Novel nano-drug for treating in-situ malignant melanoma and metastatic tumor thereof as well as preparation method and application of novel nano-drug

Abstract

The invention discloses a novel nano-drug for treating in-situ malignant melanoma and metastatic tumors thereof as well as a preparation method and application of the novel nano-drug, and belongs to the technical field of drug preparation. The novel nano-drug is obtained by loading OsCo coated with a lipid layer on the surface of anaerobic bacteria. During preparation, the nano material OsCo is firstly prepared, then the surface of the nano material OsCo is modified with the lipid layer, and finally the nano material OsCo modified with the lipid layer is loaded to the surface of anaerobic bacteria. According to the novel nano-drug, an OsCo-coated lipid layer capable of efficiently producing ROS is carried on the surface of anaerobic bacteria, a targeted multifunctional oncolytic bacterium (Bac-coated OsCo-L) is built, MM is targeted by utilizing the hypoxia taxis characteristic of the anaerobic bacteria, anaerobic glycolysis is carried out in a tumor to adjust the tumor microenvironment (reduce pH), the ROS yield is increased, CDT is achieved, and the novel nano-drug is a novel nano-drug. Meanwhile, the passive state and the immunosuppression environment of tumor-associated antigens are reversed, and CDT / immune multi-mode treatment is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and specifically relates to a novel nano-drug for treating in-situ malignant melanoma and its metastatic tumors, as well as a preparation method and application thereof. Background Art

[0002] Tumors are a major public health problem worldwide and have become an important factor threatening human health. Melanoma (MM) is the most common malignant skin tumor, which can occur in any part of the skin, with a high incidence rate, strong invasiveness, easy early metastasis, difficult treatment, and high mortality rate, bringing a heavy burden to society and families. Although there are currently many treatment methods, such as chemotherapy, surgical resection, immunotherapy, and targeted therapy, etc., however, it is still not possible to completely eradicate MM. Therefore, researchers have been constantly seeking new treatment models.

[0003] In the 1980s, it was found that using nanomaterials as carriers for anti-cancer drugs helps to enhance the penetration and retention of drugs in cells (EPR effect). Later, it was found that many nanomaterials can simulate biological enzymes to regulate cytokines and reactive oxygen species (ROS) for tumor treatment with significant curative effects. However, after nearly 20 years of research, there are very few nano-drugs that can be clinically translated. Among them, the targeting of nano-drugs and the precise and efficient release of active substances have become one of the main bottlenecks hindering the development of cancer nano-drugs and even clinical translation.

[0004] Currently, tumor nano-medicine mainly focuses on the research and development of ROS regulation in tumor cells. Most of these nano-drugs utilize the characteristics of overexpression of H2O2 and weak acidity in the tumor microenvironment to generate Fenton or Fenton-like reactions and produce excessive ROS. However, relying solely on the generation of ROS in the body's internal environment for tumor treatment has limited efficacy. Summary of the Invention

[0005] The purpose of the present invention is to provide for the first time a novel nano-drug for treating in-situ malignant melanoma and its metastatic tumors, as well as a preparation method and application thereof, aiming to explore an efficient, novel, and low-side-effect treatment strategy for advanced MM tumor patients who have lost the opportunity for surgery, and is expected to solve the key scientific problems faced by cancer nano-medicine. Specifically, this novel nano-drug consists of OsCo with high ROS production wrapped in a lipid layer and carried on the surface of anaerobic bacteria to form a targeted multi-functional oncolytic bacterium (Bac@OsCo-L). Utilizing the hypoxic-taxis characteristic of anaerobic bacteria to target MM, anaerobic glycolysis is carried out in the tumor to regulate the tumor microenvironment (reduce pH), increase ROS production, achieve CDT, and at the same time reverse the passive state of tumor-associated antigens and the immunosuppressive environment to achieve CDT / immune multi-modal therapy.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first technical solution: A novel nano-drug for treating in-situ malignant melanoma and its metastatic tumors, which is obtained by loading OsCo wrapped with a lipid layer onto the surface of anaerobic bacteria.

