Bacterial assembly as well as preparation method and application thereof

Abstract

The invention discloses a bacterial assembly as well as a preparation method and application thereof. The bacterial assembly is prepared from facultative anaerobic bacteria, double-terminated maleimide polyethylene glycol and cationic albumin, reducing disulfide bonds on the surfaces of bacterial cell walls through a reducing agent so as to expose sulfydryl, carrying out Michael addition reaction on double-terminal maleimide polyethylene glycol and sulfydryl, and connecting a plurality of bacteria with sulfydryl exposed on the surfaces to form the bacterial assembly. According to the reaction system, the cationic albumin is added to increase bacterial adhesion so as to enhance aggregation, meanwhile, bacterial surface antigens are effectively covered, and removal of immune cells is reduced. The bacterial assembly provided by the invention reduces the distribution of bacteria in normal tissues while increasing lung enrichment, has higher lung tumor targeting, and effectively improves the lung tumor treatment effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a bacterial assembly and a preparation method and application thereof. Background Art

[0002] Solid tumors are characterized by abnormal vascular proliferation and local tissue hypoxia and necrosis. This microenvironment is suitable for the colonization and growth of obligate or facultative anaerobes, such as Streptococcus, Clostridium, Bifidobacterium, Salmonella, Escherichia coli, and Listeria. These bacteria can be enriched and proliferated in tumor tissues, and their number can reach more than 1,000 times that of tissues such as the liver or spleen. Among them, Salmonella typhimurium has been widely studied due to its easy culture and genetic manipulation. Salmonella typhimurium can grow under aerobic and anaerobic conditions, so it can colonize in various solid tumor tissues. In order to improve the therapeutic effect, bacterial treatment strategies have been developed in combination with radiotherapy and chemotherapy, or designed to deliver anticancer drug molecules through bacteria. In addition to being a delivery system for antitumor therapeutic drugs, it also has intrinsic antitumor effects, which is largely attributed to its immunomodulatory activity. Tumors have immunosuppressive effects and escape immune surveillance by limiting the maturation and infiltration of immune cells. Intravenous injection of Salmonella typhimurium can effectively stimulate the immune system, leading to increased production of systemic proinflammatory cytokines such as interleukins (IL-1β, IL-18), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), and causing activation of innate and adaptive immune cells. These activated immune cells will form an immune microenvironment that is unfavorable to tumor growth.

[0003] VNP20009 is an attenuated strain of Salmonella typhimurium (hereinafter referred to as VNP), which has good safety compared with the wild type. The msbB mutant significantly reduces the level of TNF-α induced by the mutant, reducing the septic shock caused by TNF-α. In order to improve the tumor-specific colonization ability, VNP further knocked out the purI gene, making its growth ability dependent on the purine level in the environment, making it easier to colonize and proliferate in a high-purine environment, such as tumor tissue. VNP has been extensively studied in a variety of animal tumor models and Phase I clinical trials in cancer patients, including melanoma, breast cancer, and colon cancer.

[0004] Studies have shown that the tumor tissue specificity of VNPs comes from their targeting of the tumor microenvironment. The main mechanisms are as follows: (1) Abnormal tumor growth leads to the formation of hypoxic and necrotic areas in the tumor. VNPs chemotaxis the tumor hypoxic microenvironment and specifically colonize in the tumor site; (2) The flagella of VNPs increase their motility, which helps them penetrate and migrate in tumor tissues; (3) The tumor immunosuppressive microenvironment can help VNPs avoid being cleared by macrophages and neutrophils. Based on this, its enrichment in tumors is 100 to 1000 times higher than that in major organs such as the liver. Even if tumor-targeting genes are introduced into the traditional anti-tumor drug delivery system, less than 1% of the total amount of drugs is enriched in the tumor, and up to 80% of the drugs are retained in organs such as the liver and kidneys.

[0005] It cannot be ignored that there are few reports on the use of VNPs in the treatment of lung cancer. Tissue distribution data show that VNPs are less colonized in the lungs of mice, which is 1 / 10 of the total colonization in the liver and spleen; in normal crab-eating macaques, the lung enrichment of VNPs is only 1 / 3 of that in the liver. The reason is that, on the one hand, the oxygen content in the lung tissue is 13-14%, which is much higher than the oxygen content in other tissues of the human body, which is not conducive to the colonization of VNPs; on the other hand, VNPs are gradually surrounded and cleared by infiltrating neutrophils in the tumor site, which also greatly reduces the enrichment of VNPs in the lungs. The lungs are one of the most important organs in the human body. In 2020, lung cancer ranked first in both the number of cases and the number of deaths. This is because malignant tumors of different pathological types in different parts of the human body can metastasize to the lungs. This recurrence and metastasis leads to the death of 90% of cancer patients. Therefore, if a strategy based on VNP-specific targeting of the lungs can be developed, it can not only solve the problem of VNPs being difficult to enrich in the lungs, expand the types of tumors for which VNPs are applied, but also be expected to be used to improve the treatment effect of metastatic tumors in the lungs. Summary of the invention

[0006] One of the objects of the present invention is to provide a bacterial assembly, which is prepared from facultative anaerobic bacteria, double-terminal maleimide polyethylene glycol and cationic albumin.

