that achieves dual benefits of treatment and immune memory in the anti-tumor process through synergistic effects. The composition can utilize the synergistic and enhanced effects caused by combined drug use to promote the function of antigen-presenting cells, enhance T cell activity, strengthen T cell memory formation, induce deep remodeling of the immunosuppressive microenvironment and train immunity of the innate immune system, generate strong immune memory, achieve comprehensive killing of homologous or heterologous tumors, and prevent primary tumor recurrence, the occurrence of secondary primary cancers, and other new cancer attacks.

[0011] In order to achieve the above purpose, the following technical solutions are adopted:

[0012] A composition achieving dual benefits of treatment and immune memory, consisting of 5-carboxy-8-hydroxyquinoline (IOX1) or a pharmaceutically acceptable salt thereof and an anti-tumor drug nanoformulation.

[0013] Pharmaceutically acceptable salts of 5-carboxy-8-hydroxyquinoline (IOX1) refer to salts formed between IOX1 and an inorganic base, an organic base, or a basic amino acid. Inorganic bases include sodium carbonate, potassium carbonate, and sodium bicarbonate; organic bases include tromethamine and triethylamine; and basic amino acids include L-lysine, L-histidine, and L-arginine.

[0014] Tumors refer to malignant tumors, including blood cancers, colorectal cancer, breast cancer, melanoma, brain cancer, pancreatic cancer, lung cancer, liver cancer and bile duct cancer.

[0015] Anti-tumor drugs are clinically used anti-tumor drugs, including doxorubicin (DOX), epirubicin, anlotinib, idarubicin, mitoxantrone, belotecan, bortezomib, paclitaxel, cyclophosphamide, cisplatin, carboplatin, oxaliplatin, 5-fluorouracil (5-FU), cytarabine, methotrexate, gemcitabine, capecitabine, paclitaxel (Taxol), docetaxel, vincristine, and irinotecan.

[0016] Anti-tumor drug nanoformulations have been functionally targeted and can be preferentially internalized by tumor-associated immune cells, such as MDSCs, M2 cells, N2 cells, DC2 cells, Th2 cells, and Treg cells. These cells then release the anti-tumor drug nanoformulations, thereby killing tumor-associated immune cells.

[0017] At the same time, functional tumor suppressor immune cells, such as NK cells, M1 cells, N1 cells, DC1 cells, Th1 cells and CD8 + The internalization efficiency of anti-tumor drug nanoformulations by T cells and other cells is low, so the number of functional tumor-suppressing immune cells with anti-tumor effects killed by anti-tumor drug nanoformulations is relatively small, thereby retaining the immune system's ability to fight tumors to a certain extent.

[0018] Anti-tumor drug nanoformulations can selectively kill tumor-associated immune cells while retaining functional tumor-suppressing immune cells. As a result, the inhibitory effect of tumor-associated immune cells on functional tumor-suppressing immune cells is also relieved, and immune homeostasis is reshaped. This in turn reduces the degree of tumor immune infiltration, stem cell characteristics, metastatic potential, and proliferation activity.

[0019] The anti-tumor drug nanoformulation is selected from any one of solid preparations, liquid preparations, and semi-solid preparations. Further, the nanoformulation dosage form is any one of liposomes, polyion complexes, phospholipid complexes, nanoparticles, nanocapsules, and polymer micelles.

[0020] Since its advent in the 1960s, liposome technology has rapidly become a hot topic in drug carrier research due to its excellent biocompatibility, diverse drug loading capabilities, and tunable particle size. In the early 1970s, Gregoriadis's team pioneered its potential for drug delivery, thus initiating its industrialization as a drug carrier. After decades of technological advancements, this carrier system has evolved into a clinically validated, highly effective delivery platform.

[0021] Sialic acid is a class of nine-carbon acidic monosaccharides widely found at the termini of cell membrane glycoproteins and glycolipids, with N-acetylneuraminic acid (Neu5Ac) being its most common form. Sialic acid receptors include sialic acid-binding immunoglobulin-like lectins (Siglecs) and the selectin family. Siglecs are a class of immunoglobulin superfamily receptors with extracellular domains containing sialic acid-binding domains and intracellular immunoreceptor tyrosine-based inhibitory motifs, regulating immune cell activity by transmitting inhibitory signals. Siglec-1 is primarily expressed on macrophages and dendritic cells (DCs) and participates in pathogen capture; Siglec-2 is a B cell-specific receptor responsible for regulating B cell receptor signaling; Siglec-7 / 9 are expressed on NK cells and myeloid cells, inhibiting cytotoxicity; and Siglec-10 is a surface marker on regulatory T cells and macrophages, promoting immune tolerance. Tumor cells reshape the microenvironment through abnormal sialylation. Sialic acid on the tumor surface binds to Siglecs receptors, inhibiting the anti-tumor activity of immune cells.

[0022] Furthermore, the anti-tumor drug nanoformulation of the present invention is preferably doxorubicin liposomes modified with sialic acid (SA) functional groups.

[0023] Furthermore, the present invention provides a composition that achieves dual benefits of treatment and immune memory, comprising IOX1 or a pharmaceutically acceptable salt thereof and doxorubicin liposomes modified with sialic acid (SA) functional groups. The composition is used in anti-tumor treatment, and the dosage of IOX1 in the composition is 10.0 mg / kg. -1 ~30.0 mg·kg -1 The dosage of doxorubicin liposomes modified with sialic acid (SA) functional groups was 5.0 mg·kg -1 .

[0024] The present invention provides a composition that achieves dual benefits of treatment and immune memory, which can release the inhibition of effector T cells, induce the expansion and differentiation of tumor antigen-specific CTLs, and form memory T cells with a phenotype of CD3 + CD8 + CD44 + .

[0025] The present invention provides a composition that achieves dual benefits of treatment and immune memory, causing extensive tumor cell death and releasing more neoantigens. It establishes long-term protection through neoantigen diversity and memory T cell pools, strengthens immune memory, and can achieve cross-tumor protection.

