Construction and application of intratumoral self-proliferation enhanced oncolytic microbial delivery system

Abstract

Construction and application of an intratumoral self-proliferation enhanced oncolytic microbial delivery system belong to the technical field of pharmaceutical preparations, a hydrogen sulfide donor is endowed with a membrane-insertable regional structure through a synthetic reaction, then a lipid membrane containing the membrane-insertable hydrogen sulfide donor is coated with bacteria through a self-assembly effect, and the membrane-insertable hydrogen sulfide donor is obtained. An oncolytic microbial delivery system with enhanced intratumoral self-proliferation is prepared. After the delivery system is intravenously injected, the delivery system is enriched in a tumor in a targeted manner, hydrogen sulfide released by a donor can be converted into tetrathionate in a tumor inflammation microenvironment, tetrathionate respiration belongs to specific respiration of salmonella, and the specific respiration belongs to specific respiration of salmonella. The proliferation of a salmonella tumor part is specifically promoted through two respiratory chains of aerobic respiration and tetrasulfate respiration, so that the pyroptosis of tumor cells is enhanced, and the anti-tumor purpose is achieved. The invention provides a brand new theory and an advanced solution for cancer treatment, also provides a promising strategy and exploration thought for clinical application of attenuated salmonella, and has important scientific research value.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and relates to a combination of a hydrogen sulfide donor with a membrane-insertable property and a lipid membrane-coated attenuated Salmonella, and specifically relates to a preparation method of an oncolytic microorganism delivery system with enhanced intratumoral self-proliferation and its application in the treatment of cancer models. Background Art

[0002] Globally, there are more than ten million newly diagnosed cancer patients every year. However, existing conventional therapies are usually insufficient to contain the development of cancer. More importantly, the severe non-targeted systemic distribution of conventional chemotherapy methods has strong toxic and side effects on cancer patients. Therefore, there is an urgent need to develop chemotherapy drugs with better anti-cancer targeting effects.

[0003] In bacterial therapy, it has been found that bacteria can treat diseases by stimulating the body's immune response, and it has been found that they have high targeting to the tumor microenvironment. Bacterial therapy provides a new strategy for disease treatment, meeting the urgent clinical need for "highly effective and low-toxic" microbial preparations. The rapid development of genetic engineering and synthetic material technologies at home and abroad has laid a foundation for the improvement of bacterial therapy, expanding its function in treating refractory diseases. The bacterial genome is relatively simple and relatively easy to modify. VNP20009 (VNP) is an attenuated Salmonella formed by knocking out toxic genes based on wild-type Salmonella typhimurium, and it is also the earliest strain applied in clinical trials. However, it did not show good therapeutic effects in clinical phase I trials, and there are problems such as dose-dependence and low tumor response rate, which limit the application of this therapy. Therefore, constructing an oncolytic microorganism delivery system with enhanced intratumoral self-proliferation has great clinical application value. Summary of the Invention

[0004] To solve the above problems, the present invention designs and prepares an intravenous injectable oncolytic microorganism delivery system with enhanced intratumoral self-proliferation for the treatment of various complex and refractory cancers. First, a hydrogen sulfide donor is given a membrane-insertable regional structure through a synthesis reaction, and then a lipid membrane containing the membrane-insertable hydrogen sulfide donor is coated on bacteria through self-assembly to prepare an oncolytic microorganism delivery system with enhanced intratumoral self-proliferation. After intravenous injection of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation, it targets and accumulates in tumors. In the tumor inflammatory microenvironment, the hydrogen sulfide released by the donor can be converted into tetrathionate. Tetrathionate respiration is a unique respiration of Salmonella. Through two respiratory chains of aerobic respiration and tetrathionate respiration, it specifically promotes the proliferation of Salmonella at the tumor site, thereby enhancing tumor cell pyroptosis and achieving the anti-tumor purpose.

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

[0006] The oncolytic microorganism delivery system with enhanced in-tumor self-proliferation described in the present invention is specifically an injectable lipid membrane-coated oncolytic bacterium, mainly composed of a membrane-insertable hydrogen sulfide donor, a phospholipid membrane, and an oncolytic bacterium (such as attenuated Salmonella).