[0007] Further, the facultative anaerobic bacteria are selected from the group consisting of Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, Proteus, Enterobacter, Salmonella typhi, Salmonella, Shigella, etc.), Staphylococcus and Streptococcus, preferably Salmonella, more preferably attenuated Salmonella typhimurium VNP20009.

[0008] Furthermore, the molecular weight of the polyethylene glycol in the double-terminated maleimide polyethylene glycol is 1K, 2K, 5K, 10K, 20K or 40K, preferably 2K.

[0009] Furthermore, the cationic albumin is cationized bovine serum albumin.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned bacterial assembly, comprising the following steps:

[0011] Step 1: After activating the bacteria, culture them until the OD value is 0.6-0.8, collect the bacterial solution and centrifuge it, resuspend the bacteria in the culture medium and adjust the OD value to 2.0 (the bacterial concentration is 8×10 8 CFU / mL), the concentrated bacterial solution was centrifuged, the bacteria were resuspended in sterile PBS and centrifuged again;

[0012] Step 2, mixing the bacterial solution obtained in step 1 with a reducing agent, and then adding cationic albumin and double-terminated maleimide polyethylene glycol to react to obtain a bacterial assembly.

[0013] Furthermore, in step 2, every 8×10 7 CFU bacteria were mixed with 100 μL 0.3-50 mM reducing agent, wherein the reducing agent, cationic albumin and double-terminated maleimide polyethylene glycol were used in the following proportions: 10 μL 10 mg / mL cationic albumin and 10 μL 10-200 mg / mL double-terminated maleimide polyethylene glycol were added to every 100 μL 0.3-50 mM reducing agent.

[0014] Furthermore, the reducing agent is a protein reducing agent selected from dithiothreitol (DTT), β-mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, sodium dodecyl sulfate (SDS), preferably TCEP.

[0015] Preferably, the reaction conditions in step 2 are 20-37° C., 50-200 rpm, and stirring for 0.25-2 h.

[0016] Preferably, the concentration of the reducing agent is 3 mM, and the concentration of the double-terminated maleimide polyethylene glycol is 50 mg / L.

[0017] The third object of the present invention is to provide the use of the above-mentioned bacterial assembly in the preparation of tumor therapeutic drugs.

[0018] Furthermore, the tumor is a metastatic tumor originating from the lung or outside the lung, including lung cancer, breast cancer, colon cancer, colorectal cancer, thyroid cancer, liver cancer, lymphoma, kidney cancer, esophageal tumor or uterine tumor.

[0019] The cell assembly can be used alone to prepare anti-tumor drugs, or to use bacteria to express proteins, polypeptides, RNA with anti-tumor effects, or to form a composition with existing anti-tumor drugs, or to be used in combination with existing chemical drug treatment, traditional Chinese medicine treatment, physical therapy, and biological treatment methods in the anti-tumor process; increase the titer of bacteria in lung tumors, reduce the number of bacteria in other tissues, reduce the side effects of oncolytic bacterial treatment, and improve the tumor inhibition effect.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention focuses on bacterial therapy, a cutting-edge field of tumor treatment. In view of the difficulty of bacteria (especially VNPs) to be enriched in the lungs, the physiological characteristics of the lungs are used as the target, and based on the characteristics of the micron system passively enriched in the lungs by deposition in the pulmonary capillaries, an assembly specifically targeting the lungs is constructed. Specifically, the disulfide bonds on the surface of the bacterial cell wall are reduced by the reducing agent TCEP to expose the thiol groups, and the double-end maleimidized polyethylene glycol is used to react with the thiol groups to form an assembly by connecting multiple VNPs with exposed thiol groups on the surface. The reaction system also adsorbs the negatively charged surface of bacteria by adding cationic albumin, reduces electrostatic repulsion, thereby strengthening aggregation, and effectively masks bacterial surface antigens, reducing the clearance of immune cells. This assembly mechanism has not been reported in the literature, and the research is groundbreaking.