[0026] Furthermore, the present invention also provides a method for preparing doxorubicin liposomes, comprising the following steps:

[0027] The membrane material required for preparing liposomes was dissolved in anhydrous ethanol by stirring, and then a portion of the anhydrous ethanol was evaporated, and a citric acid-sodium citrate solution was slowly injected therein to obtain a blank liposome preliminary product. The blank liposome preliminary product was extruded through a carbonate membrane to obtain blank liposomes. Sodium phosphate solution was added to the blank liposomes to adjust the pH of the extraliposome aqueous phase to 7.0. The blank liposomes were mixed with a DOX solution, and the mixture was stirred and incubated to obtain DOX liposomes.

[0028] In the above preparation method, the membrane materials required for preparing liposomes include phospholipids, cholesterol and modifiers. The modifiers include DSPE-PEG, DSPE-PEG 2000 -SH, DSPE-PEG-COOH, sialic acid, sialic acid-cholesterol derivatives.

[0029] In the above preparation method, citric acid is dissolved in sterile water for injection, and the pH is adjusted to 4.0 with sodium hydroxide solution, and then the volume is fixed with sterile water for injection to obtain a citric acid-sodium citrate buffer solution with a pH of 4.0 and a concentration of 200 mM.

[0030] In the above preparation method, sodium phosphate is dissolved in sterile water for injection to prepare a 500 mM sodium phosphate solution.

[0031] In the above preparation method, DOX was dissolved in sterile water for injection to obtain a concentration of 8.0 mg mL -1 of DOX solution.

[0032] In the above preparation method, a liposome extruder is used to sequentially pass the blank liposomes through an 800 nm carbonate membrane 7 times, a 400 nm carbonate membrane 7 times, a 200 nm carbonate membrane 9 times, a 100 nm carbonate membrane 9 times and an 80 nm carbonate membrane 7 times.

[0033] The present invention also provides a composition that achieves the dual benefits of treatment and immune memory, and its use in the preparation of an anti-tumor drug. The tumors include: acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, and liver cancer.

[0034] The present invention has the following beneficial effects:

[0035] The composition provided by the present invention exhibits synergistic anti-tumor effects, achieving dual benefits of treatment and immune memory. The sialic acid-modified anti-tumor drug nanoformulation can achieve targeted effects on tumor-associated immune cells and tumor cells. This nanoformulation also possesses epigenetic regulatory functions, capable of regulating related genes. Therefore, it can be applied to treat solid tumors and achieve comprehensive destruction of homologous or heterologous tumors, effectively preventing the recurrence of primary cancers and the development of secondary primary cancers, while also reducing the risk of developing other new cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the inhibitory effect of each 10X1 salt type on cells in the present invention.

[0037] Figure 2 Schematic diagram of the Transwell cell migration experiment in the present invention.

[0038] Figure 3 It is the tumor cell migration inhibition rate in the present invention.

[0039] Figure 4 Schematic diagram of the Transwell cell invasion assay in the present invention.

[0040] Figure 5 It is the tumor cell invasion inhibition rate in the present invention.

[0041] Figure 6 The changes in the body weight of mice during the acute toxicity study of 10X1-Na in the present invention were set at 10.0, 20.0, 40.0, 50.0, 100.0, 160.0, 320.0, and 500.0 mg kg -1 The patients were given 10X1-Na solution every other day according to the dose gradient.

[0042] Figure 7 In the present invention, the mice were given 10×1-Na solution according to a dose gradient for 8 consecutive days, and then observed for 10 days, and the changes in the weight of the mice were recorded within 18 days.

[0043] Figure 8 It is the survival rate of mice during the acute toxicity study in the present invention.

[0044] Figure 9 The tumor growth curve within 21 days in the present invention is shown in FIG. (a) is the tumor volume change curve, and (b) is the local magnification curve.

[0045] Figure 10 It is the body weight of each group of mice in the present invention within 21 days.

[0046] Figure 11 It is the net body weight of each group of mice in the present invention within 21 days.

[0047] Figure 12 This is the tumor inhibition index curve within 19 days in the present invention.

[0048] Figure 13 This is the survival curve of each group of mice in the present invention during the 40-day observation period.

[0049] Figure 14 In the present invention, on the 40th day, the tumors of mice in each group fell off and were completely healed.

[0050] Figure 15 This is the immunofluorescence staining experiment in the present invention.

[0051] Figure 16 For the second challenge of S180 tumor in the present invention, on the 150th day of the pharmacodynamic test, the surviving experimental mice were stimulated with S180 tumor cells again, 20 days after the second tumor stimulation.

[0052] Figure 17 The immunofluorescence sections of the tumor sites in the present invention were used to evaluate the changes in TAMs in the tumor tissues of these groups of mice.

[0053] Figure 18 This is the third attack of B16F10 melanoma tumor in the present invention, and the tumor growth of mice 20 days after injection of B16F10 tumor cells.

[0054] Figure 19 CD3 in mouse spleen + CD8 + T cell levels.

[0055] Figure 20 This is the flow cytometric analysis of the distribution of tumor cell subpopulations under different treatment conditions in the present invention.

[0056] Figure 21 CD3 in mouse spleen + CD8 + CD44 +T cell levels.

[0057] Figure 22 The immunofluorescence staining technique of the present invention was used to analyze CD68 positive cells in tumor tissues under different treatment conditions. DETAILED DESCRIPTION

[0058] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely illustrative and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0059] The main reagents, instruments, cells and animals used in this invention are:

[0060] Instruments: BS124s electronic analytical balance (Beijing Sartorius Scientific Instrument Co., Ltd.); vernier caliper (Harbin Measuring Tools Co., Ltd.); L400 centrifuge (Xiangyi Centrifuge Instrument Co., Ltd.); SpectraMax M3 microplate reader (MeiGu Molecular Instrument Co., Ltd.); flow cytometer (FACScan, BD Biosciences, USA); 370 series carbon dioxide cell culture incubator (Thermo Fisher Scientific, USA); inverted microscope (Chongqing Optoelectronic Instrument Co., Ltd.).

