Construction and application of an intratumorally self-proliferating oncolytic microbial delivery system

Abstract

The construction and application of an intratumorally self-proliferating enhanced oncolytic microbial delivery system belongs to the field of pharmaceutical formulation technology. Through a synthetic reaction, a hydrogen sulfide donor is endowed with a membrane-intercalable regional structure. Then, a lipid membrane containing the membrane-intercalable hydrogen sulfide donor is used to encapsulate bacteria through self-assembly, thus preparing an intratumorally self-proliferating enhanced oncolytic microbial delivery system. After intravenous injection of the delivery system, it accumulates within the tumor. In the tumor inflammatory microenvironment, the hydrogen sulfide released by the donor can be converted into tetrathionate. Tetrathionate respiration is a unique form of respiration in Salmonella. Through both aerobic and tetrathionate respiration, the system specifically promotes the proliferation of Salmonella in the tumor site, thereby enhancing tumor cell pyroptosis and achieving anti-tumor effects. This invention provides a novel theory and advanced solution for cancer treatment, and also offers promising strategies and exploratory ideas for the clinical application of attenuated Salmonella, possessing significant scientific research value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and relates to a combination of membrane-intercalable hydrogen sulfide donor and lipid membrane-coated attenuated Salmonella, specifically to the preparation method of an intratumorally self-proliferating and enhanced oncolytic microbial delivery system and its application in cancer model treatment. Background Technology

[0002] Globally, over ten million new cancer patients are diagnosed each year; however, existing conventional therapies are often insufficient to curb the progression of the disease. More importantly, the severe non-targeted systemic distribution of conventional chemotherapy methods produces significant toxic side effects for cancer patients. Therefore, there is an urgent need to develop chemotherapy drugs with better targeted anti-cancer effects.

[0003] Bacterial therapy has revealed that bacteria can treat diseases by stimulating the body's immune response and possess high targeting specificity to the tumor environment. Bacterial therapy offers new strategies for disease treatment, meeting the urgent clinical need for "highly effective and low-toxicity" microbial agents. Rapid advancements in genetic engineering and synthetic materials technology both domestically and internationally have laid the foundation for improving bacterial therapy and expanding its capabilities in treating refractory diseases. Bacterial genomes are relatively simple and relatively easy to modify. VNP20009 (VNP), an attenuated Salmonella strain created by knocking out the virulence gene from wild-type Salmonella typhimurium, was one of the earliest strains used in clinical trials. However, it did not demonstrate good therapeutic efficacy in Phase I clinical trials, exhibiting dose-dependent effects and low tumor response rates, limiting the application of this therapy. Therefore, constructing an intratumorally self-proliferating and enhanced oncolytic microbial delivery system has significant clinical application value. Summary of the Invention

[0004] To address the aforementioned problems, this invention designs and prepares an intravenously injectable oncolytic microbial delivery system with enhanced intratumoral proliferation for the treatment of various complex and refractory cancers. First, a hydrogen sulfide donor is synthesized to possess a membrane-intercalable region. Then, a lipid membrane containing the membrane-intercalable hydrogen sulfide donor is self-assembled to encapsulate bacteria, thus preparing the oncolytic microbial delivery system with enhanced intratumoral proliferation. After intravenous injection of the oncolytic microbial delivery system, it accumulates within the tumor. In the tumor inflammatory microenvironment, the hydrogen sulfide released by the donor is converted into tetrathionate. Tetrathionate respiration is a unique form of Salmonella respiration. Through both aerobic and tetrathionate respiration, the system specifically promotes the proliferation of Salmonella at the tumor site, thereby enhancing tumor cell pyroptosis and achieving antitumor effects.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] The tumor-intratumor self-proliferation enhanced oncolytic microbial delivery system of the present invention is specifically an intravenously injectable lipid membrane-coated oncolytic bacteria, mainly composed of a membrane-intercalable hydrogen sulfide donor, a phospholipid membrane, and oncolytic bacteria (such as attenuated Salmonella).