[0022] (2) On this basis, the present invention will regulate Mal-PEG 2k -Mal(PEG) and TCEP concentrations were adjusted to construct VNP assemblies of different sizes, revealing the size-dependent pattern of VNP assembly enrichment in the lungs and enriching the design ideas of the system targeting lung metastases.

[0023] The bacterial assembly of the present invention increases lung enrichment while reducing the distribution of bacteria in normal tissues, has higher lung tumor targeting, and effectively improves the therapeutic effect of lung tumors. Cationic albumin is covered on the surface of bacteria and connected to form an assembly, which reduces the surface antigens exposed to the outside of the bacteria, thereby reducing the clearance and immune response of antigen-presenting cells. The application effect of the present invention does not significantly reduce the weight loss, splenomegaly and liver inflammatory lesions of tumor models induced by oncolytic bacteria therapy, and effectively avoids the toxic and side effects of oncolytic bacteria therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a scanning electron microscope photograph of Poly-VNP in Example 1.

[0025] Figure 2 The graph shows the change in the proliferation ability of Poly-VNP in Example 1, and the graph shows the 45-hour growth curve of Poly-VNP.

[0026] Figure 3 These are fluorescence microscope photos of different bacterial assemblies in Example 1.

[0027] Figure 4 In the figure, A is Poly-VNP with different particle sizes. GFP A is a fluorescence microscope photo of Poly-VNP with different particle sizes. GFP Particle size statistics.

[0028] Figure 5 In the figure, A is Poly-VNP with different particle sizes. Lux Bioluminescence and bright field superposition imaging of lung tissue coated plate (I.8.16μm, II.8.45μm, III.9.14μm, V.10.42μm); B is Poly-VNP with different particle sizes Lux Fluorescence statistics of the enrichment quantity in lung tissue (n=3, ns indicates no significant difference, ***P<0.001).

[0029] Figure 6 In the example, A is Poly-VNP. RFP Disintegration in mouse serum; B is Poly-VNP RFP The relationship between particle size and time (n>9, ns indicates no significant difference, *P<0.1, ****P<0.0001).

[0030] Figure 7 In the figure, A is the flow chart of the Poly-VNP in vivo lung targeting experiment (the prepared VNP LUX or Poly-VPN LUX The cells were injected into 4T1 tumor-bearing mice via the tail vein. The mice were killed 2 hours after the injection, and the organs were removed by dissection. The tissues were ground, diluted and spread on LB plates with resistance. B is VNP Lux and Poly-VPN Lux Bioluminescence and bright field superimposition imaging of tissues of heart, liver, spleen, lung and kidney (n=3), 1 is the image of each organ tissue after homogenization and coating on LB plate, 2 is the image of each organ directly after dissection; C is VNP Lux and Poly-VPN Lux Statistics of the ratio of enrichment number to total bacterial number in heart, liver, spleen, lung and kidney (n=3).

[0031] Figure 8 In the figure, A is a comparison of the enrichment quantity of VNP and Poly-VNP in lung melanoma. The tumor tissue was homogenized and spread on a resistant LB plate for photography; B is the statistics of the enrichment quantity of VNP and Poly-VNP in lung melanoma (n=3, *P<0.1, **P<0.01, ***P<0.001).

[0032] Fig. 9 In the figure, A shows the comparison of spleen enlargement after 6 days of Poly-VNP treatment; B shows the comparison of organ weights of B16F10 tumor-bearing mice after 6 days of Poly-VNP treatment (n=8, ns indicates no significant difference). Mice were injected with PBS, VNP, and Poly-VNP via the tail vein.

[0033] Fig.10 In the figure, A is the comparison of serum liver function index AST / ALT in B16F10 tumor-bearing mice after 22 days of Poly-VNP treatment (n=3, ns indicates no significant difference); B is the comparison of serum leukocyte count in B16F10 tumor-bearing mice after 22 days of Poly-VNP treatment (n=3). Mice were injected with PBS, VNP and Poly-VNP through the tail vein, respectively, and control indicates non-tumor-bearing mice without treatment.

[0034] Fig.11 H&E-stained sections of various organs of H22 tumor-bearing mice 22 days after Poly-VNP treatment. Scale bar is 200μm.

[0035] Fig.12 In the figure, A is the experimental process of Poly-VNP in preventing lung metastasis in 4T1 tumor-bearing mice (the prepared VNP or Poly-VNP was injected into BALB / c mice through the tail vein, and 4T1-luci cells were injected through the tail vein 2 days after the injection, and in vivo imaging was performed 9 and 12 days after the injection, respectively); B is the in vivo imaging analysis of designated time points during the Poly-VNP treatment of 4T1 tumor-bearing mice, and the figure shows the superposition of bioluminescence and bright field imaging on the 9th and 12th days; C is the analysis of the severity of tumor lung metastasis based on bioluminescence intensity at different time points in the lung tumor site (n=3, ns indicates no significant difference, *P<0.1, ***P<0.001).