[0061] Reagents: Mouse spleen lymphocyte separation fluid kit (Tianjin Haoyang Biological Products Technology Co., Ltd.); FITC-conjugated monoclonal anti-mouse CD3 antibody (FITC-CD3, Thermo Fisher Scientific); APC-conjugated monoclonal anti-mouse CD8 antibody and APC-conjugated monoclonal anti-mouse CD44 antibody (APC-CD8, APC-CD44, Thermo Fisher Scientific); mouse monoclonal Ki67 antibody (Thermo Fisher Scientific); 5-carboxy-8-hydroxyquinoline (5-carboxy-8-hydroxyquinoline, 10X1, purity ≥97%, Bid Pharmaceutical Technology Co., Ltd.); sodium carbonate, potassium carbonate, tromethamine, lysine, histidine, and arginine (Shenyang Yuwang Chemical Instrument Co., Ltd.); standard fetal bovine serum, penicillin, and streptomycin (Dalian Meilun Technology Co., Ltd.); and MTT (Sigma-Aldrich Chemical Company).

[0062] Cells and animals: Mouse sarcoma S180 cell line and mouse B16F10 cell line (Shanghai Cell Bank, Chinese Academy of Sciences); Kunming mice (18g-22g, Shenyang Pharmaceutical University Experimental Animal Center).

[0063] Example 1: Screening of pharmaceutically acceptable salts of IOX1

[0064] 1. Preparation of IOX1 salts: Inorganic bases (sodium carbonate, potassium carbonate), organic bases (tromethamine, triethylamine) and basic amino acids (L-lysine, L-histidine, L-arginine) were selected as salt-forming reagents and used in a salt type screening experiment with IOX1. Sodium carbonate (Na2CO3), potassium carbonate (K2CO3), tromethamine (Tris), L-lysine (Lys), L-histidine (His), L-arginine (Arg), and triethylamine (TEA) were prepared into a 0.1 mol·L -1 Accurately weigh 10.0 mg of 10X1 into a 10 mL volumetric flask. First, add the stock solutions of each salt-forming reagent at a molar ratio of 1:1. Then, continue to add the above salt-forming reagent stock solutions to the volumetric flask in small amounts and multiple times, and ultrasonically shake until 10X1 is completely dissolved. Redissolve the solid 10X1 salt in water until it is completely dissolved. The solubility of 10X1-Na (pH 7.6), 10X1-K (pH 7.6), 10X1-Lys (pH 7.3), 10X1-Arg (pH 7.8), and 10X1-Tris (pH 7.5) is greater than 100.0 mg·mL -1 The solubility of IOX1-His (pH 7.1) is approximately 5.6 mg mL -1 ; The solubility of 10X1-TEA is about 0.1 mg·mL -1 .

[0065] 2. MTT assay for toxicity of each 10X1 salt: dilute each 10X1-salt solution (10X1-Na, 10X1-K, 10X1-Lys, 10X1-His, 10X1-Arg, 10X1-Tris, 10X1-TEA) with complete culture medium to prepare 10X1 at concentrations of 0.01, 0.10, 0.25, 0.50, 0.75, and 1.00 mg mL -1 Digest and collect B16F10 tumor cells, and adjust the density of B16F10 tumor cells to 5×10 with complete culture medium. 4 cells·mL -1, inoculate 90.0 μL of B16F10 cell suspension in each test well and control well, add 90.0 μL of complete culture medium to the zeroing well, fill the edge wells of the 96-well plate with sterile PBS, and culture overnight in a 37°C, 5% CO2 incubator. Set up experimental wells, control wells (add culture medium and cells, without drugs), and zeroing wells (add culture medium and equal doses of drugs, without cells), and set three replicates for each sample concentration. Add the diluted 10X1 salts to the test wells and zeroing wells, 10.0 μL per well, that is, incubate the final 10X1 concentrations of 1.0, 10.0, 25.0, 50.0, 75.0, and 100.0 μg mL -1 10 μL of complete culture medium was added to the control wells, and the 96-well plate was placed in a 37°C, 5% CO2 incubator for 48 h. After the incubation, the culture medium in the 96-well plate was discarded, and 10 μL of MTT solution (5.0 mg mL -1 ), incubated in an incubator for 4 h, and added 100.0 μL of triple solution (10% SDS / 5% isobutanol / 0.012 mol·L -1 HCl, wt / v / v) and cultured in a 37°C, 5% CO2 incubator for 12 h. Optical density (OD) was measured at 570 nm using a microplate reader. Cell viability at each concentration was calculated according to the formula: Cell viability (%) = (OD value of experimental well - OD value of zero-adjusted well) / (OD value of control well - OD value of zero-adjusted well) × 100%.

[0066] Various salt forms of IOX1 were only slightly toxic to B16F10 cells, indicating that IOX1 could not inhibit the growth of the above two cell lines through direct cytotoxicity, and there was no significant difference in the inhibitory effect of various IOX1 salt forms on cells ( Figure 1 ).

[0067] 3. Transwell cell migration assay: Dilute 10X1 saline solution (10X1-Na, 10X1-K, 10X1-Lys, 10X1-His, 10X1-Arg, 10X1-Tris, 10X1-TEA) to 20 μg mL in serum-free culture medium (0.5% penicillin / streptomycin, 99.5% DMEM). -1 After starvation for 24 h, B16F10 cells were digested and collected, and the density of B16F10 tumor cells was adjusted to 5×10 5 cells·mL -1. Add 600.0μL of complete culture medium to the lower chamber of the Transwell, add 100.0μL of diluted 10X1 saline solution and 100.0μL of B16F10 cell suspension to the upper chamber, and add 200.0μL of serum-free culture medium to the upper chamber of the control group. Use tweezers to carefully place the upper chamber into the lower chamber containing complete culture medium, ensuring that there are no bubbles on the polycarbonate membrane of the upper chamber to avoid affecting cell migration. Incubate in a cell culture incubator at 37°C and 5% CO2 for 36 hours. After the incubation period, remove the Transwell chamber, discard the culture medium in the upper chamber, gently wipe the cells in the upper chamber with a cotton swab, and place the chamber in 4% paraformaldehyde for 20 minutes. After fixation, place the chamber in 0.1% crystal violet dye for 10 minutes. After staining, wipe the upper side of the chamber with a cotton swab to remove the dye non-specifically attached to the upper surface of the chamber, and rinse the chamber with clean water several times. Remove the chamber, dry the liquid in the upper chamber, and carefully peel off the chamber membrane with tweezers, making sure the bottom is facing up. Wait for the chamber membrane to air dry before taking pictures.

