Bacterial surface modification method based on atom transfer radical polymerization reaction mediation, modified bacteria and application of modified bacteria

Abstract

The invention discloses a bacteria surface modification method based on atom transfer radical polymerization reaction mediation, obtained bacteria and application. The modification method comprises the following steps: carrying out acryloylation on the surfaces of the bacteria by using N-hydroxysuccinimide acrylate, and introducing double bonds on the surfaces of the bacteria to obtain acryloylated bacteria; the method comprises the following steps: by taking acrylamide, N-(3-aminopropyl) methacrylamide hydrochloride and N, N '-methylene bisacrylamide as monomers, carrying out polymerization reaction on the surfaces of acrylated bacteria in the presence of an initiator and a catalyst; the mass ratio of the acrylamide to the N-(3-aminopropyl) methacrylamide hydrochloride to the N, N '-methylene bisacrylamide is (11.9 to 17.5) to (0.125 to 1) to (0.1 to 0.5). According to the method, bacteria can be camouflaged to reduce immunogenicity and pathogenicity of the bacteria, the safety of bacteria treatment is improved under the condition that the viability and multiplication capacity of the bacteria are not affected, the resistance of the bacteria to environmental threats is enhanced, and the bacteria are endowed with no exogenous functions.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a bacterial surface modification method mediated by atom transfer radical polymerization, the modified bacteria and applications thereof. Background Art

[0002] Immunotherapy is considered the fourth pillar of cancer treatment. It targets the activation and enhancement of the immune system and kills tumor cells by blocking inhibitory immune checkpoint pathways or triggering antigen-specific immune responses in tumors. Cancer immunotherapy includes cytokine therapy, immune cell therapy, tumor vaccines, immune checkpoint inhibitors, etc. Among them, immune checkpoint inhibitors are the most widely used immunotherapy methods. Since the first clinical trial of anti-PD-1 monoclonal antibody in 2006, the US Food and Drug Administration approved the first batch of anti-PD-1 monoclonal antibodies (pembrolizumab and nivolumab) for clinical treatment in 2014. Since then, the treatment of tumors has officially entered the era of immunotherapy from chemotherapy and targeted therapy.

[0003] However, the therapeutic efficiency and patient response population of single immunotherapy are limited. Immunotherapy is often combined with other conventional cancer therapies, especially radiotherapy and chemotherapy. Therefore, targeted delivery of anti-tumor drugs and the development of drug resistance are two major challenges in the treatment of cancer. At the same time, some bacteria are able to selectively grow in tumors in hypoxic environments, and people are increasingly interested in bacterial preparations as anti-cancer drugs. With the development of genetic engineering and synthetic biology technologies, bacterial-mediated therapy has developed rapidly in the biomedical field in the past few decades. Bacteria can be used as therapeutic agents to treat diseases by exerting their inherent functions, such as activating immunity and secreting bacteriocins. On the other hand, bacteria are widely used as carriers to deliver classic clinical drugs to hypoxic lesions, increase drug utilization at the disease site, and further improve the therapeutic effect. Although bacteria have shown great potential in tumor treatment, the early application of bacterial therapy is often accompanied by instability such as septic shock and septic death due to its reliance on the inherent toxicity and immunogenicity of bacteria. At the same time, bacteria also release special protein toxins to inhibit the immune response of the infected host, which not only weakens the therapeutic effect, but also destroys the body's own immune response to the original tumor. In addition, bacteria can inhibit the migration of macrophages and reduce the integrity of the barrier established by epithelial cells, which leads to the spread of tumors and invasion of adjacent healthy tissues. In order to retain the anti-tumor therapeutic effect of natural live bacteria and reduce their own toxicity, in recent years, modern material surface engineering technology has provided an important technical means to overcome the above problems and develop new live bacterial drugs and their advanced delivery platforms. Material surface engineering is to design the surface of the material and the body as a system, and use different surface technologies to enable the material to obtain new properties that it does not have or improve the disadvantages of the material in application. The development of bacterial surface modification strategies mainly includes three aspects: 1) Without affecting the survival and proliferation ability of bacteria, by modifying the surface of bacteria, reducing the immunogenicity and pathogenicity of bacteria, and improving the safety of bacterial therapy; 2) protecting bacteria, enhancing the resistance of bacteria to environmental threats, and improving the bioavailability or drug availability of bacteria at the disease site; 3) giving bacteria exogenous functions that they do not have, so as to effectively and accurately control bacterial activity and biological behavior, thereby improving the therapeutic effect. However, it is a huge challenge to develop a strategy that meets all three aspects at the same time. Existing technologies still have defects such as complex synthesis process, difficulty in achieving modular standardized production; poor coating coverage; coating shell thickness of hundreds of nanometers causing bacterial cells to be dormant; and poor biocompatibility and biosafety after bacterial surface modification. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to improve the safety of bacterial carriers and their resistance to environmental threats.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] A bacterial surface modification method based on atom transfer radical polymerization, comprising the following steps:

[0007] S1. acrylylating the surface of bacteria using N-hydroxysuccinimide acrylate to introduce double bonds on the surface to obtain acrylylated bacteria;

[0008] S2. Using acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride and N,N'-methylenebisacrylamide as monomers, a polymerization reaction is carried out on the surface of acrylylated bacteria in the presence of an initiator and a catalyst to obtain bacteria wrapped in a thin polymer shell; wherein the mass ratio of acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride and N,N'-methylenebisacrylamide is 11.9-17.5:0.125-1:0.1-0.5.

[0009] Preferably, in S1, the bacteria are bacteria containing amino groups on the surface of the cell membrane.

[0010] Preferably, in S1, the bacteria is Escherichia coli.

[0011] Preferably, in S1, the steps are specifically included: mixing the DMSO solution of N-hydroxysuccinimide acrylate with the bacterial culture liquid, vortexing, centrifuging, and washing.

[0012] Preferably, every 10 8 The mass of N-hydroxysuccinimide acrylate used for each colony was 0.01 mg.

[0013] Preferably, in S2, the initiator is ammonium persulfate; and the catalyst is tetramethylethylenediamine.

[0014] Preferably, in S2, the mass ratio of the initiator to N,N'-methylenebisacrylamide is 1:1; the mass ratio of the catalyst to N,N'-methylenebisacrylamide is 7.75:1.

[0015] Preferably, in S2, every 10 8 The mass of acrylamide used for each colony is 11.9-17.5 mg.

[0016] Preferably, in S2, the concentration of acrylamide in the system is 11.9-17.5 mg / mL.

[0017] Preferably, in S2, the following steps are specifically included: resuspending the acrylylated bacteria with PBS, then adding Aam aqueous solution, Apm aqueous solution and Bis DMSO solution, adding Aps aqueous solution and TMEDA aqueous solution after incubation, shaking the reaction, centrifuging and washing.

[0018] Preferably, the method further comprises modifying the surface of the bacteria obtained after the polymerization reaction with a tumor targeting polypeptide.

[0019] Preferably, the bacteria obtained after the polymerization reaction and the tumor targeting polypeptide are oscillated in a solution to perform surface modification of the tumor targeting polypeptide.

[0020] Preferably, the tumor targeting polypeptide is NHS-PEG3400-RGD.

[0021] Preferably, the molar ratio of the tumor targeting polypeptide to the N-(3-aminopropyl)methacrylamide hydrochloride used is 5-20:1.

[0022] The present invention also provides a bacterium wrapped in a thin polymer shell, which is prepared by using the bacterial surface modification method mediated by atom transfer radical polymerization.

[0023] The present invention also provides an application of the bacteria wrapped by the thin polymer shell as a drug delivery carrier.