[0036] Fig.13 A is the experimental process of Poly-VNP in preventing lung metastasis in B16F10 tumor-bearing mice (the prepared VNP or Poly-VNP was injected into BALB / c mice through the tail vein, and B16F10 cells were injected through the tail vein 3 days after the injection. On the 25th day, the mice were dissected and the lung tissue sections were stained); B is the comparison of lung metastasis in 4T1 tumor-bearing mice treated with Poly-VNP; C is the H&E staining section of lung tissue in 4T1 tumor-bearing mice treated with Poly-VNP, and the right area is the detail display of the corresponding area on the left; D is the analysis of the severity of lung metastasis in 4T1 tumor-bearing mice treated with Poly-VNP based on the ratio of tumor nodule area to total lung area (n=3, **P<0.01, ****P<0.0001).

[0037] Fig.14A is the experimental process of Poly-VNP treatment of B16F10 tumor-bearing mice and inhibition of lung metastasis (4T1-luci cells were injected into the mammary fat pad of BALB / c mice to form subcutaneous tumors, and the tumors were surgically removed 13 days after tumor implantation. In vivo imaging was performed on the 22nd day, and the mice were evenly divided into groups according to the severity of tumor recurrence. On the 23rd day, the prepared VNP or Poly-VNP was injected into the tumor-bearing mice through the tail vein. In vivo imaging was performed on the 29th and 38th days, and the mice were dissected for lung tissue analysis); B is the experimental process of Poly-VNP treatment of 4T1 tumor-bearing mice In vivo imaging analysis of mice at designated time points during the process, the figure shows the superposition of bioluminescence and bright field imaging on days 22, 29, 12, and 38; C is the analysis of the severity of lung metastasis in 4T1 tumor-bearing mice treated with Poly-VNP based on the ratio of tumor nodule area to total lung area (n=3, **P<0.01, ****P<0.0001); D is the comparison of in situ tumor recurrence and hepatosplenomegaly in 4T1 tumor-bearing mice treated with Poly-VNP; E is the comparison of organ weights in 4T1 tumor-bearing mice treated with Poly-VNP (n=5, ns indicates no significant difference). DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods in the following examples of the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0039] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0041] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0042] The attenuated Salmonella in the following examples is attenuated Salmonella typhimurium VNP20009 and its derivatives or genetically modified strains, including: VNP20009 carrying the green fluorescent protein gene (VNP GFP ), VNP20009 carries the red fluorescent protein gene (VNP RFP ), VNP20009 carries the luciferase gene LuxCDABE (VNP Lux ), and its tumor targeting ability is not changed.

[0043] Example 1

[0044] Construction of attenuated Salmonella typhimurium assembly

[0045] The specific process of constructing the attenuated Salmonella assembly is as follows: pick a single colony of attenuated Salmonella VNP20009 (VNP) and inoculate it into 3mL of kanamycin-resistant LB liquid culture medium, and culture it in a 37°C shaker overnight; take the overnight cultured bacterial solution and inoculate it into 3mL of kanamycin-resistant LB liquid culture medium at a ratio of 1:50, and culture it in a 37°C shaker until the OD600 is between 0.6-1.0, that is, the bacteria grow to the logarithmic growth phase. Collect 2mL of bacterial solution in a 2mL sterile EP tube, centrifuge at 5000rpm at room temperature for 5min, discard the culture supernatant, resuspend the bacteria with 800μL of LB liquid culture medium, measure the OD600 value and add appropriate LB liquid culture medium until the OD600 value is 2.0 (the bacterial concentration is 8×10 8 CFU / mL). Pipette 100 μL of concentrated bacterial solution into a 2 mL sterile EP tube, centrifuge at 5000 rpm at room temperature for 5 min, and discard the culture medium supernatant; resuspend the bacteria with 1 mL sterile PBS buffer, centrifuge at 5000 rpm at room temperature for 5 min, and discard the PBS buffer; resuspend the bacterial solution with 100 μL TCEP (3 mM) solution, mix well, add a 3×5 mm magnetic stir bar, and stir magnetically at 60 rpm for 10 minutes in a 37°C water bath to expose the thiol groups on the bacterial surface; add 10 μL cBSA (10 mg / mL) and 10 μL PEG (Mal-PEG 2k -Mal, 50 mg / mL), fully mixed, added a 3×5 mm magnetic stirring bar, and magnetically stirred at 60 rpm for 30 minutes in a 37°C water bath to obtain the attenuated Salmonella assembly (Poly-VNP).