[0068] 4. Transwell Cell Invasion Assay: Dilute Matrigel 8-fold with pre-chilled serum-free medium. Add 100 μL of the diluted Matrigel to the upper chamber of the Transwell (keep on ice to prevent solidification). Incubate at 37°C for 4 h to form a gel layer. Slowly discard the precipitated liquid from the upper chamber. Subsequently, treat the upper and lower chambers as in the cell migration assay and incubate in a cell culture incubator at 37°C, 5% CO2 for 48 h. After incubation, carefully remove the Transwell chamber, discard the medium in the upper chamber, and gently wipe the Matrigel and cells in the upper chamber with a cotton swab. Subsequent procedures are the same as for the cell migration assay.

[0069] Image J was used to calculate the migration / invasion rate and migration / invasion inhibition rate: migration / invasion rate (%) = cell area in the field of view / total field of view × 100%, migration / invasion inhibition rate (%) = (migration / invasion rate of the control group - migration / invasion rate of the experimental group) / migration / invasion rate of the control group × 100%.

[0070] The results of cell migration and cell invasion experiments demonstrated that IOX1 salts have the ability to inhibit tumor cell migration and invasion, and that the basic amino acid salts of IOX1 have a stronger ability to inhibit tumor cell migration / invasion ( Figures 2 to 5 ).

[0071] Example 2: Acute toxicity study of 10X1-Na

[0072] 1. Prepare the solution with sterile water for injection at a concentration of 1.0 mol·L -1Accurately weigh 300.0 mg of 10X1 into a 5 mL volumetric flask. Add the prepared NaHCO3 solution in small amounts and multiple times. Ultrasonicate until the 10X1 is completely dissolved. Add 500 μL of 50% glucose solution to the volumetric flask. Add sterile water for injection to the mark and shake well to obtain a concentration of 60.0 mg mL -1 , 10X1-Na solution with a pH of 7.7, diluted with 5% glucose injection to 10X1 concentrations of 1.0, 2.0, 4.0, 5.0, 10.0, 16.0, 32.0, and 50.0 mg mL -1 .

[0073] 2. Six healthy Kunming mice (half male and half female) were used and the doses were set at 10.0, 20.0, 40.0, 50.0, 100.0, 160.0, 320.0, and 500.0 mg kg -1 The mice were given 10×1-Na solution every other day according to a dose gradient until the mice died or the upper limit of the dose was reached, and the toxic reactions of the animals were recorded in detail.

[0074] 3. During the toxicity test, observe whether the mice breathe normally, including symptoms such as dyspnea (abdominal breathing, wheezing), rapid breathing, nasal discharge, and apnea; observe whether the mice have restricted muscle movement, including symptoms such as general stiffness and paralysis, and whether they have startle reflex and touch reflex; observe the eye symptoms of the mice, including tearing and squinting; observe whether the mice have incontinence; observe whether the mice have normal body hair and whether they have rough fur.

[0075] 4. Analysis of the results of the acute toxicity study of IOX1-Na: When the injection dose of IOX1-Na was 10.0 mg·kg -1 ~160.0 mg·kg -1 The mice were not affected and their body weight showed an increasing trend, indicating that the IOX1-Na dose was lower than 160.0 mg·kg -1 It has no effect on Kunming mice; when the IOX1-Na injection dose is 160.0 mg kg -1 ~500.0mgkg -1 When the IOX1-Na injection dose reached 320.0 mg·kg, the body weight of Kunming mice began to decrease slightly with the increase of IOX1-Na dose. -1 When the dose of 10X1-Na reached 500.0 mg·kg, the mice began to experience shortness of breath and limited muscle movement. The touch reflex disappeared with increasing doses. Eye symptoms began to appear with increasing doses. The fur became rough with increasing doses. However, there was no effect on body fluids. The mice recovered after 0.5 h. When the dose of 10X1-Na reached 500.0 mg·kg-1 When the mice were injected with 500.0 mg·kg-1, they had difficulty breathing (abdominal breathing, panting), lost touch reflex, squinted eyes, became limp, and had rough fur. On the 8th day, some mice died (mortality rate was 16.67%). The mice that survived still had difficulty breathing (abdominal breathing, panting), lost touch reflex, squinted eyes, became limp, and had rough fur 10 hours later, and gradually recovered after 20 hours. -1 When the cumulative injection of 10X1-Na reached 1200.0 mg·kg -1 Considering the survival status and body weight changes, the subsequent single injection dose of IOX1-Na should be controlled at 160.0 mg kg -1 , and the total injection dose should not exceed 380.0 mg·kg -1 , which ensures that IOX1-Na does not cause toxic damage to Kunming mice. Continuous observation of the mice revealed that after stopping the injection of IOX1-Na, the mice's weight gradually recovered. On the 18th day, their weight was higher than the initial weight level, and their fur was shiny and their survival was good, indicating that the damage caused by IOX1-Na to Kunming mice is reversible and recoverable. Figures 6 to 8 .

[0076] Example 3: Preparation of DOX liposomes

[0077] S1. Citric acid-sodium citrate buffer solution: Accurately weigh 4.20 g of citric acid into a 100 mL volumetric flask, dissolve it with sterile water for injection, and adjust the pH to 4.0 with 0.1 M NaOH solution. Then, make up to volume with sterile water for injection and mix thoroughly to obtain a citric acid-sodium citrate buffer solution with a pH of 4.0 and a concentration of 200 mM.

[0078] S2. Sodium phosphate solution: Accurately weigh 9.50 g of sodium phosphate into a 50 mL volumetric flask, dissolve it and dilute to volume with sterile water for injection, and mix thoroughly to obtain a 500 mM sodium phosphate solution.

[0079] S3.DOX solution: Accurately weigh 80.0 mg of DOX into a 10 mL volumetric flask, dissolve it in sterile water for injection and adjust to volume. Mix well to obtain 8.0 mg mL -1 of DOX solution.

[0080] S4. Preparation of blank liposomes: Add 10% (v / v) anhydrous ethanol to the membrane material required for liposome preparation and stir to dissolve in a 60°C-65°C water bath. After the membrane material is completely dissolved, open the system and continue stirring for 10 minutes to evaporate most of the anhydrous ethanol. Slowly inject citric acid-sodium citrate solution preheated to the same temperature into the membrane material and continue stirring in a 65°C water bath for 20 minutes to obtain two blank liposome preliminary products. Use a liposome extruder to pass the blank liposome preliminary products through an 800nm carbonate membrane 7 times, a 400nm carbonate membrane 7 times, a 200nm carbonate membrane 9 times, a 100nm carbonate membrane 9 times, and an 80nm carbonate membrane 7 times to obtain blank liposomes.