[0007] Furthermore, in order to provide Salmonella with the basic conditions for tetrathionine respiration and enhance the bacteria's self-replication ability at tumor sites, this invention synthesizes a membrane-intercalable hydrogen sulfide donor via an amidation reaction, which is then intercalated onto the lipid membrane of oncolytic bacteria to construct oncolytic bacteria with a lipid membrane-coated layer containing the hydrogen sulfide donor.

[0008] The oncolytic bacteria capable of tetrathionate respiration are one or more of VNP20009, Salmonella-IL2, Salmonella YB1, and ΔppGpp Salmonella.

[0009] The aforementioned membrane-intercalable hydrogen sulfide donor is selected primarily as a compound or organism capable of releasing hydrogen sulfide, and is one or more of GYY4137, DADS, and thioaminobenzamide. Its membrane-intercalable portion is a compound that can provide insertion into the phospholipid membrane structure, and is one or more of fatty acids such as lauric acid, linoleic acid, palmitic acid, and stearic acid, cholesterol-containing compounds, and phospholipid-containing compounds.

[0010] The oncolytic bacteria with a lipid membrane coating containing a hydrogen sulfide donor, as described in this invention, are prepared by the following method:

[0011] (1) Synthesis and confirmation of membrane-intercalable hydrogen sulfide donors

[0012] 4-Aminothiobenzamide, N,N-diisopropylethylamine and anhydrous tetrahydrofuran were added to the reaction flask. Lauric acid, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added while stirring. The reaction was continued.

[0013] After the reaction was completed, the reaction solution was poured into pre-cooled double-distilled water, mixed well, and extracted with pre-cooled ethyl acetate until the aqueous layer was colorless. The solution was washed, and the organic layer was evaporated and concentrated to obtain the initial product.

[0014] After separation and purification, the final product 4-aminothiobenzamide-lauric acid (ACL) was obtained, and its structure was confirmed.

[0015] The ACL structure is as follows:

[0016]

[0017] (2) Construction and confirmation of oncolytic bacteria coated with lipid membranes containing hydrogen sulfide donors

[0018] Lipids, cholesterol, and ACL were dissolved in a mixed solution of dichloromethane and methanol, and the mixture was spun into a film using a rotary evaporator for 15–20 min.

[0019] Take 5×10 7 Centrifuge at CFU / mL VNP at 3000 rpm at 4°C, discard the supernatant, resuspend the bacterial cells in sterile PBS solution, hydrate the resuspended bacterial solution for 15-20 min, and the oncolytic bacteria with lipid membrane coating of hydrogen sulfide donor is prepared.

[0020] Oncolytic bacteria coated with a lipid membrane and intercalated with hydrogen sulfide donors were characterized using dynamic light scattering, transmission electron microscopy, and confocal microscopy.

[0021] In step (1), 4-aminothiobenzamide, N,N-diisopropylethylamine, lauric acid, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are mixed in a molar ratio of 1:3:1:1:1.

[0022] In step (2), the molar ratio of lipids to cholesterol is 4:1;

[0023] In step (2), in order to prolong the circulation time of the bacteria in vivo, 1% total molar ratio of DSPE-PEG is added along with lipids and cholesterol. 2K ;

[0024] In step (2), dichloromethane and methanol are mixed at a volume ratio of 9:1.

[0025] The present invention relates to the application of the intratumoral self-proliferation enhanced oncolytic microbial delivery system in the preparation of drugs for treating tumors. The tumors include breast cancer and melanoma.