[0007] Furthermore, in order to provide the basic conditions for tetrathionate respiration to Salmonella and improve the self-proliferation ability of bacteria at the tumor site. In the present invention, a membrane-insertable hydrogen sulfide donor is synthesized through an amidation reaction and inserted into the lipid membrane coating the oncolytic bacterium to construct an oncolytic bacterium with a lipid membrane coating inserted with a hydrogen sulfide donor.

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

[0009] The selected main body of the membrane-insertable hydrogen sulfide donor is a compound or organism capable of releasing hydrogen sulfide, which is one or more of GYY4137, DADS, and thioaminobenzamide. Its membrane-insertable part is a compound capable of providing a region for inserting into the phospholipid membrane structure, which 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 bacterium with a lipid membrane coating inserted with a hydrogen sulfide donor described in the present invention is prepared by the following method:

[0011] (1) Synthesis and confirmation of the membrane-insertable hydrogen sulfide donor

[0012] 4-Aminothiobenzamide, N,N-diisopropylethylamine, and anhydrous tetrahydrofuran are added to a reaction flask. Under stirring, lauric acid, 1-hydroxybenzotriazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, and the mixture is stirred and reacted.

[0013] After the reaction is completed, the reaction solution is poured into pre-cooled double-distilled water, mixed evenly, and extracted with pre-cooled ethyl acetate until the water layer is colorless, washed, and the organic layer is evaporated and concentrated to obtain a crude product.

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

[0015] The structural formula of ACL is as follows:

[0016]

[0017] (2) Construction and confirmation of the oncolytic bacterium with a lipid membrane coating inserted with a hydrogen sulfide donor

[0018] Lipids, cholesterol, and ACL are dissolved in a mixed solution of dichloromethane and methanol, and the solution is rotated and filmed for 15 - 20 minutes with a rotary evaporator.

[0019] Take 5×10 7 CFU / mL of VNP, centrifuge at 3000 rpm under low temperature conditions of 4°C, discard the supernatant, resuspend the bacterial cells with sterile PBS solution, hydrate the resuspended bacterial solution for 15 - 20 min, and the oncolytic bacteria coated with lipid membrane inserted with hydrogen sulfide donor are thus prepared;

[0020] Characterize the oncolytic bacteria coated with lipid membrane inserted with hydrogen sulfide donor by dynamic light scattering, transmission electron microscopy, and confocal microscopy.

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

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

[0023] In the step (2), to extend the circulation time of the bacteria in vivo, 1% of the total molar ratio of DSPE-PEG is added while adding lipid and cholesterol 2K ;

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

[0025] Application of the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation described in the present invention in the preparation of anti-tumor drugs. The tumors include breast cancer and melanoma.

[0026] Beneficial effects of the present invention:

[0027] The oncolytic bacteria coated with lipid membrane inserted with hydrogen sulfide donor described in the present invention are simple to prepare and have a clear mechanism. The coated oncolytic bacteria prepared by low-dose injection have similar therapeutic effects to those of directly injecting high-dose oncolytic bacteria in a mouse tumor model. However, whether it is the mouse state body weight or the induction of acute immunity, the oncolytic bacteria coated with lipid membrane inserted with hydrogen sulfide donor prepared show highly effective and low-toxic therapeutic effects. In summary, the present invention will provide a new theory and advanced solution for cancer treatment, and also provide promising strategies and exploration ideas for the clinical application of attenuated Salmonella, and has important scientific research value. Brief Description of the Drawings

[0028] Figure 1 Confirmation of the hydrogen sulfide donor (ACL) that can be inserted into the membrane in Example 1 of the present invention;

[0029] Figure 2Particle size and transmission electron microscopy images of oncolytic bacteria before and after lipid membrane coating in Example 2 of the present invention; in the figure, LVA represents lipid membrane-coated bacteria containing a hydrogen sulfide donor, i.e., the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation described above;

[0030] Figure 3 Confocal images of oncolytic bacteria before and after lipid membrane coating in Example 2 of the present invention;