[0008] MM is extremely malignant, with rapid cell growth, high oxygen consumption, and a glycolysis-based metabolic mode that forms a tumor microenvironment rich in lactic acid and hypoxia, making it difficult for anti-tumor drugs to penetrate and limiting their effects. The hypoxic microenvironment is exactly a good environment for the growth of anaerobic bacteria, and anaerobic bacteria have an innate tendency towards hypoxia. Therefore, anaerobic bacteria can automatically target tumors, reach the tumors and compete with tumor cells for nutrients to restrict tumor growth, and the inherent pro-inflammatory ability of bacteria can also stimulate the body's immune system. At the same time, anaerobic respiration occurs in the tumors, producing metabolic products such as butyric acid and acetic acid, reducing the pH value in the tumors, regulating the tumor microenvironment, and facilitating the nano-drug to play its role. Therefore, anaerobic bacteria theoretically have dual functions of targeting and oncolysis, and this application calls them oncolytic bacteria. An ideal oncolytic bacterium should have characteristics such as strong targeting, low side effects, strong biocompatibility, and good stability.

[0009] Bacillus amylobacter is an anaerobic bacterium discovered earlier and is commonly used in food and livestock production. It has high safety, targets hypoxic tumor tissues, reduces the pH value, and Bacillus amylobacter gradually dies while stimulating tumor immunity. Therefore, Bacillus amylobacter as the main body of oncolytic bacteria is expected to solve the targeting problem of nano-drugs, and at the same time has the effects of regulating the tumor microenvironment and enhancing tumor immunogenicity.

[0010] Therefore, the present invention designs a novel nano-drug for the treatment of in-situ malignant melanoma and its metastatic tumors. Osmium cobalt (OsCo) with high ROS production is wrapped with a lipid layer and carried on Bacillus amylobacter to build a multifunctional oncolytic bacterium treatment platform (Bac@OsCo-L). The oncolytic bacterium targets tumor cells, undergoes anaerobic glycolysis to reduce the pH value, and then increases the ROS production to achieve enhanced CDT treatment. The generated cell debris, dead Bacillus amylobacter debris, and inflammatory factors secreted by Bacillus amylobacter reverse the passive state of tumor-associated antigens (TAAs) and the tumor immune suppression microenvironment, initiate the body's tumor immunity, effectively inhibit in-situ carcinoma of murine melanoma and its distant metastases, and achieve CDT / immune combined anti-tumor.

[0011] Preferably, the anaerobic bacterium is Bacillus amylobacter.

[0012] The second technical solution: A preparation method of a novel nano-drug for treating in-situ malignant melanoma and its metastatic tumors, comprising the following steps:

[0013] S1. Prepare the nano-material OsCo;

[0014] S2. Modify a lipid layer on the surface of the nano-material OsCo;

[0015] S3. Load the lipid layer-modified nanomaterial OsCo onto the surface of anaerobic bacteria.

[0016] The third technical solution: The application of a novel nanomedicine for the treatment of in-situ malignant melanoma and its metastatic tumors in the preparation of products for treating melanoma.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] Anaerobic bacteria can actively target the hypoxic and nutrient-rich tumor microenvironment, break through the resistance of tumor blood vessels and high interstitial pressure, and proliferate in vivo. They can induce the activation of the immune system in tumor tissues, and the immune response attacks anaerobic bacteria and tumor cells in a manner independent of tumor antigens, producing an anti-tumor effect. Therefore, the present invention designs a novel nanomedicine for the treatment of in-situ malignant melanoma and its metastatic tumors. This nanomedicine consists of OsCo with high ROS production wrapped in a lipid layer and carried on the surface of anaerobic bacteria (Butyricimonas), constructing a targeted multi-functional oncolytic bacterium (Bac@OsCo-L). Utilizing the hypoxic property of anaerobic bacteria to target MM, anaerobic glycolysis is carried out in the tumor to regulate the tumor microenvironment (reduce pH), increase the ROS production, achieve CDT, and at the same time reverse the passive state of tumor-associated antigens and the immunosuppressive environment, achieving CDT / immune multimodal therapy. It aims to explore an efficient, novel, and low-side-effect treatment strategy for advanced MM tumor patients who have lost the opportunity for surgery and is expected to solve the key scientific problems faced by cancer nanomedicine. The schematic diagram of the immunotherapy induced by Bac@OsCo-L is as Figure 32 shown. Brief Description of the Drawings

[0019] Figure 1 : Synthesis and characterization of Bac@OsCo-L.

[0020] (a) TEM of OsCo. (b) TEM of OsCo-L. (c) TEM of Bac. (d) TEM of OsCo-L@Bac. (e) Detection of hydroxyl radical content of OsCo-L by TA. (f) Detection of singlet oxygen production of OsCo-L by ESR. (g) EDX mapping images of Os, Co, O, and F elements in OsCo, scale bar: 300 nm. (h) POD activity of OsCo-L in solutions with different pH values. (i) Zeta potential of OsCo, OsCo-L, B, and Bac@OsCo-L. (j) OD600 of Bac and OsCo-L@Bac at different time points. (k) Hydrodynamic diameter of OsCo. (l) Hydrodynamic diameter of OsCo-L.