[0046] like Figure 1As shown, the untreated VNP has a particle size of about 2-3 μm, a smooth surface, and a regular cylindrical shape; the assembled Poly-VNP is composed of 40-60 VNPs, and the bacteria are tightly connected to form an irregular oval with a particle size of about 8-10 μm. The reaction conditions are mild, and the use of thiol TCEP as a reducing agent can selectively reduce disulfide bonds on the surface of bacteria, and the reaction can be completed quickly at room temperature.

[0047] like Figure 2 As shown, the 45h bacterial growth curve showed that compared with VNP, the time for the thiolated VNP to reach the plateau phase and the plateau OD value in the LB liquid culture medium had no significant difference, indicating that surface modification did not affect the proliferation ability of bacteria.

[0048] The above construction method can also be used for other strains, such as E. coli Nissle1917 (ECN), E. coli K12MG1655 (ECG), E. coli BL21 (DE3) and Bifidobacterium longum (B. longum). This example uses the above cross-linking reaction conditions to prepare ECN carrying the green fluorescent protein gene (ECN GFP ), ECN carries the green fluorescent protein gene (ECG GFP ), BL21 carrying the red fluorescent protein gene (BL21 RFP ) and Bifidobacterium longum (B. longum) assembly system.

[0049] like Figure 3 As shown, ECN GFP , ECG GFP and BL21 RFP The assembly structure is similar to Poly-VNP, with a particle size of about 8-10μm and an irregular oval shape; the size of B. longum bacteria is about 5-8μm, so the particle size of the cross-linked assembly is larger, reaching 20μm.

[0050] Example 2

[0051] Screening of the optimal particle size for lung enrichment of attenuated Salmonella typhimurium assemblies

[0052] The method of Example 1 was used to adjust the concentrations of PEG (50-100 mg / mL) and TCEP (3-30 mM) to prepare VNPs of different particle sizes. GFP Assemblies (Poly-VNP GFP ), the specific concentration ratio and grouping are shown in Table 1, the prepared Poly-VNP GFP Fluorescence images such as Figure 4 As shown in A. The area of ​​the assembly was counted by Fiji, and the irregular elliptical particle size was converted to the diameter of the equal-area circle to obtain the Poly-VNPGFP The particle size distribution is 8-10.5μm, such as Figure 4 As shown in B.

[0053] Table 1 Poly-VNP with different particle sizes GFP The construction conditions

[0054] Group Name I II III IV V VI VII VIII PEG concentration (mg / mL) 50 100 50 100 50 100 50 100 TCEP concentration (mM) 3 3 5 5 10 10 30 30

[0055] The experimental animals were 6-8 week old female BALB / c mice or C57BL / 6 mice (Changzhou Cavens Laboratory Animal Co., Ltd.), and the mice were randomly divided into different groups. Lux Poly-VNP was prepared by cross-linking as described above. Lux -I(8.16μm),Poly-VNP Lux -II(8.45μm),Poly-VNP Lux -III (9.14 μm) and Poly-VNP Lux -V (10.42 μm) were injected into BALB / c mice via the tail vein at a dose of 1×10 7 CFU / mouse. After 2 hours, the mice were dissected, the lung tissues were cut into small pieces, resuspended with sterile PBS and 1‰ Triton X-100, and the tissues were fully ground by Tissuelyser-24; the suspension was diluted ten times with sterile PBS, 100 μL was evenly applied to LB solid medium with kanamycin resistance, and cultured overnight at a constant temperature until obvious single colonies appeared, and then the bioluminescence and bright field superimposed images were taken by PE IVIS Spectrum in vitro live imaging system ( Figure 5 The results show that the particle size of Poly-VNP is 8 μm. Lux -I has the highest enrichment efficiency in the lungs, which is 3 times that of other assemblies ( Figure 5 Therefore, the Poly-VNP obtained under this reaction condition was used for subsequent studies.

[0056] Example 3

[0057] Detection of the ability of attenuated Salmonella assemblies to disintegrate and release bacteria in serum

[0058] About 1 mL of blood was collected from the eyeball of healthy C57BL / 6 mice and placed in a 1.5 mL EP tube. After standing at room temperature for 2 hours, the tube was centrifuged at 3000 rpm for 10 minutes at room temperature. The upper serum was aspirated and an equal volume of sterile PBS was added. RFP Poly-VNP was prepared according to the conditions in Example 2 RFP , added to the resulting serum, the bacterial concentration was 1×10 8CFU / mL, cultured at 37°C, and the disintegration of the assembly was observed under a fluorescence microscope on the 1st, 2nd, 3rd and 4th days.