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

[0032] Figure 5 Confirmation of hydrogen sulfide release of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation in Example 4; in the figure, LVNP represents lipid membrane-coated bacteria without a hydrogen sulfide donor;

[0033] Figure 6 For the in vitro generation of S 2 O 3 2- and S 4 O 6 2- confirmation in Example 5 of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation;

[0034] Figure 7 Colony plating image of the in vitro proliferation of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation in Example 6;

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

[0036] Figure 9 Confocal bright field images of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation inducing pyroptosis of tumor cells at the cellular level in Example 8 (G1: PBS, G2: VNP, G3: LVNP, G4: LVA);

[0037] Figure 10 Protein immunoblotting images of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation activating the caspase-1 pathway at the cellular level in Example 9 (G1: PBS, G2: VNP, G3: LVNP, G4: LVA);

[0038] Figure 11 Thermogram of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation triggering the release of inflammatory factors from tumor cells at the cellular level in Example 10 (G1: PBS, G2: VNP, G3: LVNP, G4: LVA);

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

[0040] Figure 13 Plating diagrams of major organs and tumors 48 h after intravenous injection of the oncolytic microbial delivery system with enhanced in-tumor self-proliferation in Example 11. From left to right are: heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), and tumor (T);

[0041] Figure 14 Pharmacodynamic study and in vivo imaging diagrams of the oncolytic microbial delivery system with enhanced in-tumor self-proliferation on murine orthotopic breast cancer in Example 12 (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; except for special markings, all are intravenous injection equivalents of 5×10 7 CFU / mL VNP);

[0042] Figure 15 Immunofluorescence staining diagrams of the oncolytic microbial delivery system with enhanced in-tumor self-proliferation inducing tumor tissue pyroptosis in Example 12;

[0043] Figure 16 Immunofluorescence confocal diagrams of the oncolytic microbial delivery system with enhanced in-tumor 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; except for special markings, all are intravenous injection equivalents of 5×10 7 CFU / mL VNP);

[0044] Figure 17 Pharmacodynamic study and volume-body weight change diagrams of the oncolytic microbial delivery system with enhanced in-tumor self-proliferation on murine melanoma in Example 14 (G1: PBS, G2: Lip-ACL, G3: VNP, G4: LVNP, G5: intravenous injection of 1.5×10 8 CFU / mL VNP, G6: LVA; except for special markings, all are intravenous injection equivalents of 5×10 7CFU / mL VNP). Detailed implementation mode

[0045] Through the following specific examples, the above content of the present invention is further described in detail, but it does not mean that the examples limit the present invention.

[0046] Example 1: Synthesis and confirmation of hydrogen sulfide donor

[0047] Add 4-aminothiobenzamide (0.5 mmol), N,N-diisopropylethylamine (1.5 mmol) and 15 mL of anhydrous tetrahydrofuran into a 100 mL round-bottom flask. Under stirring, add lauric acid (0.5 mmol), 1-hydroxybenzotriazole (0.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.5 mmol). After reacting for 24 h, pour it into pre-cooled double-distilled water. After mixing, extract it 3 times with 100 mL of pre-cooled ethyl acetate until the water layer is colorless, and then wash it with saturated sodium bicarbonate solution. The organic layer is concentrated by rotary evaporation under reduced pressure to obtain the crude product. Then it is separated and purified by preparative liquid phase to obtain the final product: 4-aminothiobenzamide-lauric acid (ACL). The structure is determined by mass spectrometry and nuclear magnetic resonance, as Figure 1 shown.