[0021] Figure 2 : In vitro experimental study on the treatment of MM by Bac@OsCo-L.

[0022] (a) Killing effects of Control, OsCo-L, B, and OsCo-l@B groups on B16-F10 cells. (b) Flow cytometry analysis of intracellular reactive oxygen species levels after treatment under different conditions. (c) Statistical analysis of fluorescence intensity of mitochondrial membrane potential staining in B16-F10 cells after treatment under different conditions. (d) Flow cytometry analysis of apoptosis levels of B16-F10 after treatment under different conditions. (e) Statistical analysis of fluorescence intensity at different time points after B16-F10 cells were stained with pH fluorescence probe after treatment with B. (f) Statistical analysis of fluorescence intensity after B16-F10 cells were treated with pH gradient medium and stained with pH fluorescence probe. (g) Statistical analysis of calreticulin CRT and (h) HMGB-1 in tumor cells under different treatment conditions. (i) Flow cytometry analysis results of mature dendritic cells (CD11c + CD80 + CD86 + ). (j) Release of TNF-α and (k) IFN-γ in cells after different treatments. (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001).

[0023] Figure 3 : In vivo therapeutic effect and mechanism study of Bac@OsCo-L in the treatment of MM.

[0024] (a) Ex vivo tumor fluorescence images at different time points after treatment with OsCo-L and OsCo-L@B and (b) Quantitative statistical analysis of ex vivo fluorescence intensity of heart, liver, spleen, lung, and kidney tumors 12 h after injection, n = 3. (c) Statistical analysis of relative fluorescence intensity of OsCo-L and OsCo-L@B in blood at different time points. (d) Flow chart of mouse orthotopic tumor. (e) Changes in orthotopic carcinoma tumor volume in mice under different treatments and (f) Tumor mass statistics on the 8th day, n = 3. (g) tSNE maps of the distribution of various immune cells in tumors and (h) spleens.

[0025] Figure 4 : In vivo therapeutic effect and mechanism study of Bac@OsCo-L in the treatment of metastatic carcinoma of MM.

[0026] (a) Establishment and treatment flow chart of mouse metastatic carcinoma model. (b) Primary tumor volume of tumor-bearing mice during treatment, n = 3. (c) Primary tumor weight in different groups, n = 3. (d) tSNE maps of the distribution of immune cells in tumors and (e) spleens in different groups. (f) Proportion of various immune cells in tumors and (g) spleens in different groups. (h) Statistical chart of Tcm and Tem in CD4 + T cells. (i) CD8 +Statistical graphs of Tcm and Tem in T cells. (j) Statistical data of TNF-α and (k) IFN-γ in mouse serum.

[0027] Figure 5 : Hydroxyl radicals and superoxide anions were generated by OsCo-L through ESR testing.

[0028] Figure 6 : Singlet oxygen was measured by OsCo-L using SOSG.

[0029] Figure 7 : pH values of Bac and OsCo-L@Bac at different time points.

[0030] Figure 8 : Cytotoxicity of different concentrations of Bac@OsCo-L against B16-F10 cells.

[0031] Figure 9 : Live / dead fluorescence images of B16-F10 cells under different treatment methods (a). Statistical analysis of the viability of B16-F10 cells through live / dead fluorescence images (b).

[0032] Figure 10 : Apoptosis rate of cells in each group.

[0033] Figure 11 : Statistical analysis of intracellular reactive oxygen species levels after treatment of cells under different conditions by flow cytometry.

[0034] Figure 12 : Detection of intracellular reactive oxygen species in cells of different treatment groups using DCFH-DA probe (a). Statistical analysis of fluorescence intensity in cells of different treatment groups (b).

[0035] Figure 13 : Fluorescence images of mitochondrial membrane potential staining of B16-F10 cells after treatment under different conditions.

[0036] Figure 14 : Fluorescence images of B16-F10 cells at different time points after staining with pH fluorescence probe after treatment with B.

[0037] Figure 15 : Fluorescence images of B16-F10 cells after treatment with pH gradient medium and staining with pH fluorescence probe.

[0038] Figure 16 : CRT fluorescence images of tumor cells under different treatment conditions (a). HMGB-1 fluorescence images of tumor cells under different treatment conditions (b).