[0026] The beneficial effects of this invention are:

[0027] The lipid-coated oncolytic bacteria with hydrogen sulfide donors described in this invention are simple to prepare and have a clear mechanism. Low-dose injection of the prepared coated oncolytic bacteria showed similar therapeutic effects in mouse tumor models as direct high-dose injection. However, the prepared lipid-coated oncolytic bacteria with hydrogen sulfide donors exhibited highly effective and low-toxicity therapeutic effects in both mouse body weight and acute immunity induction. In summary, this invention provides a novel theory and advanced solution for cancer treatment, and also offers promising strategies and exploratory ideas for the clinical application of attenuated Salmonella, possessing significant scientific research value. Attached Figure Description

[0028] Figure 1 This confirms the membrane-intercalable hydrogen sulfide donor (ACL) in Embodiment 1 of the present invention;

[0029] Figure 2The images show the particle size and transmission electron microscopy (TEM) images of oncolytic bacteria before and after lipid membrane coating in Example 2 of this invention. In the images, LVA represents lipid membrane-coated bacteria containing hydrogen sulfide donors, i.e., the oncolytic microbial delivery system with enhanced intratumoral proliferation.

[0030] Figure 3 This is a confocal image of oncolytic bacteria before and after lipid membrane coating in Example 2 of the present invention;

[0031] Figure 4 Evaluation of ACL content in the oncolytic microbial delivery system with enhanced intratumoral self-proliferation in Example 3 of this invention;

[0032] Figure 5 This confirms the hydrogen sulfide release from the oncolytic microbial delivery system with enhanced intratumoral self-proliferation in Example 4; in the figure, LVNP represents lipid-coated bacteria without hydrogen sulfide donors;

[0033] Figure 6 S2O3 was generated in vitro using the oncolytic microbial delivery system with enhanced intratumoral self-proliferation in Example 5. 2- and S4O6 2- Confirmation;

[0034] Figure 7 This is an in vitro proliferation smear image of the oncolytic microbial delivery system with enhanced intratumoral self-proliferation in Example 6;

[0035] Figure 8 In vitro cytotoxicity assessment of the oncolytic microbial delivery system with enhanced intratumoral self-proliferation in Example 7;

[0036] Figure 9 The confocal bright-field image (G1: PBS, G2: VNP, G3: LVNP, G4: LVA) shows the pyroptosis of tumor cells induced at the cellular level by the tumor-intratumor self-proliferation enhanced oncolytic microbial delivery system in Example 8.

[0037] Figure 10 The image shows a Western blot of proteins from the intratumoral self-proliferation enhanced oncolytic microbial delivery system in Example 9 that activates the caspase-1 pathway at the cellular level (G1:PBS, G2:VNP, G3:LVNP, G4:LVA).

[0038] Figure 11 The heatmap shows how the tumor cell release of inflammatory factors was triggered at the cellular level by the tumor self-proliferation enhanced oncolytic microbial delivery system in Example 10 (G1:PBS, G2:VNP, G3:LVNP, G4:LVA).

[0039] Figure 12The images show fluorescence imaging of the major organs and tumors isolated after intravenous injection of the tumor-intratumorally self-proliferating enhanced oncolytic microbial delivery system in Example 11. From top to bottom, they represent the VNP group, LVNP group, and LVA group; from left to right, they represent: tumor (T1, T2, T3), heart (H), liver (Li), spleen (S), lung (Lu), and kidney (K).

[0040] Figure 13 This is a smear of the major organs and tumor 48 hours after intravenous injection of the tumor-intratumor self-proliferation enhanced oncolytic microbial delivery system in Example 11. From left to right: heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), and tumor (T);

[0041] Figure 14 The pharmacodynamic study and in vivo imaging of the oncolytic microbial delivery system with enhanced intratumoral proliferation in Example 12 on mouse orthotopic breast cancer (G1: PBS, G2: Lip-ACL, G3: VNP, G4: LVNP, G5: intratumoral injection of 5×10 6 CFU / mL VNP, G6: Intratumoral injection of 5×10 7 CFU / mL VNP, G7:LVA; unless otherwise specified, all are intravenous injection equivalents of 5 × 10⁻⁶. 7 CFU / mL VNP);

[0042] Figure 15 This is an immunofluorescence staining image of tumor tissue pyroptosis induced by the tumor-intratumorally self-proliferating oncolytic microbial delivery system in Example 12;