[0059] like Figure 6 As shown in A, after 1 day of culture in serum, Poly-VNP RFP The structure remained stable, gradually disintegrated on the third day, released VNP, and completely disintegrated on the fourth day ( Figure 6 (B) Combined with the growth curve, it is confirmed that the attenuated Salmonella assembly system of the present invention can gradually disintegrate in the retained blood vessels and release bacteria.

[0060] Example 4

[0061] In vivo biodistribution and tumor targeting detection of attenuated Salmonella typhimurium assemblies

[0062] After 4T1 mouse breast cancer cells were grown in 1640 cell basal medium to the exponential growth phase, they were digested with 0.5% trypsin digestion solution for 3 minutes, 1640 cell basal medium was added to terminate the digestion and the cells were collected by blowing. Then, they were centrifuged at 800 rpm for 5 minutes at room temperature, the supernatant of the culture medium was removed, and the cells were washed twice with PBS. The cells were counted using a hemocytometer, and an appropriate amount of PBS was added to adjust the cell number to 2×10 6 Each mouse was injected with 100 μL via the tail vein, i.e. 2×10 5 After inoculation, the mice were kept in a clean animal room for about 7 days until lung metastasis formed before subsequent experiments.

[0063] VPN Lux Poly-VNP was prepared according to the conditions in Example 2 Lux , respectively VPN Lux and Poly-VPN Lux VNPs obtained in previous experiments were injected into 4T1 tumor-bearing mice via tail vein. Lux Therapeutically effective dose, VNP Lux and Poly-VPN Lux The injection dose was 1×10 6 CFU / mouse, after intravenous injection, the mice were dissected, and the heart, liver, spleen, lung, kidney and blood were collected, ground and diluted by the above method, and spread on LB solid medium with kanamycin resistance and cultured at a constant temperature, among which the heart, lung, kidney and blood were diluted 10 times, the spleen was diluted 100 times, and the liver was diluted 1000 times. Figure 7 As shown in B and C, after tail vein injection, VNP Lux In comparison, Poly-VPN Lux The distribution in the heart, kidney and liver was significantly reduced, while the distribution in the lung and blood increased, proving that Poly-VNP LuxIncreased lung tumor targeting via vascular retention.

[0064] The colonization of VNP and Poly-VNP in the tumor was further compared by lung tumor tissue dilution plating method. Figure 8 As shown in A and B, Poly-VNP increased the enrichment of lung capillaries, reaching 2.3 times the number of lung tumor colonization of VNP, thereby improving the anti-tumor lung metastasis effect.

[0065] Example 5

[0066] In vivo evaluation of attenuation efficacy of attenuated Salmonella assemblies

[0067] To investigate whether Poly-VNP can reduce the toxicity and inflammatory response caused by intravenous injection, the morphological characteristics and pathological changes of major organs after tail vein injection of VNP and Poly-VNP were subsequently evaluated. After B16F10 mouse melanoma cells were grown in 1640 cell basal medium to the exponential growth phase, the cells were digested with 0.5% trypsin digestion solution for 3 minutes, 1640 cell basal medium was added to terminate the digestion and the cells were collected by blowing. Then centrifugation was performed at 800 rpm at room temperature for 5 minutes, the supernatant of the culture medium was removed and washed twice with PBS, the cells were counted by a hemocytometer, and the cell number was adjusted to 2×10 by adding an appropriate amount of PBS. 6 Each mouse was injected with 100 μL via the tail vein, i.e. 2×10 5 After inoculation, the mice were kept in a clean animal room for about 7 days until lung metastasis was formed before subsequent experiments. Poly-VNP, VNP and Poly-VNP prepared by VNP20009 according to the conditions in Example 2 were injected into B16F10 tumor-bearing mice via the tail vein, and the mice were dissected 6 days later to obtain the heart, liver, spleen, lung, kidney and blood for subsequent studies. Fig. 9 As shown in A and B, the strategy of the present invention can significantly reduce VNP-induced hepatosplenomegaly. About 1 mL of blood was collected from the mouse eyeball, of which 0.5 mL was collected in an anticoagulant tube and stored at 4°C, and 0.5 mL was collected in a 1.5 mL EP tube. After standing at room temperature for 2 hours, the upper serum was collected by centrifugation at 3000 rpm and 4°C for 10 minutes. Hematological testing was performed by Sewell Bioservices. Liver injury indicator AST / ALT test showed ( Fig.10 Middle A), Poly-VNP treatment significantly alleviated VNP-induced acute liver injury; the increase in white blood cells and lymphocytes in the blood indicated that Poly-VNP could induce immune system activation and enhance anti-tumor ability ( Fig.10 Middle B). H&E stained sections of major organs show ( Fig.11), compared with VNP, Poly-VNP caused less spleen damage area. All these results indicate that the Poly-VNP treatment strategy effectively avoids the toxic side effects caused by intravenous VNP.