[0039] Figure 17 : DC activation statistics.

[0040] Figure 18:Ex vivo fluorescence images of the heart, liver, spleen, lung, and kidney of mice treated with OsCo-L and OsCo-L@B.

[0041] Figure 19 :Plating images of tumor homogenates from the heart, liver, spleen, lung, and kidney of mice treated with OsCo-L and OsCo-L@B.

[0042] Figure 20 :Ex vivo tumor images (a) and body weight changes (b) of mice after treatment with different methods.

[0043] Figure 21 :Statistical data of TNF-α (a) and IFN-γ (b) in mouse serum.

[0044] Figure 22 :HE, Ki-67, and TUNEL images of tumors from mice in different treatment groups.

[0045] Figure 23 :Flow cytometry detection of DC maturation in each group and its statistical data.

[0046] Figure 24 :Proportion of each immune cell in the tumor in the CD45 + cell population.

[0047] Figure 25 :Proportion of each immune cell in the spleen in the CD45 + cell population.

[0048] Figure 26 :Ex vivo tumor images (a) and body weight changes (b) of mice after treatment with different methods.

[0049] Figure 27 :Flow cytometry detection of DC maturation in each group and its statistical data.

[0050] Figure 28 :Effects of different concentrations of Bac@OsCo-L on endothelial cells (a). Effects of different treatment groups on endothelial cells (b).

[0051] Figure 29 :Hemolysis rate (a) and images (b) of different concentrations of Bac@OsCo-L.

[0052] Figure 30 :HE images of the heart, liver, spleen, lung, and kidney of mice.

[0053] Figure 31 :Statistical data of blood biochemical indexes ALT, AST, CK, CREA, and UREA in mice.

[0054] Figure 32 :Schematic diagram of immunotherapy induced by Bac@OsCo-L. Detailed implementation methods

[0055] 1. Synthesis and Characterization of Bac@OsCo-L( Figure 1 )。

[0056] In the tumor microenvironment, there is a hypoxic and weakly acidic environment. The weakly acidic environment is conducive to the nanomaterials with peroxidase-like activity to play a CDT role in generating reactive oxygen species to kill tumor cells. In this application, a nanomaterial OsCo with peroxidase-like activity was prepared (Co(NO3)2·6H2O (2 mmol, 582.06 mg) + urea (7.5 mmol, 450.3 mg) + NH4F (ammonium fluoride, 6 mmol, 222.24 mg) + 35 mL deionized water, stirred for 4 h. Hydrothermal treatment at 120 °C for 6 h, vacuum drying, and air heat treatment at 300 °C for 4 h to obtain Co3O4-F. 50 mg Co3O4-F + 10 mL water, in a 20 mL vial, add 0.2 mL, 0.5 mL, 1 mL, and 2 mL OsCl3 (10 mg / ml), stir for 24 h, filter, wash, and dry)( Figure 1 a), and its hydrodynamic diameter is about 43.7 nm( Figure 1 k), and its EDX mapping image shows that the main elements are Os, Co, O, and F( Figure 1 g). To load it onto the surface of anaerobic bacteria Bac( Figure 1 c), in this application, the surface of OsCo was wrapped with a lipid layer (DPPC, DPPE-mPEG2000, DC-Chol) to obtain the material OsCo-L( Figure 1 b). Then, TA, ESR, and particle size analyzer were used to detect the performance changes of the nanomaterials before and after wrapping the lipid layer, such as Figure 1 e, 1f, 1h, 1l and Figure 5 、 Figure 6 . The results show that the hydrodynamic diameter of OsCo-L after wrapping the lipid layer is about 115 nm, and its ability to produce reactive oxygen species is reduced compared with before, but it still has a high CDT effect. Finally, OsCo-L (1 mg / mL) and Bac (1×10 8 CFU / ml) were connected together by non-covalent binding to obtain Bac@OsCo-L. By detecting the Zeta potential of OsCo, OsCo-L, B, and Bac@OsCo-L, the possibility that Bac is connected to OsCo-L through electrostatic interaction was proved, and the binding of the two was verified again by TEM( Figure 1 d).

[0057] To further verify whether OsCo-L has an impact on the growth activity and production performance of anaerobic bacteria Bac, in this application, Bac and Bac@OsCo-L were used as controls, with a concentration of 40 μg / mL×10 5CFU / ml Bac@OsCo-L and 40×10 5 CFU / ml Bac was cultured in an anaerobic incubator, and the OD600 of the solution was measured at different time points. As Figure 1 shown in j, the effect of OsCo-L on the growth activity of Bac was not obvious. Then, Bac and Bac@OsCo-L were cultured in the same way, and the pH of the solution was detected at different time points. As Figure 7 shown, the effect of OsCo-L on the acid-producing ability of Bac was not obvious.