[0081] S5. Preparation of Gradient Liposomes and DOX Loading: Prepare blank liposomes and adjust the pH of the extraliposome aqueous phase to 7.0 by adding sodium phosphate solution. Mix the blank liposomes with DOX solution at a drug-to-lipid ratio of 1:10 (w / w). Incubate the mixture in a 65°C water bath with stirring for 10 minutes. Remove the mixture and place it in an ice-water bath for 2 minutes to terminate drug loading. Two types of liposomes, DOX-CL and DOX-SAL, were prepared. The specific formulation compositions are shown in Table 1.

[0082] Table 1 Liposome formulation composition

[0083]

[0084] In Table 1, DOX-CL represents conventional doxorubicin liposomes, and DOX-SAL represents sialic acid-modified doxorubicin liposomes. The sialic acid used is a sialic acid-cholesterol derivative (SA-CH), prepared using the method described in CN113024400A. HSPC represents hydrogenated soy lecithin, and CH represents cholesterol.

[0085] Example 4: Antitumor Pharmacodynamics Study of IOX1 Combined with DOX Liposomes

[0086] 1. Inject S180 tumor cells into male Kunming mice intraperitoneally. When the mice show a significant increase in abdominal circumference and feel cystic to the touch, it indicates that tumor ascites has formed in the abdominal cavity. Collect the viscous ascites under sterile conditions and dilute it with normal saline until the number of S180 tumor cells is 1.8×10 7 cells·mL -1 The S180 cell suspension was inoculated into the subcutaneous tissue of the right anterior armpit of mice, with 0.2 mL inoculated per mouse, and a total of 44 mice were inoculated.

[0087] 2. Forty-four mice inoculated with the S180 tumor strain were randomly divided into 11 groups: Contorl group, doxorubicin solution group (DOX-S), DOX-CL, DOX-SAL, IOX1-Na 10, IOX1-Na 30, IOX1-Na 10 + DOX-CL, IOX1-Na 10 + DOX-SAL, IOX1-Na 30 + DOX-CL, IOX1-Na 30 + DOX-SAL, and IOX1 30 + Single DOX-SAL, with 4 mice in each group. The single dose of DOX in each group was 5.0 mg kg -1 , IOX1-Na 10 group represents a single dose of 10.0 mg·kg -1 , IOX1-Na 30 group represents a single dose of 30.0 mg·kg -1 Single DOX-SAL group was injected with DOX-SAL only once, and Contorl group was injected with the same volume of 5% glucose injection. 3 After that, the drugs were injected into the tail vein, and each group was given once every 2 days for a total of 5 times. During the entire pharmacodynamic test, the body weight, death events and other data were recorded.

[0088] During the experiment, tumor growth curves were plotted for each group of mice, and the area under the curve (AUC) was calculated. The AUC for each group was as follows: Control > IOX1-Na 10 > IOX1-Na 30 > DOX-S > DOX-CL > IOX1-Na 10 + DOX-CL > DOX-SAL > IOX1-Na 30 + DOX-CL > IOX1-Na 10 + DOX-SAL > IOX1-Na 30 + Single DOX-SAL > IOX1-Na 30 + DOX-SAL. The results of the anti-tumor experiment showed that compared with the control group, all treatment groups inhibited the growth of mouse tumors. The tumor growth of mice in the IOX1-Na 30 group was slightly improved, but the tumor growth of mice in the IOX1-Na 10 group was not significantly different from that in the control group. The anti-tumor efficacy of DOX liposomes was stronger than that of DOX-S, and DOX-SAL significantly improved the anti-S180 tumor efficacy compared with DOX-CL. The combined use of IOX1 further enhanced the therapeutic response of DOX. The AUC of the IOX1-Na 30+DOX-SAL group was the smallest, so the ratio of the AUC of the remaining groups to the AUC of the IOX1-Na 30+DOX-SAL group was calculated (AUC Ratio ), the calculation formula is as follows: AUC Ratio =AUC Treated AUC IOX1-Na 30+DOX-SALThe AUC of the other groups was 1.0 to 13.00 times that of the IOX1-Na 30 + DOX-SAL group. Figure 9 .

[0089] Table 2 Area under the tumor growth curve of each group of mice on the 21st day of the pharmacodynamic study

[0090]

[0091] During the experiment, the weight changes of mice were analyzed to investigate the safety of DOX liposomes and IOX1. There was no significant difference in the net body weight of mice in the control group, IOX1-Na 10 group, and IOX1-Na 30 group, indicating that IOX1-Na is non-toxic to mice. In the group injected with DOX liposomes, the net body weight of mice did not change significantly. However, the net body weight of mice in the DOX-S group gradually decreased, indicating that DOX liposomes reduced the systemic toxicity of DOX-S. The results are shown in the table. Figure 10 .

[0092] Tumor inhibition index (TI index, g·g -1 )" (formula: TI index = Body weight / Tumor weight, body weight / tumor weight) breaks through the limitations of traditional simple tumor volume assessment and can more fully characterize the effectiveness and safety of drugs. The larger the TI index, the better the treatment effect. The tumor inhibition index of each treatment group was greater than that of the Control group, and the tumor inhibition index of the DOX liposome treatment group was greater than that of the DOX-S group. The tumor inhibition index of the DOX-SAL, IOX1-Na10+DOX-CL, IOX1-Na 30+DOX-CL, IOX1-Na 10+DOX-SAL, and IOX1-Na 30+DOX-SAL groups first decreased and then showed an upward trend. The TI index curve showed a "tail-up" phenomenon, indicating that these groups had better treatment effects; IOX1-Na10, IOX1-Na 30. The tumor inhibition index of the DOX-CL and DOX-S groups continued to decline; the tumor inhibition index of the DOX-SAL group was greater than that of the DOX-CL group, indicating that the anti-tumor efficacy of the DOX-SAL group was stronger than that of the DOX-CL group. The combined use of IOX1-Na enhanced the anti-tumor effect of each DOX liposome group. The combination of IOX1-Na increased the tumor inhibition index of each DOX liposome treatment group in a dose-related manner. As the dose of IOX1-Na increased, the tumor inhibition index of each IOX1-Na combined with DOX liposome group increased, indicating that the combined treatment not only has excellent anti-tumor effect, but also has less non-specific toxicity to the body and the best overall therapeutic effect. See the results. Figures 10 to 12 .