[0043] Figure 16 Immunofluorescence confocal image of the oncolytic microbial delivery system with enhanced intratumoral self-proliferation in Example 13 (G1: PBS, G2: Lip-ACL, G3: VNP, G4: LVNP, G5: intratumoral injection of 5×10 6 CFU / mL VNP, G6: Intratumoral injection of 5×10 7 CFU / mL VNP, G7:LVA; unless otherwise specified, all are intravenous injection equivalents of 5 × 10⁻⁶. 7 CFU / mL VNP);

[0044] Figure 17 The pharmacodynamic study and volume / weight changes of the oncolytic microbial delivery system with enhanced intratumoral self-proliferation in Example 14 on mouse melanoma are shown in the figure (G1: PBS, G2: Lip-ACL, G3: VNP, G4: LVNP, G5: intravenous injection of 1.5 × 10⁻⁶). 8 CFU / mL VNP, G6:LVA; unless otherwise specified, all values ​​are equivalent to 5 × 10⁻⁶ intravenous injection doses. 7CFU / mL VNP). Detailed Implementation

[0045] The following specific examples further illustrate the above-mentioned content of the present invention in detail, but do not imply that the embodiments limit the present invention.

[0046] Example 1: Synthesis and Validation of Hydrogen Sulfide Donors

[0047] In a 100 mL round-bottom flask, 0.5 mmol of 4-aminothiobenzamide, 1.5 mmol of N,N-diisopropylethylamine, and 15 mL of anhydrous tetrahydrofuran were added. Lauric acid (0.5 mmol), 1-hydroxybenzotriazole (0.5 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.5 mmol) were added with stirring. After reacting for 24 h, the mixture was poured into pre-cooled double-distilled water, mixed well, and extracted three times with pre-cooled 100 mL of ethyl acetate until the aqueous layer was colorless. The mixture was then washed with saturated sodium bicarbonate solution. The organic layer was concentrated by rotary evaporation under reduced pressure to obtain the initial product. Further purification by preparative liquid chromatography yielded the final product: 4-aminothiobenzamide-lauric acid (ACL). The structure was confirmed by mass spectrometry and NMR. Figure 1 As shown.

[0048] Example 2: Construction and validation of an intratumorally self-proliferating and enhanced oncolytic microbial delivery system

[0049] In this invention, VNP20009 is coated with egg yolk lecithin and cholesterol, with cholesterol used to further stabilize the self-assembled lipid membrane. The molar ratio of egg yolk lecithin to cholesterol is set at 4:1. To prolong the bacterial circulation time in vivo, 1% of DSPE-PEG is added as a total molar ratio. 2K The specific method is as follows: Dissolve egg yolk lecithin, cholesterol, and ACL in a mixed solution of dichloromethane and methanol (volume ratio 9:1). Use a rotary evaporator to rotate the film for 15–20 minutes. Take 5 × 10⁻⁶ [amount missing]. 7 CFU / mLVNP was centrifuged at 3000 rpm for 12 min at 4°C, the supernatant was discarded, and the bacterial cells were resuspended in 1 mL of sterile PBS. 1 mL of the resuspended bacterial solution was hydrated for 15–20 min. The lipid-coated oncolytic bacteria with hydrogen sulfide donors, i.e., the intratumorally self-proliferating enhanced oncolytic microbial delivery system (LVA), was thus prepared. The lipid-coated oncolytic bacteria with hydrogen sulfide donors were characterized using dynamic light scattering, transmission electron microscopy, and confocal localization. Figure 2 , 3 As shown, particle size, transmission electron microscopy, and confocal microscopy images all demonstrate the successful construction of lipid-coated oncolytic bacteria.