[0048] Example 2: Construction and confirmation of an oncolytic microbial delivery system with enhanced intratumoral self-proliferation

[0049] In the present invention, VNP20009 is coated with egg yolk lecithin and cholesterol, and cholesterol is used to further stabilize the self-assembled lipid membrane. The molar ratio of egg yolk lecithin to cholesterol is set to 4:1. In order to extend the circulation time of the bacteria in vivo, we added 1% of the total molar ratio of DSPE-PEG 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). Spin-coat the film with a rotary evaporator for 15-20 min. Take 5×10 7 CFU / mL VNP, centrifuge at 3000 rpm at low temperature of 4 °C for 12 min, discard the supernatant, and resuspend the bacterial cells with 1 mL of sterile PBS solution. Take 1 mL of the resuspended bacterial solution and hydrate it for 15-20 min. The oncolytic bacteria coated with a lipid membrane inserted with a hydrogen sulfide donor, that is, an oncolytic microbial delivery system with enhanced intratumoral self-proliferation, is prepared, abbreviated as LVA. Methods such as dynamic light scattering, transmission electron microscopy, and confocal localization are used to characterize the lipid-coated oncolytic bacteria inserted with a hydrogen sulfide donor. As Figure 2 , 3 shown, the particle size, transmission electron microscopy, and confocal microscope images all prove the successful construction of the lipid-coated oncolytic bacteria.

[0050] Example 3: Determination of the Content of ACL in the Oncolytic Microorganism Delivery System with Enhanced Intratumoral Self-Proliferation

[0051] After culturing tumor cells in the lower layer of a transwell plate until they reached 80% confluence, the medium was changed to 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 culturing for 8 h, the liquid in the upper layer of the transwell plate was taken to measure the OD value at 600 nm to determine the optimal addition concentration. As Figure 4 shown, when ACL was 0.1 mg / mL, Salmonella had the best growth trend.

[0052] Example 4: Confirmation of the Ability of the Oncolytic Microorganism Delivery System with Enhanced Intratumoral Self-Proliferation to Release Hydrogen Sulfide

[0053] The production of hydrogen sulfide by the synthesized ACL in the presence or absence of dithiothreitol was evaluated using a hydrogen sulfide kit. Under simulated tumor conditions with the Solarbio hydrogen sulfide kit, the hydrogen sulfide release amount of 1 mM hydrogen sulfide donor in 4 mM dithiothreitol was measured, and samples were taken at set times. Using H 2 S reacts with zinc acetate, N,N-dimethyl-p-phenylenediamine, and ammonium ferric sulfate to form methylene blue. The optical density value was measured at 665 nm using an enzyme-linked immunosorbent assay reader according to the instructions, and then the concentration conversion was performed.

[0054] After culturing mouse breast cancer (4T1) cells in a six-well plate until they reached 80% confluence, they were washed 3 times with PBS, and the cell nuclei were identified by staining with 10 μg / mL DAPI. Then, the liquid was discarded, and the cells were incubated with PBS containing WSP-1 (15 μM) with a hydrogen sulfide detection probe for 12 min. After washing 3 times with PBS, VNP, lipid-coated VNP (LVNP), and LVA were added respectively and reacted for 10 min. Then, all samples were observed using a laser confocal microscope. The results were as Figure 5 shown, proving that LVA can release hydrogen sulfide.

[0055] Example 5: In Vitro Replication of S 2 O 3 2- and S 4 O 6 2- Verification

[0056] 4T1 cells were seeded at 1×10 5The cells / well were seeded in a 6-well plate. When the cell confluence reached 80%, the original medium was then replaced with serum-free and antibiotic-free RPMI-1640 medium, and LVA was applied thereto. After culturing for 8 h, a small amount of the liquid was taken, centrifuged to obtain the supernatant, and 100 times the volume of absolute ethanol was added to allow natural precipitation, and the precipitate was confirmed by mass spectrometry. As Figure 6 shown (sodium thiosulfate + hydrogen, sodium tetrathionate dihydrate + sodium molecular weight), it was proved that there was S 2 O 3 2- and S 4 O 6 2- production.

[0057] Example 6: Evaluation of the in vitro replication and proliferation ability of an oncolytic microorganism delivery system with enhanced intratumoral self-proliferation

[0058] 4T1 cells were seeded in the lower layer of a 6-well transwell plate at a density of 1×10 5 cells / well. When the cell confluence reached 80%, the original medium was then replaced with serum-free and antibiotic-free RPMI-1640 medium, and VNP, LVNP, and LVA were added to the upper layer of the transwell plate respectively. After culturing for 8 h, the liquid in the upper layer of the transwell plate was taken and examined at 600 nm, and the proliferation of each group was judged by comparing the OD values. The upper-layer liquid was cultured on an LB solid medium for 24 h and photographed with a camera. As Figure 7 shown, the in vitro replication and proliferation ability of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation was evaluated by comparing the number of colonies in different groups.