[0093] Table 3 Tumor inhibition index of mice in each group on the 21st day of pharmacodynamics

[0094]

[0095] Calculate the tumor volume inhibition rate (TIRv) (the formula is: TIR V (%)=(V Control -V Treated ) / V Control × 100%), and the interaction effect (synergistic, additive, or antagonistic) of DOX liposomes and IOX1 was evaluated using Jin's formula, and the synergistic effect index (Jin's Q value) was calculated as: Q = E(A+B) / (EA+EB-EA×EB), where E(A+B) is the TIR of the combined use of IOX1-Na and DOX liposomes. V EA is TIR for IOX1-Na alone V EB is TIR for DOX liposomes alone V Q > 1 indicates a synergistic effect, Q = 1 indicates an additive effect, and Q < 1 indicates an antagonistic effect. The Q values for the IOX1-Na 10 + DOX-CL group were 1.14, and those for the IOX1-Na 30 + DOX-CL group were 1.10, indicating a synergistic effect between IOX1 and DOX-CL. The Q values for the IOX1-Na 10 + DOX-SAL group and the IOX1-Na 30 + DOX-SAL group were both 1.02. According to King's formula, the combination strategy of IOX1-Na 10 + DOX-SAL and IOX1-Na30 + DOX-SAL may simply be additive in effect. However, when DOX-SAL alone approaches its maximum effect (such as EB = 0.94 in this experiment), the denominator (EA + EB - EA × EB) approaches 1. At this time, even if the combined effect is significantly enhanced (E(A+B) = 0.96 / 0.97), the Q value may still be close to 1. Due to the insufficient sensitivity of King's formula to the high-effect interval, the true synergistic effect of IOX1-Na 10 and DOX-SAL is concealed.

[0096] Table 4 Volume inhibition rate and combined effect index of mice in each group on the 21st day of pharmacodynamics

[0097]

[0098] Survival curves were drawn for each group of mice during the 40-day observation period, and the median survival time (MST) of the groups with mortality events was calculated. The survival time of mice in the Control, DOX-S, DOX-CL, IOX1-Na 10, and IOX1-Na 30 groups did not exceed 40 days. It was also observed that the DOX-S group was the first to die. Combined with the changes in body weight and net body volume in the DOX-S group, this indicated that DOX-S caused severe damage to the body and that DOX liposomes had an efficacious and toxic-reducing effect. The median survival time of mice in each group was Control1 (22.5 days), DOX-S (18 days), IOX1-Na 10 (28 days), IOX1-Na 30 (28.5 days), IOX1-Na 30 + Single DOX-SAL (35 days), and DOX-CL (36 days). During the 40-day observation period, no mortality occurred in the IOX1-Na 10+DOX-CL, IOX1-Na 10+DOX-SAL, IOX1-Na 30+DOX-CL, IOX1-Na 30+DOX-SAL, IOX1-Na10+DOX-SAL treatment groups, as well as the DOX-SAL monotherapy group (IOX130+Single DOX-SAL), with a survival rate of 100%. The survival rate of mice in the DOX-CL group was 50%, but after combined treatment with IOX1-Na, the survival rates of the IOX1-Na 10+DOX-CL and IOX1-Na 30+DOX-CL groups were 100%, indicating that IOX1-Na improved the survival rate of DOX-CL-treated mice. The results are shown in Figure 13 .

[0099] No tumor shedding occurred in the Control, DOX-S, IOX1-Na 10, and IOX1-Na 30 groups. Combining the tumor growth curves with the survival analysis, it was found that the tumors in these groups of mice gradually grew until all the mice died, and their survival rates were 0%. The tumor shedding rates of mice in the DOX-CL and IOX1-Na 10+DOX-CL groups were both 25%, and the tumor shedding rate of mice in the IOX1-Na 30+DOX-CL group was 100%, indicating that IOX1 combined with DOX-CL treatment has a higher tumor inhibitory effect than DOX-CL monotherapy. The tumor shedding rates of mice in the IOX1-Na 30+Single DOX-SAL group were both 25%, the tumor shedding rates of mice in the DOX-SAL group were both 50%, the tumor shedding rates of mice in the IOX1-Na 10+DOX-SAL group were both 75%, and the tumor shedding rates of mice in the IOX1-Na The mice in the 30+DOX-SAL group had a 100% tumor shedding rate, indicating that IOX1 combined with DOX-SAL treatment had a higher tumor inhibition effect than DOX-SAL monotherapy. Figure 14 .

[0100] Example 5: Immunofluorescence staining of tumor sites

[0101] CD68, a macrophage-specific marker, can identify tumor-associated macrophages in the tumor microenvironment, and its expression level correlates with the degree of tumor immune infiltration. CD133 is a typical surface marker of tumor stem cells, and a positive signal indicates the presence of a stem cell subpopulation with self-renewal and differentiation potential in tumor tissue. CD44, a transmembrane glycoprotein receptor, not only serves as a stem cell marker but also participates in cell-matrix interactions, and its overexpression is closely associated with tumor invasion and metastasis. Ki-67, a cell proliferation activity marker, quantifies the proliferation index of tumor cells, providing an important basis for assessing tumor malignancy. The combined detection of these four biomarkers can systematically analyze key biological characteristics of tumor tissue, including the immune microenvironment, stem cell characteristics, metastatic potential, and proliferation activity.

[0102] To examine the effects of IOX1 combined with DOX therapy on apoptosis and proliferation of mouse tumor cells, as well as changes in the tumor immune environment, tumor tissues were collected from mice in the Control, IOX1-Na 10+DOX-CL, IOX1-Na 10+DOX-SAL, DOX-CL, DOX-SAL, and IOX1-Na 30+Single DOX-SAL groups (all tumors in mice in the IOX1-Na 30+DOX-CL and IOX1-Na30+DOX-SAL groups were completely detached). Frozen sections of tumor tissues were subjected to multiple immunofluorescence staining using CD68, CD133, CD44, and Ki-67 antibodies, and sections were observed and images were collected using a fluorescence microscope. The results are shown in the table. Figure 15 .