[0050] Example 3: Determination of ACL content in an intratumorally self-proliferating oncolytic microbial delivery system

[0051] After tumor cells reached 80% confluence in the lower layer of a Transwell plate, the medium was replaced with serum-free and antibiotic-free RPMI-1640 medium. Lipid-coated oncolytic bacteria containing 0.1, 0.5, and 1 mg / mL ACL were added to the upper layer of the Transwell plate, respectively. After 8 hours of incubation, the OD value of the upper layer of the Transwell plate was measured at 600 nm to determine the optimal concentration. Figure 4 As shown, Salmonella exhibits the best growth trend when the ACL is 0.1 mg / mL.

[0052] Example 4: Confirmation of the hydrogen sulfide release capability of an intratumorally self-proliferating oncolytic microbial delivery system

[0053] The generation of hydrogen sulfide from the synthesized ACL in the presence or absence of dithiothreitol was evaluated using a hydrogen sulfide kit. Using a Solarbio hydrogen sulfide kit to simulate tumor conditions, the release of hydrogen sulfide from a 1 mM hydrogen sulfide donor in the presence of 4 mM dithiothreitol was measured, with samples taken at set times. Methylene blue was generated by reacting H₂S with zinc acetate, N,N-dimethyl-p-phenylenediamine, and ferric ammonium sulfate. The optical density was measured at 665 nm using a microplate reader according to the manufacturer's instructions, and then the concentration was calculated.

[0054] Mouse breast cancer (4T1) cells were cultured in six-well plates to 80% confluence. After washing three times with PBS, the cell nuclei were identified by staining with 10 μg / mL DAPI. The liquid was then discarded, and the cells were incubated for 12 min with PBS containing WSP-1 (15 μM) containing a hydrogen sulfide detection probe. After washing three times with PBS, VNPs, lipid-coated VNPs (LVNPs), and LVA were added and reacted for 10 min respectively. All samples were then observed using a laser confocal microscope. The results are as follows: Figure 5 As shown, this demonstrates that LVA can release hydrogen sulfide.

[0055] Example 5: In vitro replication of S2O3 by an intratumorally self-proliferating and enhanced oncolytic microbial delivery system. 2- and S4O6 2- verify

[0056] 4T1 cells were fed at a dose of 1×10 5 Cells / well were seeded in 6-well plates. When cell confluence reached 80%, the original medium was replaced with serum-free and antibiotic-free RPMI-1640 medium, and LVA was applied. After 8 hours of culture, a small amount of liquid was taken, centrifuged, and the supernatant was collected. 100 times the volume of anhydrous ethanol was added, and the supernatant was allowed to precipitate naturally. The precipitation was confirmed by mass spectrometry. Figure 6 The diagram (sodium thiosulfate + hydrogen, sodium tetrasulfate dihydrate + sodium molecular weight) demonstrates the presence of S₂O₃.2- and S4O6 2- The generation of.

[0057] Example 6: Evaluation of the in vitro replication and proliferation capacity of an intratumorally self-proliferating oncolytic microbial delivery system

[0058] 4T1 cells at 1×10 5 Cells / well were seeded in the lower layer of 6-well Transwell plates. When cell confluence reached 80%, the original medium was replaced with serum-free and antibiotic-free RPMI-1640 medium. VNP, LVNP, and LVA were added to the upper layer of the Transwell plates. After 8 hours of incubation, the upper layer of the Transwell plates was examined at 600 nm, and the proliferation status of each group was determined by comparing the OD values. The upper layer was then incubated on LB solid medium for 24 hours and photographed. Figure 7 As shown, the in vitro replication and proliferation capacity of the oncolytic microbial delivery system with enhanced intratumoral self-replication was evaluated by comparing the colony counts of different groups.

[0059] Example 7: In vitro cytotoxicity of an intratumorally self-proliferating oncolytic microbial delivery system

[0060] 4T1 and melanoma (B16-F10) cells were respectively treated at 2×10⁻⁶. 4 Cells / well were seeded in 96-well plates and cultured at 37°C until approximately 80% confluence was achieved. Subsequently, cells were treated with PBS, ACL liposomes prepared using only hydrogen sulfide donor (Lip-ACL), VNP, LVNP, and LVA for 8 h. Afterward, the original culture medium was removed, and the culture plate was incubated in 200 μL MTT at 37°C for 180 min. Next, 150 μL DMSO was added to dissolve the blue-purple formazan crystals, and after shaking for 10 min, the optical density of each well was measured at 490 nm using a microplate reader. Figure 8 As shown, the oncolytic microbial delivery system with enhanced intratumoral self-proliferation can significantly reduce cancer cell survival.

