Preparation and application of micromolecular coated oncolytic bacteria

Abstract

The invention discloses preparation and application of micromolecular coated oncolytic bacteria, and relates to the field of nano-drugs. According to the coated bacteria, the surface of attenuated salmonella VNP20009 is coated with a nano-coating formed by coordination of micantrone and calcium ions, a bacterial antigen signal is shielded, so that the peripheral clearance rate and toxicity risk of the attenuated salmonella VNP20009 in blood circulation are reduced, and the coated bacteria have good biocompatibility and can maintain natural physiological characteristics and biodegradability of the bacteria. In order to solve the problems of lack of effective molecular targets and high immunosuppression of triple negative breast cancer (TNBC), the coated oncolytic bacteria utilize the natural tropism of VNP20009 on TNBC and the physiological targeting motion characteristic to directionally deliver miltroquinone and calcium ions, and cooperate with the oncolytic bacteria to kill tumor cells; bacterial surface antigen signals are shielded, so that tumor accumulation is increased, peripheral toxicity is avoided, and a breakthrough is provided for overcoming long-term failure of oncolytic bacteria clinical tests.

Description

Technical Field

[0001] The present invention relates to the field of nano-drugs, and relates to the preparation and application of an oncolytic bacteria preparation with a bio-adaptive nano-coating technology. Background Art

[0002] Oncolytic Bacteria Therapy is a cancer treatment method that uses genetically engineered or screened bacteria to directly or indirectly kill tumor cells. Research has found that, taking advantage of the epithelial tissue malignancy and hypoxic characteristics of progressive tumors in triple-negative breast cancer (TNBC), several strains of Salmonella typhimurium (abbreviated as S.T) can utilize their natural epithelial cell adhesion and motility characteristics as well as hypoxic tropism to actively distribute to breast tumors, and use the local hypoxia, low immune clearance, and high-concentration purine environment in tumors to stably colonize and then exert an oncolytic effect. The superior efficacy of S.T has been demonstrated in at least 4 clinical studies (NCT01099631, NCT00004988, NCT00006254, and NCT00004216). Research shows that lipopolysaccharide (LPS) of Gram-negative bacteria can serve as pathogen-associated molecular patterns (PAMPs), providing strong immune stimulation signals to innate immune cells - dendritic cells (DCs) and macrophages - and binding to their pattern recognition receptors, thereby promoting the tumor infiltration and migration of immune cells. In addition, LPS exerts an anti-tumor effect by enhancing the expression of IL-18, activating the inflammasome and the signal pathway mediated by Toll-like receptor 4 (TLR4). In addition, bacterial flagellin can bind to TLR5 on the surface of DCs, enhance the cytotoxic effect of CD8+ T cells, inhibit the immunosuppressive function of T cells, and promote the transformation of M2 macrophages to M1 macrophages, thereby achieving a strong immune activation and immunosuppression reversal effect. These studies indicate that S.T relies on its unique hypoxia-epithelial cell tropism and related biological metabolic processes to colonize and exert oncolytic and immune activation effects in TNBC tissues, providing a new clinical breakthrough direction for the treatment of TNBC.

[0003] However, direct intravenous injection of ST will be easily recognized and quickly cleared by phagocytes of the circulatory system due to its surface antigen exposure, resulting in low tumor colonization efficiency, and may induce strong peripheral toxic reactions (such as acute inflammatory reactions) or even cytokine storms, which significantly limits the safety of its clinical application. Therefore, it is key to carry out attenuation modification while retaining the hypoxic-epithelial cell tropism, oncolytic properties and biological metabolic activity of bacteria. Bioadaptive nanocoating technology provides a non-destructive surface coating modification solution for bacterial surfaces, which can retain the natural physiological metabolic activity and motility of live bacteria, and shield bacterial antigen signals by forming functional nanocoatings on the bacterial surface, thereby reducing its peripheral clearance rate and toxicity risk in the blood circulation. It has good biocompatibility and can maintain the natural physiological characteristics and biodegradability of bacteria. In addition, existing clinical trials (NCTO1099631, NCT00004988N, CTO0006254, NCT00004216) have shown that VNP20009 alone is not sufficient to completely eliminate TNBC. Therefore, new strategies need to be explored to enhance the immunotherapeutic effect of ST. Summary of the invention