[0103] like Figure 15As shown in the figure, compared with the Control group, the positive rates (fluorescence signals) of CD68, CD133, CD44 and Ki-67 in the other groups were weakened to varying degrees. The magnitude of CD44 fluorescence signals in mouse tumor tissues was: Control>IOX1-Na30+Single DOX-SAL>DOX-CL, while there was almost no CD44 fluorescence signal in the IOX1-Na 10+DOX-CL, DOX-SAL and IOX1-Na 10+DOX-SAL groups, indicating that the treatment downregulated CD44 expression and had the ability to inhibit tumor invasion and metastasis; the magnitude of CD68 fluorescence signals in mouse tumor tissues was: Control>IOX1-Na 30+Single DOX-SAL>DOX-CL>IOX1-Na 10+DOX-CL, while there was almost no CD68 fluorescence signal in the tumor tissues of mice in the DOX-SAL and IOX1-Na 10+DOX-SAL groups, indicating that the treatment downregulated CD68 expression and had the ability to reduce TAMs in the tumor site; the tumor tissue in the Control group expressed weak CD133 fluorescence signals, indicating that there may be only a very small amount of CD133 in the S180 tumor. + The remaining groups barely expressed CD133 fluorescence signals, indicating that the treatment may target CD133. + The ability of cancer stem cell subpopulations to suppress malignant phenotypes; the size of the Ki-67 fluorescence signal in the tumor tissue of mice was: Control>IOX1-Na 30+Single DOX-SAL>DOX-CL>IOX1-Na 10+DOX-CL≈DOX-SAL, while there was almost no Ki-67 fluorescence signal in the tumor tissue of mice in the IOX1-Na 10+DOX-SAL group, indicating that the treatment has the ability to inhibit tumor cell proliferation.

[0104] Example 6: Second challenge of S180 tumor

[0105] On the 150th day of the pharmacodynamic study, the surviving mice were challenged with S180 tumor cells again, and the inoculation density was consistent with the initial tumor-bearing cell density (cells were prepared into 1.0×10 7 cells·mL -1 0.2 mL of S180 cell suspension was inoculated into the subcutaneous tissue under the right anterior armpit of each Kunming mouse).

[0106] After the second tumor attack, no tumor growth was observed in mice treated with IOX1-Na 10+DOX-SAL, IOX1-Na 30+DOX-SAL, IOX1-Na 30+Single DOX-SAL, or DOX-SAL, indicating that DOX-SAL treatment and IOX1-Na combined with DOX-SAL treatment induced antigen-specific immune memory. Tumor growth occurred in mice in the DOX-CL group, and the tumor continued to grow until the mice died, indicating that DOX-CL treatment could not induce antigen-specific immune memory. After tumor growth occurred in mice in the IOX1-Na 30+DOX-CL group, the tumor gradually shrank to palpable punctate connective tissue. This indicates that the combination of IOX1 and DOX-SAL has a positive regulatory effect on the number, function, location, and other factors of the body's immune cells, enabling the body to acquire antigen-specific immune memory. The results are shown in Figure 16 .

[0107] Example 7: B16F10 melanoma tumor third challenge

[0108] By second inoculation of the syngeneic tumor S180, it was observed that treatment with DOX-SAL and IOX1-Na induced antigen-specific immune memory. Therefore, mice were further inoculated with the heterogeneous tumor B16F10 to test whether this immune memory was cross-reactive. If B16F10 grew normally, it would indicate that the immune memory was specific to S180 and could not provide cross-tumor protection. However, if B16F10 was also suppressed in the treatment group, it would indicate that the treatment activated the immune memory, which was cross-reactive (e.g., innate immune training and reprogramming of the immune microenvironment), and could provide cross-tumor protection.

[0109] Thirty days after the second S180 tumor challenge, the surviving mice were challenged with B16F10 tumor cells for the third time: B16F10 melanoma cells were cultured to the logarithmic growth phase, and the cells were collected and the B16F10 cell density was adjusted to 3.5×10 6 cells·mL -1 Prepared B16F10 cells were inoculated into mice through the tail vein, with 0.2 mL injected per mouse, i.e., 7×10 cells were inoculated per mouse. 5 B16F10 cells.

[0110] Twelve days after inoculation with B16F10 tumor cells, mice in the DOX-SAL group died. Tumor growth in other mice 20 days after inoculation with B16F10 tumor cells.

[0111] A control group inoculated with only B16F10 (not treated with S180) was added to exclude the influence of mouse strain or experimental conditions on the growth of B16F10. No melanoma grew in the mice in the IOX1-Na 30+DOX-SAL group, indicating that the immune memory induced by IOX1-Na 30+DOX-SAL is cross-reactive and can achieve cross-tumor protection; one mouse in the IOX1 30+DOX-CL group grew a very small melanoma; melanoma tumors grew in the IOX1-Na 10+DOX-SAL and IOX1-Na 30+Single DOX-SAL groups, but the tumor growth rate and tumor size were smaller than those in the Control group. The tumor growth rate and tumor size were: Control>IOX1-Na 30+Single DOX-SAL>IOX1-Na 10+DOX-SAL>IOX1-Na 30+DOX-CL, indicating that the immune memory induced by IOX1-Na 30+Single DOX-SAL, IOX1-Na 10+DOX-SAL, and IOX1-Na 30+DOX-CL treatment has a weak heterologous tumor protection effect. At the same time, the comparison of IOX1-Na 30+DOX-SAL group and IOX1-Na 30+Single DOX-SAL group showed that single DOX-SAL treatment could not inhibit the third attack of B16F10 tumor cells, while multiple administration of DOX-SAL could inhibit the third attack of B16F10 tumor cells. Figure 17 .