[0061] Example 8: Assessment of the ability of an intratumorally self-proliferating oncolytic microbial delivery system to induce pyroptosis in tumor cells at the cellular level

[0062] 4T1 cells were fed at a dose of 1×10 5 Cells / well were seeded into 6-well plates. When the cell confluence reached 80%, the original medium was replaced with serum-free and antibiotic-free RPMI-1640 medium, and PBS and 5×10⁶ ppm of PBS were added. 7Cells were incubated with CFU / mL VNP, LVNP, and LVA at 37°C for another 8 hours. The original culture medium was discarded, and cells were fixed with paraformaldehyde. Cell morphology was observed using a confocal microscope, and balloon-shaped cells were considered to have undergone pyroptosis. Figure 9 As shown, no significant histological changes were observed in the PBS group. In contrast, some tumor cells in the LVNP and VNP groups showed reduced swelling and exhibited blister protrusions. The LVA group showed significantly more blistering of tumor cell membranes than the other groups.

[0063] Example 9: Assessment of the ability of an intratumorally self-proliferating oncolytic microbial delivery system to activate the caspase-1 pathway at the cellular level.

[0064] 4T1 cells were fed at a dose of 1×10 5 Cells / well were seeded into 6-well plates. When the cell confluence reached 80%, the original medium was replaced with serum-free and antibiotic-free RPMI-1640 medium, and PBS and 5×10⁶ ppm of PBS were added. 7 Cells were incubated with CFU / mL VNP, LVNP, and LVA at 37°C for 8 h. Cells were then collected and lysed for 2 s on ice with lysis buffer containing 1 mM protease inhibitor. Total protein was extracted after centrifugation at 12000g for 3 min at 4°C. Western blotting was then used to assess the expression of each protein group. Figure 10 As shown, the intratumoral self-proliferation enhanced oncolytic microbial delivery system can activate the caspase-1 pathway and induce pyroptosis in tumor cells.

[0065] Example 10: Assessment of the ability of an intratumorally self-proliferating oncolytic microbial delivery system to trigger the release of inflammatory factors from tumor cells

[0066] 4T1 cells were fed at a dose of 1×10 5 Cells / well were seeded into 6-well plates. When the cell confluence reached 80%, the original medium was replaced with serum-free and antibiotic-free RPMI-1640 medium, and PBS and 5×10⁶ ppm of PBS were added. 7 CFU / mL of VNP, LVNP, and LVA were added and incubated at 37°C for another 8 hours. The culture medium from each group was collected, and the supernatant was obtained by centrifugation. The release of inflammatory factors from cells in each group was detected using an ELISA kit. Figure 11 As shown, the intratumoral self-proliferation enhanced oncolytic microbial delivery system can enhance the release of inflammatory factors from tumor cells.

[0067] Example 11: Assessment of the tumor colonization and replication capabilities of an intratumorally self-proliferating oncolytic microbial delivery system.

[0068] Qualitative studies on in vivo biodistribution involved subcutaneous injection of 4T1 cells into female Balb / c mice (8-10 weeks old) to construct a 4T1 orthotopic mammary tumor model and investigate the colonization and replication capacity of LVA tumors. Tumor-bearing mice were randomly divided into three groups (n=3). Each group received an intravenous injection of 5×10⁻⁶ cells. 7 CFU / mL of VNP, LVNP, and LVA. Salmonella strains expressing GFP fluorescent protein, genetically engineered, were visualized using a small animal in vivo imaging system. Mice were euthanized by cervical spondylosis at a predetermined time, and their organs were isolated. The fluorescence signal of GFP was observed, and the results are as follows: Figure 12 The intratumorally self-proliferating enhanced oncolytic microbial delivery system exhibited a strong fluorescent signal, demonstrating its excellent colonization and proliferation capabilities. Then, at the final set time point, 48 hours after intravenous injection of each group's formulation, the tumors and major organs of each group using the intratumorally self-proliferating enhanced oncolytic microbial delivery system were weighed and homogenized. The supernatant of the homogenate was diluted to the set factor and spread onto solid LB medium, incubated at 37°C for 24 hours, and so on. Figure 13 As shown.