[0058] In summary, a nanomaterial OsCo with high efficiency in producing reactive oxygen species was synthesized in this application, and it was successfully loaded onto the surface of anaerobic bacteria by modifying the lipid layer on its surface, synthesizing a novel and efficient reactive oxygen species-producing and acid-producing bacterial hybrid Bac@OsCo-L.

[0059] 2. In vitro effect and mechanism study of Bac@OsCo-L in the treatment of MM ( Figure 2 ).

[0060] To evaluate the in vitro effect and mechanism of Bac@OsCo-L in the treatment of MM. In this application, an anti-tumor experiment of Bac@OsCo-L was carried out using B16-F10 cells as a representative model to detect its biocatalytic killing ability. The catalytic anti-tumor ability of Bac@OsCo-L against B16-F10 cells was confirmed by CCK-8 experiment, and the pH 6.5 acidic complete medium was used for culture to simulate the TME conditions; the results showed that the half-inhibitory concentration value of the Bac@OsCo-L group against B16-F10 cells was about 40 μg / mL×10 5 CFU / ml ( Figure 8 ). Therefore, a dose of 40 μg / mL×10 5 CFU / ml was used in the subsequent anti-tumor research of B16-F10 cells. As Figure 2 a and Figure 9 shown, the control group (Control) hardly showed any cell damage, while a large number of dead cells appeared in the Bac@OsCo-L group. Thereafter, this application detected and evaluated the intracellular ROS level by 2,7-dichlorofluorescein diacetate (DCFH-DA) and flow cytometry (FCM); as Figure 2 b and Figure 11 , Figure 12 shown, the Bac@OsCo-L group showed the highest ROS intensity, and flow cytometry (FCM) detection gave similar results, thus proving that the ROS produced by Bac@OsCo-L could effectively kill tumor cells ( Figure 2 d, Figure 10 ).

[0061] In this application, a mitochondrial membrane potential detection kit (JC-1) was used to test the damage of Control, OsCo-L, B, and Bac@OsCo-L to the mitochondrial membrane potential of tumor cells. The results showed that the Bac@OsCo-L group had the greatest degree of reduction in mitochondrial membrane potential, thus having the strongest killing effect on tumor cells. Figure 2 c, Figure 13 ) After that, in order to observe the fluctuation of intracellular pH value after treatment with B. As Figure 2 e and Figure 14 shown, as the incubation time extended, the intracellular pH value seemed to decrease. To further study the effect of extracellular acidification induced by B on intracellular pH value, B16-F10 cells were treated with a pH gradient medium. The intracellular pH value also decreased. Figure 2 f, Figure 15 )

[0062] The results showed that the decrease in pH value of cancer cells after treatment with B was due to the acidification of the culture medium by B. To test the ICD activation potential of Bac@OsCo-L, this application separately examined two key markers in B16-F10 cells, namely CRT and HMGB1. CRT releases the "eat me" signal, exposes the surface of apoptotic cells, and stimulates dendritic cells (DCs) to phagocytose apoptotic cells and their fragments. HMGB1 also plays a key role in inducing the tumor ICD effect. It acts as a Toll-like receptor 4 agonist to stimulate antigen presentation to T cells. CRT and HMGB1 were measured by confocal laser scanning microscopy (CLSM). As Figure 2 g and Figure 16 a shown, the exposure level of CRT after treatment with Bac@OsCo-L was much higher than that of other groups. At the same time, after treatment with Bac@OsCo-L, HMGB1 was released from the nucleus, and the amount of HMGB1 in tumor cells decreased, while the naked OsCo-L and B groups showed limited release effects. Figure 2 h and Figure 16 b) DCs are one of the main antigen-presenting cells (APCs) and play a crucial role in innate and adaptive immunity. This application further examined the activation of bone marrow-derived DCs by the tumor cell culture medium after different treatments. CD80 + and CD86 + are considered representative markers of DC maturation. Figure 2 i, Figure 17 revealed that compared with other groups, Bac@OsCo-L could effectively activate the maturation of DCs. Mature DCs can release inflammatory factors. This application used an Elisa kit to detect the cell supernatant after incubating bone marrow-derived DCs with the tumor cell culture medium after different treatments. Figure 2 k, 2j shown, the contents of inflammatory factors TNF-α and IFN-γ after treatment with Bac@OsCo-L were much higher than those of other treatment groups.