[0059] Example 7: In vitro cytotoxicity of an oncolytic microorganism delivery system with enhanced intratumoral self-proliferation

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

[0061] Example 8: Evaluation of the ability of the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation to induce pyroptosis of tumor cells at the cellular level

[0062] Seed 4T1 cells at a density of 1×10 5 cells / well in a 6-well plate. When the cell confluence reaches 80%, then replace the original medium with serum-free and antibiotic-free RPMI-1640 medium, and add PBS and 5×10 7 CFU / mL of VNP, LVNP, and LVA respectively, and continue to incubate at 37°C for 8 h. Discard the original culture medium, fix the cells with paraformaldehyde, observe the cell morphology of each group using a confocal microscope, and balloon-like cells are considered to have undergone pyroptosis. As Figure 9 shown, no obvious histological changes were observed in the PBS group. In the LVNP group and the VNP group, some tumor cells showed reduced swelling and bubble protrusions. The tumor cell membranes in the LVA group showed significantly more bubbling than the other groups.

[0063] Example 9: Evaluation of the ability of the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation to activate the caspase-1 pathway at the cellular level

[0064] Seed 4T1 cells at a density of 1×10 5 cells / well in a 6-well plate. When the cell confluence reaches 80%, then replace the original medium with serum-free and antibiotic-free RPMI-1640 medium, and add PBS and 5×10 7 CFU / mL of VNP, LVNP, and LVA respectively, and continue to incubate at 37°C for 8 h. Subsequently, collect the cells and lyse them on ice with lysis buffer containing 1 mM protease inhibitor for 2 s. Centrifuge at 12,000 g for 3 min at 4°C, and extract the total protein. Then, use Western blotting to evaluate the protein expression of each group. As Figure 10 shown, the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation can activate the caspase-1 pathway and trigger pyroptosis of tumor cells.

[0065] Example 10: Evaluation of the ability of the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation to trigger the release of inflammatory factors from tumor cells

[0066] Seed 4T1 cells at a density of 1×10 5 cells / well in a 6-well plate. When the cell confluence reaches 80%, then replace the original medium with serum-free and antibiotic-free RPMI-1640 medium, and add PBS and 5×10 7 CFU / mL of VNP, LVNP, and LVA respectively, and continue to incubate at 37°C for 8 h. Collect the culture supernatants of each group, centrifuge to obtain the supernatant, and use an ELISA kit to detect the release of inflammatory factors from the cells of each group. AsFigure 11 As shown, the oncolytic microorganism delivery system with enhanced self-proliferation in tumors can enhance the release of inflammatory factors by tumor cells.

[0067] Example 11: Evaluation of the colonization and replication ability of the oncolytic microorganism delivery system with enhanced self-proliferation in tumors

[0068] Qualitative study of in vivo biodistribution: 4T1 cells were subcutaneously injected into Balb / c mice (female, 8 - 10 weeks old) to construct a 4T1 orthotopic breast tumor model to study the colonization and replication ability of LVA in tumors. The tumor-bearing mice were randomly divided into three groups (n = 3). Each group was intravenously injected with 5×10 7 CFU / mL of VNP, LVNP, and LVA. Salmonella that self-expresses GFP fluorescent protein after genetic engineering transformation can be used for visualization research in a small animal in vivo imaging system. At the set time, the mice were sacrificed by cervical dislocation, and their organs were isolated to observe the fluorescence signal of GFP. The results are as Figure 12 , the oncolytic microorganism delivery system with enhanced self-proliferation in tumors has a strong fluorescence signal, demonstrating that this system has excellent colonization and proliferation abilities. Then, at the last set time point, 48 hours after intravenous injection of each group of preparations, the tumors and the main organs of the group with the oncolytic microorganism delivery system with enhanced self-proliferation in tumors of each group were weighed and homogenized. The supernatant of the homogenate was diluted by a set multiple and spread on solid LB medium, and cultured at 37°C for 24 hours, as Figure 13 shown.