[0069] Example 12: Pharmacodynamic study of mouse orthotopic breast cancer by an intratumorally self-proliferating oncolytic microbial delivery system.

[0070] First, an orthotopic breast cancer model was established in mice using mouse breast cancer cells labeled with luciferase (Luc-4T1). Tumor-bearing mice were randomly divided into 7 groups (n=5) and treated with PBS (G1), Lip-ACL (G2), VNP (G3), LVNP (G4), and LVA (G7) at doses equivalent to 5 × 10⁻⁶ cells / year. 7 VNP levels of CFU / mL. Low-dose group (5×10⁻⁶) was administered directly into the tumor. 6 CFU / mL VNP (G5) and intratumoral injection of 5×10 7 Mice were treated in groups with CFU / mL VNP (G6). Tumor volume and weight were recorded during treatment (n=5). Mice were imaged using an in vivo imaging system. Figure 14 As shown, the intratumoral self-proliferating and enhanced oncolytic microbial delivery system can effectively inhibit the development of mammary cancer in mice. Tumor tissues from groups G1, G3, G4, and G7 were collected, prepared into tumor sections, and subjected to immunofluorescence staining. Figure 15 As shown, the intratumoral self-proliferating enhanced oncolytic microbial delivery system amplifies tumor pyroptosis by activating the caspase-1 pathway.

[0071] Example 13: Study on the activation of the body's immune system by an intratumorally self-proliferating and enhanced oncolytic microbial delivery system

[0072] First, an orthotopic breast cancer model was established in mice by subcutaneous injection of 4T1 cells. Tumor-bearing mice were randomly divided into 7 groups (n=4) and treated according to the above grouping method. After treatment, all mice were euthanized, and samples were collected from the tumors, plasma, and spleen. Single-cell suspensions were then prepared and stained with fluorescently labeled antibodies. Flow cytometry was used to measure the populations of T cells and dendritic cells in the tumors and spleen, as well as the number of T cells in the blood.

[0073] In addition, tumor tissue was prepared into paraffin sections, and the sections were restored with antigen retrieval solution at 85°C for 6 min. Next, the sections were blocked at 37°C using 5% goat serum. One hour later, the sections were incubated with antibodies at 4°C for 12 h. After washing with PBS, the sections were incubated with fluorescent secondary antibody for 90 min. Subsequently, the sections were incubated with DAPI for 15 min, and then observed using a confocal microscope. The results are as follows. Figure 16 As shown, the oncolytic microbial delivery system with enhanced intratumoral self-proliferation activates the body's immune system to a greater extent than other groups.

[0074] Example 14: Investigation of the improvement of clinical dose-dependent toxicity of VNP20009 by an intratumorally self-proliferating oncolytic microbial delivery system