[0068] Example 6

[0069] Detection of the effect of attenuated Salmonella assembly in preventing tumor lung metastasis

[0070] Subsequently, the effects of different treatment groups on preventing lung metastasis of tumors were evaluated. Poly-VNP, VNP and Poly-VNP prepared by VNP20009 according to the conditions in Example 2 were injected into BALB / c mice via the tail vein at a dose of 1×10 6 CFU / mouse. Two days later, 100 μL of 4T1 cell suspension expressing luciferase was injected into the tail vein to simulate tumor lung metastasis. The injection dose was 2×10 6 / mouse, and bioluminescence imaging was performed using the PE IVIS Spectrum in vitro live imaging system on the 9th and 12th days. The chest and abdominal hair of the mice were shaved before imaging, and 100 μL of luciferase substrate D-luciferin potassium salt was injected intraperitoneally at a dose of 3 mg / mouse. After 10 minutes, the mice were placed in a gas anesthesia chamber and anesthetized with isoflurane. After the mice were completely anesthetized, they were placed flat on the live imaging platform with their chests facing up to detect the fluorescence signal. Fig.12 As shown in B and C, fluorescence signals first appeared in the lungs of mice in the control group on the 9th day after treatment. At this time, the fluorescence signals in the lungs of mice in the VNP and Poly-VNP treatment groups were weak, and no obvious lung metastasis had occurred. On the 12th day after treatment, fluorescence signals appeared in the lungs of mice in each group. Compared with the control group, the fluorescence in the lungs of mice after VNP treatment was reduced to 0.41 times. The Poly-VNP treatment had the best effect, and the fluorescence intensity in the lungs of mice was only 0.34 times that of the VNP treatment group.

[0071] The same effect of preventing tumor lung metastasis was observed in the B16F10 experimental metastasis model. Poly-VNP, VNP and Poly-VNP prepared by VNP20009 according to the conditions in Example 2 were injected into C57BL / 6 mice via the tail vein at a dose of 1×10 6 CFU / mouse, and B16F10 cell suspension was injected into the tail vein 3 days later to simulate tumor lung metastasis. The injection dose was 2×10 6 / mouse, and lung tissue was dissected on the 25th day to analyze the severity of lung metastasis. The results showed that a large number of melanoma metastatic nodules appeared in the lungs of untreated mice, and the lungs were dark black with white blocky tissue lesions. The corresponding melanoma nodules were significantly reduced in the mice treated with VNP, and the lungs returned to bright red. Consistent with previous results, Poly-VNP treatment had the best effect, with almost no obvious black nodules ( Fig.13 (B). H&E stained lung sections showed that the area of ​​lung metastatic melanoma nodules was counted by Fiji. The area of ​​lung nodules in the control group was 3.8 times that of the VNP treatment group, while that in the Poly-VNP treatment group was only 0.3 times that of the VNP treatment group ( Fig.10 The above results strongly prove that the attenuated Salmonella assembly Poly-VNP treatment has a better effect in preventing tumor lung metastasis while reducing toxicity compared with VNP alone.

[0072] Example 7

[0073] Detection of the effect of attenuated Salmonella assemblies on the treatment of lung metastasis from orthotopic tumors