[0004] In view of the current limitations of oncolytic bacteria in the treatment of malignant tumors, we designed a "small molecule coat" that can adapt to the appearance of bacteria - MTO@Ca, which effectively shields bacterial surface antigens. This small molecule coated oncolytic bacteria MTO@Ca-VNP20009 can maintain the bacteria's tumor hypoxia-epithelial cell tropism, oncolytic properties and biological metabolic activity, while achieving attenuation and modification to increase circulation in the body.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The present invention discloses a method for preparing small molecule coated oncolytic bacteria, which is characterized by: coordinating attenuated salmonella with mitoxantrone and calcium ions in a 1:1 molar ratio to form a nano coating to coat the surface of the bacteria.

[0007] Furthermore, the concentration of mitoxantrone is 0.6 mM and the concentration of calcium chloride is 0.6 mM.

[0008] A specific step of the method for preparing the above-mentioned small molecule coated oncolytic bacteria: (1) Centrifuge the attenuated Salmonella in the logarithmic growth phase, collect the precipitate and fully resuspend it in ddH2O, add mitoxantrone and calcium chloride, incubate at room temperature with shaking for 10 minutes, and centrifuge at 3000 rpm to remove free mitoxantrone and calcium chloride; (2) Resuspend the precipitate obtained in (1) after removing free mitoxantrone and calcium chloride in ddH2O, and then add mitoxantrone hydrochloride and calcium chloride again. Incubate with shaking at room temperature for 1 h; collect the mixture by centrifugation at 3000 rpm to obtain MTO@Ca-VNP20009.

[0009] Further, the concentration of mitoxantrone is 0.6 mM, and the concentration of calcium chloride is 0.6 mM.

[0010] Further, the mixing ratio of mitoxantrone and calcium chloride is 1:1.

[0011] The present invention also discloses a small molecule-coated oncolytic bacterial preparation, characterized in that the preparation is obtained by the method described in any one of the above.

[0012] Further, the application of the preparation in the preparation of a drug for treating triple-negative breast cancer.

[0013] The present invention also discloses a pharmaceutical composition, comprising the above-mentioned preparation and a pharmaceutically acceptable carrier.

[0014] Further, the pharmaceutical composition is in the form of an intravenous injection preparation.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] Oncolytic bacteria are a novel immunotherapy, currently used for tumors in hypoxic and immunosuppressive microenvironments, such as triple-negative breast cancer, pancreatic cancer, and colorectal cancer. However, direct intravenous injection is easily recognized by phagocytes in the circulatory system due to the exposure of its surface antigens, resulting in a high peripheral clearance rate (affecting tumor colonization efficiency) and possible peripheral toxicity, severely weakening the clinical practical accessibility of this therapy. Coordinating calcium ions with mitoxantrone can enable oncolytic bacteria to load more mitoxantrone. Forming a nano-coating to coat the surface of the bacteria can shield the surface antigens of the bacteria, avoid recognition by neutralizing antibodies, prolong the in vivo circulation time, and increase tumor colonization. This coating has pH responsiveness and dissociates in the acidic microenvironment of the tumor. The released mitoxantrone and calcium ions can up-regulate the expression of caspase-3, and then activate the expression of GSDME, amplifying the tumor cell pyroptosis induced by VNP20009. The pyroptotic tumor cells release immune-related molecular patterns, which together with VNP20009 activate the body's immune response and effectively inhibit distant metastasis of tumors. This small molecule coating strategy provides a breakthrough for overcoming the long-term failure of oncolytic bacteria clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1For the condition optimization experiment in Example 1 and the transmission electron microscopy of VNP20009 and MTO@Ca-VNP20009, scale bar: 1 μm. Among them, A is the bar graph of the optimization results of particle size at different drug concentrations; B is the transmission electron micrograph of VNP20009; C is the transmission electron micrograph of MTO@Ca-VNP20009.

[0018] Figure 2 For the particle size and surface potential of VNP20009 (A) and MTO@Ca-VNP20009 (B) in Example 1.

[0019] Figure 3 For the results graphs of the release rates of mitoxantrone and calcium ions under different pH environments in Example 3. Among them, A is the release rate of mitoxantrone of MTO@Ca-VNP20009 under different pH environments; B is the release rate of calcium ions of MTO@Ca-VNP20009 under different pH environments.