[0069] Example 12: Pharmacodynamic study of the oncolytic microorganism delivery system with enhanced self-proliferation in tumors on murine orthotopic breast cancer

[0070] First, a murine orthotopic breast cancer model was constructed using murine breast cancer cells - luciferase labeled (Luc - 4T1). The tumor-bearing mice were randomly divided into 7 groups (n = 5), and PBS (G1), Lip - ACL (G2), VNP (G3), LVNP (G4), LVA (G7) were used, with a dose equivalent to 5×10 7 CFU / mL of VNP. The low-dose group of 5×10 6 CFU / mL VNP (G5) was directly injected into the tumor and the group injected with 5×10 7 CFU / mL VNP (G6) were grouped for treatment. In addition, the tumor volume and weight of the mice were recorded during the treatment period (n = 5). The mice were imaged on an in vivo imaging system. As Figure 14 shown, the oncolytic microorganism delivery system with enhanced self-proliferation in tumors can effectively inhibit the development of murine breast cancer. The tumor tissues of groups G1, G3, G4, and G7 were collected, made into tumor sections respectively, and subjected to immunofluorescence staining. As Figure 15As shown, the oncolytic microbial delivery system with enhanced in-tumor self-proliferation strengthened tumor pyroptosis by activating the caspase-1 pathway.

[0071] Example 13: Study on the activation of the body's immunity by the oncolytic microbial delivery system with enhanced in-tumor self-proliferation

[0072] First, an orthotopic breast cancer model of mice was constructed by subcutaneous injection of 4T1 cells. The tumor-bearing mice were randomly divided into 7 groups (n = 4), and the above-mentioned grouping was used for treatment. After treatment, all mice were euthanized, and the tumors, plasma, and spleens of the mice were sampled. Then, single-cell suspensions were prepared, and the cells were stained with fluorescently labeled antibodies. Flow cytometry was used to measure the populations of T cells and DC cells in the tumors and spleens and the T cells in the blood.

[0073] In addition, tumor tissues were made into paraffin sections, and the tumor sections were restored with an antigen repair solution at 85°C for 6 min. Next, the sections were blocked with 5% goat serum at 37°C. After one hour, the sections were incubated with the antibody at 4°C for 12 h. After washing with PBS, the sections were incubated with a fluorescent secondary antibody for 90 min. Subsequently, the sections were incubated with DAPI for 15 min, and then observed with a confocal microscope. The results are as Figure 16 shown, the oncolytic microbial delivery system with enhanced in-tumor self-proliferation activated the body's immunity to a greater extent than other groups.

[0074] Example 14: Investigation on the improvement of the clinical dose-dependent toxicity of VNP20009 by the oncolytic microbial delivery system with enhanced in-tumor self-proliferation

[0075] First, a B16 mouse model was constructed, and 1×10 6 cells / well B16 cells were subcutaneously injected into the right side of female C57 mice. When the tumor volume reached ~100 mm 3 , the 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), and the VNP equivalent dose was 5×10 7 CFU / mL. The G5 group was intravenously injected with a dose of 1.5×10 8 CFU / mL of VNP. The tumor volume and body weight of the mice (n = 5) were recorded during the treatment process. As Figure 17 shown, G5 and G6 had similar therapeutic effects, but the body weight of the mice in the G5 group decreased significantly, while the body weight change in the G6 group was small. This indicates that the intravenous injection of 1.5×10 8 CFU / mL of VNP had greater toxicity, while the oncolytic microbial delivery system with enhanced in-tumor self-proliferation could achieve the effect of high efficiency and low toxicity.