[0063] The above results confirmed that the tumor cell culture medium after different treatments could effectively induce the ICD effect by its effect on the production of ROS by bone marrow-derived DCs, thereby stimulating the activation of APCs.

[0064] 3. Research on the tumor targeting ability of Bac@OsCo-L in vivo and its in vivo therapeutic effect and mechanism on treating in situ MM( Figure 3 )。

[0065] According to previous literature, the anaerobic tropism of some bacteria can drive them to actively migrate towards tumors because tumors provide a perfect hypoxic environment for their growth.

[0066] To verify the tumor targeting ability of Bac@OsCo-L, the biodistributions of free OsCo-L and Bac@OsCo-L in excised organs and tumors after intravenous injection (i.v.) were examined by an in vivo imaging system, and the near-infrared dye IR820 was loaded on OsCo-L. As Figure 3 shown in a, a weak fluorescence signal was observed in the tumors of mice injected with free OsCo-L, indicating that free OsCo-L showed limited tumor targeting ability. After injection of Bac@OsCo-L, a significantly enhanced fluorescence signal was observed at the tumor site, and the fluorescence intensity gradually increased over time and reached a peak 12 hours after injection( Figure 3 a, 3b and Figure 18 ), and the fluorescence intensities of the main organs and tumors of the two groups of mice at 12 hours were statistically analyzed( Figure 2 b). These results indicated that Bac@OsCo-L had the ability to selectively target tumors and effectively deliver the loaded cargo.

[0067] In addition, blood samples were collected at different time points after intravenous injection of OsCo-L and Bac@OsCo-L to detect the fluorescence intensity for in vivo pharmacokinetic analysis( Figure 3 c). According to the published literature, Bac@OsCo-L showed normal blood circulation behavior and retention ability of Bac@OsCo-L. To demonstrate the selective colonization of Bac. 72 hours after intravenous injection of OsCo-L and Bac@OsCo-L, the tumor tissues and main organs were excised, ground and diluted and spread on agar plates. A large number of bacterial colonies were observed in the tumor area collected in the Bac@OsCo-L treatment group, while few bacterial colonies were detected in other main organs( Figure 3 i and Figure 19), further indicating that Bac@OsCo-L can selectively colonize tumor sites. Inspired by the effective tumor targeting and penetration ability of the bacterial hybrid, the present application next evaluated its anti-tumor ability in a subcutaneous tumor-bearing mouse B16-F10 tumor model. When the tumor volume reached 50-100 mm 3 , the mice were randomly divided into four groups (n = 3): 1) Control; 2) OsCo-L; 3) Bac; 4) Bac@OsCo-L. As Figure 3 shown in d, on days 0, 2, 4, 6, and 8, OsCo-L, Bac, and Bac@OsCo-L were intravenously injected into the mice with an equivalent dose of bacteria (about 10 9 CFU / kg) and the corresponding OsCo-L (10 mg / kg per mouse). The tumor size and body weight of the mice were measured every two days as Figure 3 shown in e, 3f, and 20a. The tumors of the mice in the Control group or the OsCo-L group grew rapidly, indicating that the effect of no treatment or single OsCo-L injection on tumor growth was negligible. The tumor growth of the mice treated with Bac was slightly inhibited, which might be due to the growth and reproduction of anaerobic bacteria at the tumor site competing with tumor cells for nutrients.

[0068] As expected, the tumor growth of the mice treated with Bac@OsCo-L was significantly reduced. The excellent therapeutic effect achieved by Bac@OsCo-L should be attributed to the enhanced accumulation and penetration of OsCo-L carried by Bac at the tumor site, combined with the reduction of the tumor microenvironment pH by Bac metabolites, thereby enhancing the peroxidase-like ability of OsCo-L.

[0069] In addition, no obvious changes in the body weight of the mice in each group were observed during the whole treatment process ( Figure 20 b), indicating that the side effects caused by the bacterial hybrid developed in the present application were negligible.