[0020] Figure 4 For the results graph of the stability of MTO@Ca-VNP20009 in blood in Example 4. Among them, A is the particle size of MTO@Ca-VNP20009 in blood for 24 h; B is the surface potential of MTO@Ca-VNP20009 in blood for 24 h.

[0021] Figure 5 For the results graph that the small molecule coating can shield bacterial surface antigens and increase the circulation time of oncolytic bacteria in blood in Example 5. Among them, A is the IgG concentration in mouse serum after injecting VNP20009 and MTO@Ca-VNP20009; B is the circulation time of VNP20009 and MTO@Ca-VNP20009 in mice.

[0022] Figure 6 For the distribution of VNP20009 and MTO@Ca-VNP20009 in the main organs of mice in Example 6.

[0023] Figure 7 For the microscopic photos of VNP20009 and MTO@Ca-VNP20009 causing pyroptosis in Example 7.

[0024] Figure 8 For the anti-tumor efficacy of MTO@Ca-VNP20009 in breast cancer mice in Example 8. Among them, A is the photo of the tumor of breast cancer mice in different treatment groups after the treatment; B is the weight of the tumor of breast cancer mice in different treatment groups after the treatment. Detailed implementation manners

[0025] The following is a further detailed description of the present invention with specific embodiments. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following embodiments. All technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0026] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0027] In the following examples, Salmonella typhimurium VNP20009 was purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0028] In the following examples, mitoxantrone hydrochloride was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0029] In the following examples, calcium chloride was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0030] Example 1 Preparation of MTO@Ca-VNP20009 and optimization of the preparation process.

[0031] I. Optimization process.

[0032] Salmonella typhimurium VNP20009 was cultured in 100 mL of LB medium and shaken at 220 rpm at 37 °C for 12 hours. When the optical density of Salmonella typhimurium at 600 nm (OD600) reached 0.8 - 1.0, it was centrifuged at 3000 rpm for 5 minutes. The bacterial pellet was resuspended in ddH2O for use in coating the nanocoating below.

[0033] To prepare MTO@Ca-VNP20009 with the highest drug loading efficiency, different concentrations of calcium chloride and mitoxantrone hydrochloride were added to the above-mentioned bacterial suspension in a 1:1 molar concentration ratio to coat the bacteria. When the concentrations of mitoxantrone hydrochloride and calcium chloride reached 0.6 mM, the particle size of MTO@Ca-VNP20009 reached the maximum value, and further increasing the drug concentration did not increase the particle size (as Figure 1 A).

[0034] II. Preparation of MTO@Ca-VNP20009.

[0035] Step 1. Preparation of bacterial suspension: Salmonella typhimurium VNP20009 was cultured in 100 mL of LB medium and shaken at 220 rpm at 37 °C for 12 hours. When the optical density of Salmonella typhimurium at 600 nm (OD600) reached 0.8 - 1.0, it was centrifuged at 3000 rpm for 5 minutes, and the bacterial pellet was resuspended in ddH2O to obtain a bacterial suspension for use in coating the nanocoating below.

[0036] Step 2. Preparation of MTO@Ca-VNP20009.

[0037] Add 0.6 mM mitoxantrone hydrochloride and 0.6 mM calcium chloride to the bacterial suspension and shake at room temperature for 10 minutes. Then centrifuge at 3000 rpm for 5 minutes to remove free mitoxantrone hydrochloride and calcium chloride. The precipitate is completely resuspended in ddH2O, and 0.6 mM mitoxantrone hydrochloride and 0.6 mM calcium chloride are added again. Subsequently, centrifuge at 3000 rpm for 5 - 10 minutes. Collect the obtained MTO@Ca-VNP20009, wash it twice with PBS, resuspend it in PBS, and store it at 4°C.

[0038] The morphologies of naked bacteria VNP20009 and coated bacteria MTO@Ca-VNP20009 were observed using a transmission electron microscope (FEI, Tecnai G2 20 S-TWIN, 200 kV, USA). Dynamic light scattering (DLS) was used to analyze the sizes and surface potentials of VNP20009 and MTO@Ca-VNP20009. As Figure 1 shown, where Figure 1 B is the transmission electron micrograph of VNP20009, Figure 1 C is the transmission electron micrograph of MTO@Ca-VNP20009.