[0112] Example 8: Detection of memory T cell infiltration in mouse spleen

[0113] To elucidate the mechanism of tumor-specific immune memory, we further explored the infiltration of memory T cells in the spleen of mice, and also added a healthy group (Normal) that had not received tumor inoculation and treatment. 20 days after inoculation of B16F10 tumor cells, serum was collected from each group of mice, and spleen cell suspensions were obtained from each group of mice. Splenic lymphocytes were isolated and the obtained single cell suspensions were co-incubated with fluorescent-labeled conjugated antibodies. Memory T cells (phenotype CD3) in spleen lymphocytes were stained using FITC-conjugated anti-mouse CD3 antibodies, APC-conjugated anti-mouse CD44 antibodies, and CD8 antibodies. + CD8 + CD44 + ), first select cytotoxic T cells (phenotype is CD3 + CD8 + ).

[0114] The expression of CD3 in spleen of mice in IOX1-Na 30+Single DOX-SAL, IOX1-Na 10+DOX-SAL, IOX1-Na 30+DOX-CL and IOX1-Na 30+DOX-SAL groups was significant. + CD8 + T cells and CD3 + CD8 + CD44 + The levels of T cells in the spleen of mice in the IOX1-Na 30+DOX-SAL group were higher than those in the control group and even exceeded those in healthy mice, indicating that the above treatment groups successfully induced long-term protective immunity. + CD8 + T cells and CD3 + CD8 + CD44 + The highest level of T cells, CD3 + CD8 + CD44 + The T cell level was 6.17 times that of the control group mice, 2.27 times that of the healthy mice, 1.94 times that of the IOX1-Na 30+Single DOX-SAL group mice, 1.76 times that of the IOX1-Na 10+DOX-CL group mice, and 1.70 times that of the IOX1-Na 10+DOX-SAL group mice, indicating that the immune memory ability induced by IOX1-Na 30+DOX-SAL treatment was the strongest. Figures 18 to 21 .

[0115] Example 9: Detection of TAMs infiltration in tumor sites

[0116] Melanoma growth occurred in mice in the IOX1-Na 30+Single DOX-SAL, IOX1-Na 10+DOX-SAL, IOX1-Na 30+DOX-CL, and Control groups, and the changes in TAMs in the tumor tissues of these groups of mice were evaluated by immunofluorescence sections of the tumor sites.

[0117] The changes in CD68 fluorescence intensity in tumor sections of different groups can reflect the colonization of TAMs in mouse tumors. TAMs infiltration was more in the control group, and the TAMs infiltration density in the IOX1-Na 30+Single DOX-SAL and IOX1-Na 10+DOX-SAL groups decreased to a certain extent; TAMs infiltration was almost not observed in the IOX1-Na 30+DOX-CL group. Figure 22 .

[0118] In the above experiments, we replaced IOX1-Na with IOX1-Arg and the results were similar to those of the experiment using IOX1-Na 30 + DOX-SAL.

Claims

1. A composition for achieving dual benefits of treatment and immune memory, characterized in that: The invention consists of 5-carboxyl-8-hydroxyquinoline or a pharmaceutically acceptable salt thereof and an anti-tumor drug nano preparation.

2. A composition for achieving dual benefits of treatment and immune memory according to claim 1, characterized in that: The pharmaceutically acceptable salt of 5-carboxy-8-hydroxyquinoline refers to a salt formed by 5-carboxy-8-hydroxyquinoline and an inorganic base, an organic base or a basic amino acid; the inorganic base includes sodium carbonate, potassium carbonate, and sodium bicarbonate; the organic base includes tromethamine and triethylamine; and the basic amino acid includes L-lysine, L-histidine, and L-arginine.

3. A composition for achieving dual benefits of treatment and immune memory according to claim 1, characterized in that: Anti-tumor drugs include doxorubicin, epirubicin, anlotinib, idarubicin, mitoxantrone, belotecan, bortezomib, paclitaxel, cyclophosphamide, cisplatin, carboplatin, oxaliplatin, 5-fluorouracil, cytarabine, methotrexate, gemcitabine, capecitabine, paclitaxel, docetaxel, vincristine, and irinotecan; anti-tumor drug nanoformulations are selected from any one of liposomes, polyion complexes, phospholipid complexes, nanoparticles, nanocapsules, and polymer micelles.

4. A composition for achieving dual benefits of treatment and immune memory according to claim 1, characterized in that: The composition is composed of 5-carboxy-8-hydroxyquinoline or a pharmaceutically acceptable salt thereof and doxorubicin liposomes modified with sialic acid functional groups; the dosage of 5-carboxy-8-hydroxyquinoline is 10.0 mg·kg -1 ~30.0 mg·kg -1 The dosage of sialic acid functional group-modified doxorubicin liposomes was 5.0 mg kg -1 .

5. A composition for achieving dual benefits of treatment and immune memory according to claim 4, characterized in that: The preparation method of doxorubicin liposomes comprises the following steps: The membrane material required for preparing liposomes was dissolved by stirring in anhydrous ethanol, and then a portion of the anhydrous ethanol was evaporated, and a citric acid-sodium citrate solution was slowly injected therein to obtain a blank liposome preliminary product. The blank liposome preliminary product was extruded through a carbonate membrane to obtain blank liposomes, and a sodium phosphate solution was added to the blank liposomes to adjust the pH of the extraliposome aqueous phase to 7.

0. The blank liposomes were mixed with a doxorubicin solution, and the mixture was stirred and incubated to obtain doxorubicin liposomes.

6. A composition for achieving dual benefits of treatment and immune memory according to claim 5, characterized in that: The membrane materials required for preparing liposomes include phospholipids, cholesterol and modifiers; the modifiers include DSPE-PEG, DSPE-PEG 2000 -SH, DSPE-PEG-COOH, sialic acid, sialic acid-cholesterol derivatives; using a liposome extruder, the blank liposome sample was passed through an 800 nm carbonate membrane 7 times, a 400 nm carbonate membrane 7 times, a 200 nm carbonate membrane 9 times, a 100 nm carbonate membrane 9 times and an 80 nm carbonate membrane 7 times.

7. Use of the composition according to any one of claims 1 to 6 for achieving the dual benefits of treatment and immune memory in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The tumors include: acute leukemia, malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, and liver cancer.

9. Use of 5-carboxy-8-hydroxyquinoline or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

10. Use of 5-carboxy-8-hydroxyquinoline or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting tumor cell invasion and / or migration, characterized in that: The pharmaceutically acceptable salt is a salt formed by 5-carboxy-8-hydroxyquinoline and lysine, arginine or histidine.

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