[0075] First, a B16 mouse model was constructed by subcutaneously injecting 1×10⁻⁶ of the substance into the right side of female C57 mice. 6 cells / well of B16 cells. When the tumor volume reaches ~100 mm. 3 Mice were randomly divided into 7 groups. C57 tumor-bearing mice were intravenously injected with PBS (G1), Lip-ACL (G2), VNP (G3), LVNP (G4), and LVA (G6), with a VNP equivalent dose of 5 × 10⁻⁶. 7 CFU / mL. Group G5 received 1.5 × 10⁻⁶ CFU / mL intravenously. 8 VNP at a dose of CFU / mL. Tumor volume and body weight of mice (n=5) were recorded during treatment. Figure 17 As shown, G5 and G6 had similar therapeutic effects, but the G5 group mice experienced a significant decrease in body weight, while the G6 group showed little change in body weight. This indicates that intravenous injection of 1.5 × 10⁻⁶ mg / L of the drug may be more effective than intravenous injection of 1.5 × 10⁻⁶ mg / L of the drug. 8 VNP at a dose of CFU / mL has significant toxicity, while an intratumoral self-proliferation enhanced oncolytic microbial delivery system can achieve a highly efficient and low-toxicity effect.

Claims

1. An intratumoral self-propagation enhanced oncolytic microbe delivery system, characterized in that, The delivery system is a intravenous injectable liposome-coated oncolytic bacteria, which is mainly composed of a membrane-insertable hydrogen sulfide donor, a phospholipid membrane and an oncolytic bacteria; the membrane-insertable hydrogen sulfide donor is synthesized by an amidation reaction, and is inserted into the liposome coating the oncolytic bacteria to construct the liposome-coated oncolytic bacteria with the inserted hydrogen sulfide donor; wherein the oncolytic bacteria is VNP20009, and the membrane-insertable hydrogen sulfide donor is 4-aminothiobenzamide-lauric acid, which is prepared by the reaction of 4-aminothiobenzamide and lauric acid; after intravenous injection of the delivery system, it is targeted and enriched in the tumor, and under the tumor inflammatory microenvironment, the hydrogen sulfide released by the donor is converted into tetrathionate, which specifically promotes the proliferation of the bacteria at the tumor site through the two respiratory chains of aerobic respiration and tetrathionate respiration, and further enhances pyroptosis of the tumor cells.

2. A method of constructing the intratumoral self-propagation enhanced oncolytic microbial delivery system of claim 1, characterized by, The method comprises the following steps: (1) Synthesis and confirmation of the membrane-insertable hydrogen sulfide donor A reaction bottle is added with 4-aminothiobenzamide, N,N-diisopropylethylamine and anhydrous tetrahydrofuran, and lauric acid, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added under stirring, and the reaction is stirred; After the reaction is completed, the reaction solution is poured into pre-cooled double-distilled water, mixed, and then extracted with pre-cooled ethyl acetate until the water layer is colorless, washed, and the organic layer is evaporated and concentrated to obtain the initial product; After separation and purification, the final product 4-aminothiobenzamide-lauric acid, abbreviated as ACL, is obtained, and the structure is confirmed; (2) Construction and confirmation of the liposome-coated oncolytic bacteria with the inserted hydrogen sulfide donor The lipids, cholesterol and ACL are dissolved in a mixed solution of dichloromethane and methanol, and a rotary evaporator is used to spin the membrane; Take 5 x 10 7 CFU / mL VNP, centrifuged at 3000 rpm under low temperature 4℃, discard the supernatant, resuspend the bacteria with sterile PBS solution, hydrate the resuspended bacteria for 15~20 min, and the oncolytic bacteria coated with lipid film inserted with hydrogen sulfide donor are prepared. The liposome-coated oncolytic bacteria with the inserted hydrogen sulfide donor is characterized by dynamic light scattering, transmission electron microscopy and confocal microscopy.

3. The method of constructing an intratumoral self-propulsion-enhanced oncolytic microorganism delivery system according to claim 2, wherein, In the step (2), the molar ratio of the lipids to the cholesterol is 4:

1.

4. The method of constructing an intratumoral self-propulsion-enhanced oncolytic microorganism delivery system according to claim 2, wherein The step (2) is performed by adding 1% of DSPE-PEG in total molar ratio at the same time of adding the lipid and cholesterol 2K .

5. Use of the intratumoral self-propulsion-enhanced oncolytic microorganism delivery system of claim 1 for the manufacture of a medicament for treating a tumor, characterized in that, The tumor is breast cancer and melanoma.

Images