[0039] Example 2 characterized the sizes and surface potentials of naked bacteria and coated bacteria.

[0040] Prepare MTO@Ca-VNP20009 according to the optimal method described in Example 1. The sizes and surface potentials of VNP20009 and MTO@Ca-VNP20009 were detected by dynamic light scattering (DLS). Figure 2 A is the size comparison of VNP20009 and MTO@Ca-VNP20009, Figure 2 B is the surface potential comparison of VNP20009 and MTO@Ca-VNP20009.

[0041] Example 3 verified the release rates of mitoxantrone and calcium ions of MTO@Ca-VNP20009 in different pH environments.

[0042] (1) Take equal amounts of MTO@Ca-VNP20009 (15×10 9 CFU) and add them to 15 mL of PBS at pH 7.4 (n = 3) and PBS at pH 6.5 (n = 3) respectively. Place them in a shaker at 37°C. Take 1 mL of the suspension in the two groups of test tubes at 10 min, 30 min, 1 h, 2 h, and 6 h respectively.

[0043] (2) Centrifuge the suspension at 3000 rpm for 10 min, take 150 μL of the supernatant and transfer it to a 96-well plate. For the detection of mitoxantrone, use an ELISA reader to detect the fluorescence intensity at an excitation wavelength of 605 nm and an emission wavelength of 695 nm. For calcium ions, use a calcium ion content probe kit for detection and use an ELISA reader to detect the absorbance at OD575. The results are as Figure 3 A, more mitoxantrone is released at pH 6.5 than at pH 7.4.

[0044] (3) Calculate the content of mitoxantrone and calcium ions obtained from the results of the ELISA reader. The ratio of the content of mitoxantrone and calcium ions at each time point to the content of mitoxantrone and calcium ions carried by each milliliter of MTO@Ca-VNP20009 (1×10 9 CFU / mL) is the release rate. The results are as Figure 3 B, more calcium ions are released at pH 6.5 than at pH 7.4.

[0045] Example 4 verifies the stability of MTO@Ca-VNP20009 in blood.

[0046] (1) Take the blood of Balb / c mice and put it into an anticoagulant tube, add MTO@Ca-VNP20009, and shake it in a shaker at 37 °C for 24 h.

[0047] (2) Use a 100 kDa ultrafiltration centrifuge tube to centrifuge at 5000 rmp for 5 minutes to obtain MTO@Ca-VNP20009.

[0048] (3) For the same batch of MTO@Ca-VNP20009, use dynamic light scattering (DLS) to detect the particle size and surface potential of MTO@Ca-VNP20009 before and 24 h after adding blood respectively. The results are as Figure 4 A particle size and Figure 4 B surface potential show no obvious changes, proving its stability in blood.

[0049] Example 5 verifies that the small molecule coating can shield the bacterial surface antigen and increase the circulation time of oncolytic bacteria in blood.

[0050] (1) To explore whether repeated injection of exogenous components can induce antibody-dependent clearance reactions, randomly divide Balb / c mice into two groups with 3 mice in each group, and intravenously inject naked bacteria VNP20009 and MTO@Ca-VNP20009 (1×10 7 CFU / mouse) on days 0, 2, and 4 respectively. On days 0, 2, 4, 6, and 8 after injection, use a mouse immunoglobulin G (IgG) ELISA kit to detect the level of IgG in the serum. The results are as Figure 5As shown in A, the serum IgG concentration increased significantly on the fourth day, and VNP20009 was significantly higher than that in the MTO@Ca-VNP20009 group, indicating that the surface antigen exposure was significantly reduced after coating.

[0051] (2)To study the circulation time of naked bacteria and MTO@Ca-VNP20009 in the blood, balb / c mice were randomly divided into two groups of 3 each, and injected intravenously with naked bacteria VNP20009 and MTO@Ca-VNP20009 expressing GFP (1×10 8 CFU / mouse). The fluorescence intensity in the blood was measured using an enzyme-linked immunosorbent assay (ELISA) at 0, 2, 4, 6, and 8 hours. The results are as Figure 5 shown in B, and the oncolytic bacteria after coating increased the blood circulation time.

[0052] Example 6 evaluated the distribution of MTO@Ca-VNP20009 in a mouse breast cancer model.