[0070] The mouse tumor tissues obtained from this experiment in the present application were sent for pathological examination. The HE, ki-67, and Tunel results of the tumor tissues showed ( Figure 22)Consistent with the previous experimental results. Considering the inherent immune-stimulating properties of bacteria and the potential of CDT to activate immune responses, this application has conducted a thorough study to clarify the potential mechanism underlying the significantly enhanced CDT therapeutic effect mediated by Bac@OsCo-L. In this application, B16-F10 tumor-bearing mice were randomly divided into four groups and received the same treatment as described above. Three days after different treatments, the mice were euthanized, and their tumors, spleens, and tumor-draining lymph nodes were harvested for different analyses. First, the maturation status of dendritic cells (DCs) in tumor-draining lymph nodes, which are key antigen-presenting cells for immune system activation, was measured. The results showed that this treatment based on bacterial biohybrids to enhance CDT (Bac@OsCo-L) could significantly enhance DC maturation (approximately 52.2%), much higher than that induced by Control (approximately 25.2%), OsCo-L (25.5%), and Bac (26%) ( Figure 23 ). In addition to DCs, the proportions of tumor- and spleen-infiltrating lymphocytes (TILs, CD3 + T), including cytotoxic T lymphocytes (CTLs, CD8 + T), helper T cells (CD4 + T), and regulatory T cells (Treg T) among CD45 + T cells were also measured. Through tSNE analysis of the results of polychromatic flow cytometry, it was found that the Bac@OsCo-L group could significantly increase the abundance of total T cells (CD3 + ) in the tumor and promote the infiltration of CD8 + T cells ( Figure 3 g and Figure 24 ), along with its high efficiency in reducing intratumoral immunosuppressive cells such as regulatory T cells (Treg; Figure 3 g and Figure 24 ). The spleen detection results were consistent with those of the tumor ( Figure 3 h and Figure 25 ). These results indicate that CDT based on Bac@OsCo-L may contribute to a significant increase in the ratio of CD4 - CD8 + T cells and a reduction in the proportion of Tregs, demonstrating the successful initiation of adaptive anti-tumor immunity. In addition to different immune cells in the tumor, the levels of multiple pro-inflammatory cytokines in the sera of mice treated with Bac@OsCo-L also increased significantly, including TNF-α and IFN-γ ( Figure 21 a, 21b).

[0071] All of the above results strongly demonstrate that this bacterial bihybrid can jointly trigger a powerful anti-tumor immune response.

[0072] 4. In Vivo Therapeutic Efficacy and Mechanism of Bac@OsCo-L in Treating Metastatic MM( Figure 4 )

[0073] To evaluate the distant tumor suppressive activity of Bac@OsCo-L, a bilateral tumor model was adopted. B16-F10 cancer cells were subcutaneously inoculated into the right hip of Balb / c live mice, and a second inoculation was performed on the left hip of the same mice 5 days later.

[0074] Bac@OsCo-L was injected into the mice via the tail vein( Figure 4 a). After different treatments, the distant tumors and primary tumors were excised on the 8th day. All mice behaved normally, and the weight changes in all groups were negligible( Figure 26 b), indicating few side effects when the immune system was activated. The weight of the distant metastatic tumors in the Bac@OsCo-L treatment group was the lightest( Figure 4 c), and the volume of the distant metastatic tumors was the smallest( Figure 4 b). Photos of the distant metastatic tumors are shown in Figure 26 a. All these results directly demonstrated that Bac@OsCo-L treatment significantly inhibited the growth of primary and metastatic tumors.

[0075] Subsequently, this application investigated whether Bac@OsCo-L treatment could effectively activate the immune response in a bilateral orthotopic tumor model. The maturation of DCs and the proliferation of CTLs in peripheral immune organs were preliminarily studied. As shown in Figure 27 , tumor-draining lymph nodes were collected for analysis, which indicated the maturation of DCs. The proportions of cytotoxic T lymphocytes (CTLs, CD8 + T), helper T cells (CD4 + T) in CD45 + T cells were also measured( Figure 4 d-4g), and the results showed that the degree of CD8 + T cell infiltration in the Bac@OsCo-L group was higher than that in other groups; meanwhile, the CD4 + T cells in the Bac@OsCo-L group decreased, indicating the further activation of CD8 + T cells in the tumor spleen. Therefore, it is believed that Bac@OsCo-L has great potential in activating the immune response through the expansion and proliferation of CTLs in peripheral immune organs.