Claims

1. An oncolytic microorganism delivery system with enhanced in-tumor self-proliferation, characterized in that, it is an injectable intravenous oncolytic microorganism delivery system with enhanced in-tumor self-proliferation; prepared by endowing the region structure of the membrane-insertable area with a hydrogen sulfide donor through a synthesis reaction, and then coating the bacteria with a lipid membrane containing the membrane-insertable hydrogen sulfide donor through self-assembly; after intravenous injection of the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation, it targets and enriches in tumors. In the tumor inflammatory microenvironment, the hydrogen sulfide released by the donor is converted into tetrathionate, and the proliferation of bacteria at the tumor site is specifically promoted through two respiratory chains of aerobic respiration and tetrathionate respiration, thereby enhancing tumor cell pyroptosis.

2. The oncolytic microorganism delivery system with enhanced in-tumor self-proliferation according to claim 1, characterized in that, the system is an injectable intravenous lipid membrane-coated oncolytic bacterium, mainly composed of a membrane-insertable hydrogen sulfide donor, a phospholipid membrane and an oncolytic bacterium; the membrane-insertable hydrogen sulfide donor is synthesized through an amidation reaction and inserted into the lipid membrane coating the oncolytic bacterium to construct an oncolytic bacterium with a lipid membrane coating inserted with a hydrogen sulfide donor.

3. The oncolytic microorganism delivery system with enhanced in-tumor self-proliferation according to claim 1, characterized in that, the oncolytic bacterium capable of tetrathionate respiration is one or more of VNP20009, Salmonella-IL2, Salmonella YB1, and ΔppGpp Salmonella.

4. The oncolytic microorganism delivery system with enhanced in-tumor self-proliferation according to claim 2, characterized in that, for the membrane-insertable hydrogen sulfide donor, the main body selected is one or more of GYY4137, DADS, and thioaminobenzamide, and its membrane-insertable part is a compound that can provide a region structure for inserting into the phospholipid membrane, which is one or more of lauric acid, linoleic acid, palmitic acid, stearic acid, cholesterol-containing compounds, and phospholipid-containing compounds.

5. A construction method of the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation according to claim 1 or 2, characterized in that, it includes the following steps: (1) Synthesis and confirmation of the membrane-insertable hydrogen sulfide donor Add 4-aminothiobenzamide, N,N-diisopropylethylamine, and anhydrous tetrahydrofuran to a reaction flask, stir and add lauric acid, 1-hydroxybenzotriazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir for reaction; After the reaction is completed, pour the reaction solution into pre-cooled double-distilled water, mix well, extract with pre-cooled ethyl acetate until the water layer is colorless, wash, and evaporate and concentrate the organic layer to obtain a crude product; Through separation and purification, the final product 4-aminothiobenzamide-lauric acid, abbreviated as ACL, is obtained and its structure is confirmed; (2) Construction and confirmation of an oncolytic bacterium with a lipid membrane coating inserted with a hydrogen sulfide donor Dissolve lipids, cholesterol, and ACL in a mixed solution of dichloromethane and methanol, and spin-coat with a rotary evaporator; Take 5×10 7 CFU / mL of VNP, centrifuge at 3000 rpm under low temperature conditions of 4 °C, discard the supernatant, resuspend the bacterial cells with sterile PBS solution, hydrate the resuspended bacterial solution for 15 - 20 min, and the oncolytic bacteria with a lipid membrane coating inserted with a hydrogen sulfide donor are then prepared; Characterize the oncolytic bacterium with a lipid membrane coating inserted with a hydrogen sulfide donor by dynamic light scattering, transmission electron microscopy, and confocal microscopy.

6. The construction method of the oncolytic microorganism delivery system with enhanced in-tumor self-proliferation according to claim 5, characterized in that, In the step (2), the molar ratio of lipid to cholesterol is 4:

1.

7. The method for constructing an oncolytic microorganism delivery system with enhanced intratumoral self-proliferation as claimed in claim 5, characterized in that In the step (2), while adding lipids and cholesterol, add DSPE-PEG with a total molar ratio of 1%. 2K .

8. The application of the oncolytic microorganism delivery system with enhanced intratumoral self-proliferation as claimed in claim 1 or 2 in the preparation of a medicament for treating tumors.

9. The application as claimed in claim 8, characterized in that the tumors are breast cancer and melanoma.

Images