[0053] (1)An orthotopic breast cancer model was established by injecting 4T1 cells (1×10 6 cells) into the right mammary fat pad of female mice. When the 4T1 tumor volume reached 200-300 mm³, the tumor-bearing mice were randomly divided into two groups (3 each).

[0054] (2)One group was injected intravenously with naked bacteria expressing GFP, and the other group was injected intravenously with the same number of MTO@Ca-VNP20009. At 6, 12, and 24 hours after injection, the mice were euthanized by cervical dislocation, and their hearts, livers, spleens, lungs, and kidneys were rapidly collected.

[0055] (3)Then, fluorescence imaging and biodistribution quantification were performed using an in vivo imaging system (IVIS) to statistically analyze the distribution of VNP20009 and MTO@Ca-VNP20009 in tumor-bearing mice over time. The results showed that the coated bacteria significantly increased tumor accumulation.

[0056] Example 7 verified the effect of MTO@Ca-VNP20009 in inducing pyroptosis of 4T1 cells in vitro.

[0057] (1)4T1 cells were seeded in 12-well culture plates at an initial density of 1×10 5 cells per well. After incubation for 24 hours, the cells were treated in five groups: PBS, VNP20009, MTO, MTO-VNP20009, and MTO@Ca-VNP20009, and then cultured for another 6 hours.

[0058] (2)Subsequently, the cell morphology was observed using a confocal laser scanning microscope (CLSM). Cells showing balloon-like swelling were considered to be undergoing pyroptosis. The results are as Figure 7As shown, the oncolytic bacteria after coating have the effect of amplifying tumor cell pyroptosis.

[0059] Example 8 verified the anti-tumor efficacy of MTO@Ca-VNP20009 in breast cancer mice.

[0060] (1) Four 4T1 cells (1×10 6 ) were injected into the right mammary fat pad of female mice. When the 4T1 tumor volume reached 200 mm³, the balb / c mice were randomly divided into 5 treatment groups, PBS, VNP20009, MTO, MTO-VNP20009, and MTO@Ca-VNP20009, with 5 mice in each group.

[0061] (2) Different treatment components (1×10 7 CFU / mouse) were injected on days 8, 10, and 12. On day 20, after the treatment ended, the mice were euthanized, and the tumor tissues of each group were collected for photographing ( Figure 8 A) and weighing ( Figure 8 B). The results showed that MTO@Ca-VNP20009 had the best anti-tumor efficacy.

[0062] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a small molecule-coated oncolytic bacterium, characterized in that: The attenuated Salmonella is coordinated with mitoxantrone and calcium ions at a molar ratio of 1:1 to form a nano-coating to coat the bacterial surface.

2. The preparation method according to claim 1, wherein the concentration of mitoxantrone is 0.6 mM and the concentration of calcium chloride is 0.6 mM.

3. Specific steps of a method for preparing a small molecule-coated oncolytic bacterium according to claim 1: (1) Centrifuge the attenuated Salmonella VNP20009 in the logarithmic growth phase. After collecting the precipitate and resuspending it sufficiently in ddH2O, add mitoxantrone hydrochloride and calcium chloride, and incubate with shaking at room temperature for 10 minutes. Centrifuge at 3000 rpm to remove free mitoxantrone and calcium chloride; (2) After sufficiently resuspending the precipitate obtained in (1) that has removed free mitoxantrone and calcium chloride in ddH2O, add mitoxantrone hydrochloride and calcium chloride again, and incubate with shaking at room temperature for 1 h; Centrifuge the mixture at 3000 rpm to collect it to obtain MTO@Ca-VNP20009.

4. The preparation method according to claim 3, wherein the concentration of mitoxantrone is 0.6 mM and the concentration of calcium chloride is 0.6 mM.

5. The preparation method according to claim 4, wherein the mixing ratio of mitoxantrone and calcium chloride is 1:

1.

6. A small molecule-coated oncolytic bacterial preparation, characterized in that, The preparation is obtained by the method according to any one of claims 1-5.

7. Use of the preparation according to claim 6 in the preparation of a drug for treating triple-negative breast cancer.

8. A pharmaceutical composition comprising the preparation according to claims 6-7 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical preparation form is an intravenous injection preparation.

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