[0076] Subsequently, the present application further investigated the mechanism of immunosuppression in distant tumors. As a hallmark of adaptive immunity, the immune memory response is crucial for tumor prevention. Effector memory T cells (Tem) are commonly found in peripheral tissues and can induce immediate protection by producing various cytokines when encountering the same antigen. Meanwhile, central memory T cells (Tcm) preferentially reside in secondary lymphoid organs to enhance the response to antigen restimulation. Tcm provides immunity only after a series of processes such as trained expansion, differentiation, and migration. Mouse spleens were collected after Bac@OsCo-L treatment to determine the changes in Tcm and Tem. FCM data showed that in the B16-F10 bilateral orthotopic tumor model, Tcm (CD44 + CD62L + ) was downregulated, while Tem (CD44 + CD62L - ) was upregulated ([[]] Figure 4 Figure 4 h-4i). Therefore, the ability of Bac@OsCo-L to convert Tcm cells into the Tem phenotype further indicates the potential to generate a more effective anti-tumor immune response. Meanwhile, the secretion levels of TNF-α and IFN-γ in mouse serum were also measured by enzyme-linked immunosorbent assay to evaluate the synergistic cancer immune response. Figure 4 j-4k showed that the two cytokines were upregulated after Bac@OsCo-L treatment, indicating that Bac@OsCo-L can induce a strong T cell-mediated immune response.

[0077] Overall, the above results provide key evidence that a strong anti-tumor immune response and immune memory have been established after treatment with the proposed bacterial hybrid.

[0078] 5. In vitro and in vivo biosafety.

[0079] To investigate whether the nanodrug would cause unnecessary damage to healthy cells, at the cellular level, the present application tested the biosafety of different concentration gradients of Bac@OsCo-L and Control, OsCo-L, B, and Bac@OsCo-L at a concentration of 40 μg / ml×10 5 CFU / ml using a CCK8 kit. HUVEC cells were treated with Bac@OsCo-L at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml. The results showed that no significant cytotoxicity was detected under different concentrations and different group treatments ([[]] Figure 28 Figure 28 a, 28b). The hemolysis test showed that although the concentration of Bac@OsCo-L was 100 μg / mL, no obvious hemolysis occurred ([[]] Figure 29 Figure 29a, 29b). After performing in-situ tumor experiments on mice, the hearts, livers, spleens, lungs, kidneys and sera of the mice were sent for pathological examination.

[0080] The results showed that the treatments in different groups did not cause obvious harm to the hearts, livers, spleens, lungs and kidneys of the mice ( Figure 30 ), and the serum biochemical indexes were all within the normal range ( Figure 31 ).

[0081] Conclusion: In this application, OsCo-L was obtained by wrapping a lipid layer on the surface of OsCo with peroxidase-like activity, and then OsCo-L was loaded onto the anaerobic bacterium Bac in a non-covalent binding manner to construct a hybrid Bac@OsCo-L. It can aggregate in tumors and reduce the acidity of the tumor microenvironment under the targeting and metabolism of anaerobic bacteria, achieving enhanced nanozyme CDT effect and ultimately realizing the anti-tumor in vivo treatment effect. At a dose of 40 μg / ml × 10 5 CFU / ml, Bac@OsCo-L can effectively eliminate 70% of B16-F10 tumor cells, which is achieved by enhancing immune activation through the combined action of OsCo-L and Bac. Intravenous injection of Bac@OsCo-L (10 mg / kg, 10 9 CFU / kg) in vivo can significantly inhibit the progression of malignant tumors, which is mainly due to the destruction of immunosuppression and the reversal of the immunosuppressive microenvironment of tumors. DC and CD8 + T cells with enhanced immune activity, reduced immunosuppressive Treg T cells, and activation of effector T cells and memory T cells.

[0082] This research work prepared an example of an engineered hybrid based on the combination of bacteria and nanodrugs for tumor treatment, stimulating both innate and adaptive immunity, and is expected to be beneficial to the design and development of novel high-performance anti-tumor therapies.

Claims

1. A novel nano-drug for treating in-situ malignant melanoma and its metastatic tumors, characterized in that, The novel nano-drug is obtained by loading OsCo with a lipid layer on its surface onto the surface of anaerobic bacteria.

2. The novel nano-drug for treating in-situ malignant melanoma and its metastatic tumors according to claim 1, characterized in that, The anaerobic bacteria is Bacillus butyricum.

3. The preparation method of a novel nano-drug for treating in-situ malignant melanoma and its metastatic tumors according to claim 1 or 2, characterized in that, It includes the following steps: S1. Prepare the nano-material OsCo; S2. Modify a lipid layer on the surface of the nano-material OsCo; S3. Load the nano-material OsCo modified with a lipid layer onto the surface of anaerobic bacteria.

4. Use of a novel nano-drug according to claim 1 or 2 for treating in-situ malignant melanoma and its metastatic tumors in the preparation of a product for treating melanoma.

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