[0074] In the orthotopic tumor mouse model, the most common treatment for early-stage tumor patients was simulated, that is, surgical removal of the tumor followed by drug therapy. 100 μL of luciferase-expressing 4T1 cell suspension was inoculated into the mammary fat pad of BALB / c mice, and the injection dose was 2×10 6 / mouse. After inoculation, the mice were kept in a clean animal room. Thirteen days after tumor implantation, the tumor volume of the mice grew to about 200 mm 3 When the tumor is removed by surgery. Tumor cells remaining during the operation may continue to grow, forming in situ recurrence, or enter the circulation through damaged blood vessels during the operation, forming tumor metastasis. Among them, lung metastasis, as the most common form of tumor metastasis, causes the death of 20-30% of cancer patients. Live imaging was performed on the 22nd day, and the mice were evenly divided into groups according to the severity of tumor recurrence and the fluorescence intensity of the lungs. Then on the 23rd day, VNP and Poly-VNP (VNP20009 was prepared according to the conditions in Example 2) were injected into tumor-bearing mice through the tail vein, and live imaging was performed on the 29th and 38th days. The mice were dissected and lung tissue was analyzed. The results showed that after 6 days of treatment (day 29), fluorescent signals appeared in the lungs of the control group, proving that lung metastasis had begun to appear, and some mice had head and neck metastasis. In contrast, almost no fluorescent signals were found in the lungs in the VNP or Poly-VNP treatment groups. On the 38th day, the degree of lung metastasis in the control group was serious, while only a small amount of lung metastasis was observed in the VNP treatment group. The Poly-VNP treatment group had the best effect, and almost no fluorescent signals were observed in the lungs ( Fig.14In order to further analyze the therapeutic effect, mice were dissected on the 38th day, and the lungs were taken out and bioluminescence superimposed bright field images were taken in the in vivo imaging system. The results showed that the lung fluorescence signal of the VNP-treated mice was only 0.07 times that of the control mice. Poly-VNP treatment further enhanced the effect. Compared with the VNP-treated group, the lung fluorescence signal was only 0.3 times that of the control group ( Fig.14 C), demonstrating its enhanced anti-tumor lung metastasis ability. It is worth noting that VNP treatment significantly inhibited the growth of in situ tumors while inhibiting lung metastasis. Poly-VNP treatment even caused a decrease in tumor volume ( Fig.14 D), which is consistent with the results of previous VNP treatment studies on solid tumors. The unexpected therapeutic effect of Poly-VNP may be due to the fact that tumor tissue is rich in new blood vessels and has poor continuity, which is similar to the capillary microenvironment of the lungs, and can intercept large-size bacterial assemblies in the blood circulation, so that Poly-VNP can also achieve an improved in situ tumor enrichment effect after injection, thereby significantly inhibiting the growth and development of in situ tumors. In addition, the Poly-VNP treatment in the aforementioned experiment caused an increase in immune cells in the blood, which can also inhibit the growth of in situ tumors. Consistent with previous results, Poly-VNP treatment alleviated VNP treatment-induced hepatosplenomegaly ( Fig.14 (E) These results strongly demonstrate that compared with the use of VNP alone, the attenuated Salmonella assembly Poly-VNP treatment has a better and more unexpected effect on the treatment of postoperative tumor recurrence and lung metastasis while reducing toxicity.

[0075] The attenuated Salmonella assembly can be used alone to prepare anti-tumor drugs, or can be combined with existing anti-tumor drugs for treatment, or used in combination with existing chemical drug treatment, biological treatment, traditional Chinese medicine treatment or physical treatment method in anti-tumor treatment.

[0076] The above description shows and describes several preferred embodiments of the present invention, but as mentioned above, the present invention is not limited to the application forms listed herein, and should not be regarded as excluding other embodiments. The present invention can be used in various other combinations and methods, and can be modified within the scope of the present invention through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims

1. A bacterial assembly, characterized in that Made from facultative anaerobic bacteria, double-terminated maleimide polyethylene glycol, and cationic albumin.

2. The bacterial assembly according to claim 1, characterized in that The facultative anaerobic bacteria are selected from the group consisting of Enterobacteriaceae, Staphylococcus and Streptococcus.

3. The bacterial assembly according to claim 1, characterized in that The molecular weight of the polyethylene glycol in the double-terminated maleimide polyethylene glycol is 1K, 2K, 5K, 10K, 20K or 40K.

4. The bacterial assembly according to claim 1, characterized in that The cationic albumin is cationized bovine serum albumin.

5. The method for preparing the bacterial assembly according to claim 1, characterized in that: The following steps are involved: Step 1, activating the facultative anaerobic bacteria and culturing them to an OD value of 0.6-0.8, collecting the bacterial solution and centrifuging it, resuspending the bacteria in a culture medium to adjust the OD value to 2.0, centrifuging the concentrated bacterial solution, resuspending the bacteria in sterile PBS and centrifuging again; Step 2, mixing the bacterial solution obtained in step 1 with a reducing agent, and then adding cationic albumin and double-terminated maleimide polyethylene glycol to react to obtain a bacterial assembly.

6. The preparation method according to claim 5, characterized in that In step 2, each 8×10 7 CFU bacteria are mixed with 100 μL 0.3-50 mM reducing agent, wherein the reducing agent, cationic albumin and double-terminal maleimide polyethylene glycol are used in the following ratio: 10 μL 10 mg / mL cationic albumin and 10 μL 10-200 mg / mL double-terminal maleimide polyethylene glycol are added to every 100 μL 0.3-50 mM reducing agent.

7. The preparation method according to claim 5, characterized in that: The reducing agent is a protein reducing agent selected from dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine, reduced glutathione, and sodium dodecyl sulfate.

8. Use of the bacterial assembly according to claim 1 in the preparation of tumor therapeutic drugs.

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