[0024] The advantages of the present invention are:

[0025] In the present invention, a thin polymer shell composed of multiple functional monomers is formed on the surface of the bacteria by in situ free radical polymerization to protect the bacteria from being degraded by enzymes or cleared by immune cells, thus overcoming multiple physiological barriers. At the same time, the preparation conditions and process are simple and convenient, and modular standardized production can be achieved, with the advantages of low cost, low toxicity, and high efficiency. At the same time, the bacterial coating effect is good, and by controlling the number and type of monomers, the bacteria are given better biocompatibility and biosafety, thereby optimizing its multi-stage drug delivery process, including blood circulation and tumor accumulation, so it may become a universal platform for cross-barrier drug delivery and effective cooperation of multiple drugs, such as cells, bacteria, proteins, nucleic acids and small molecules.

[0026] The thin polymer shell prepared by the monomer and preparation method of the present invention can disguise bacteria to reduce their immunogenicity and pathogenicity, improve the safety of bacterial treatment without affecting the survival and proliferation capabilities of bacteria, enhance the resistance of bacteria to environmental threats, and improve the bioavailability of bacteria in diseased areas. At the same time, it can also give bacteria some exogenous functions that they do not have, effectively and accurately control the activity and biological behavior of bacteria, and improve the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of bacterial surface modification in an embodiment of the present invention;

[0028] Figure 2 This is a schematic diagram of the in vivo delivery mechanism of modified bacteria in an embodiment of the present invention, and the image material is from BioRender;

[0029] Figure 3 Transmission electron micrographs of free bacteria, bacteria wrapped in the prepared thin polymer shell, and bacteria with tumor-targeting polypeptides modified on the surface of the thin polymer shell according to Example 3 of the present invention, with a scale bar of 500 nm;

[0030] Figure 4 The transmission electron micrographs of free bacteria and bacteria wrapped in a thin polymer shell resisting lysozyme from degrading bacterial cell walls in Example 4 of the present invention, with a scale bar of 500 nm;

[0031] Figure 5 The particle size and distribution of free bacteria used in Example 3 of the present invention;

[0032] Figure 6 The particle size and distribution of bacteria encapsulated by a thin polymer shell prepared in Example 3 of the present invention;

[0033] Figure 7 The particle size and distribution of bacteria with tumor-targeting polypeptide modified on the surface of thin polymer shell prepared in Example 3 of the present invention;

[0034] Figure 8 The bacterial activity of free bacteria, bacteria wrapped in the prepared thin polymer shell, and bacteria with tumor-targeting polypeptides modified on the surface of the thin polymer shell after resisting lysozyme to degrade the bacterial cell wall in Example 3 of the present invention;

[0035] Fig. 9 The free bacteria in Example 5 of the present invention enter the body of mice through the tail vein, and the distribution of bacteria in various organs on the plate after the mice were killed at different time points;

[0036] Fig.10 The distribution of the bacteria in various organs on the plate after the mice were killed at different time points after the bacteria encapsulated by the polymer shell in Example 5 of the present invention entered the mice through the tail vein;

[0037] Fig.11 The bacteria with tumor-targeting polypeptide modified on the surface of thin polymer shell in Example 5 of the present invention enter the body of mice through the tail vein, and the distribution of bacteria in various organs on the plate after the mice are killed at different time points;

[0038] Fig.12 The activity change of the bacteria before and after in-situ polymerization in Example 6 of the present invention;

[0039] Fig.13 This is the growth curve of the bacteria in Example 3 of the present invention before and after in-situ polymerization over a period of time;

[0040] Fig.14The present invention uses a laser scanning confocal fluorescence microscope to observe the survival and death of the bacteria in Example 3 before and after in-situ polymerization using a live and dead bacteria staining reagent. The scale bar is 10 μm.

[0041] Fig.15 The enrichment of free bacteria in various organs of mice at different time points after injection of free bacteria into mice of the present invention;

[0042] Fig.16 The enrichment of the bacteria wrapped in the thin polymer shell prepared in Example 3 in various organs of the mice at different time points after the mice were injected with the present invention;

[0043] Fig.17 The enrichment of the bacteria with the tumor-targeting polypeptide modified on the surface of the thin polymer shell prepared in Example 3 in various organs of the mice at different time points after the mice were injected with the bacteria;

[0044] Fig.18 The figure shows the formation of a thin polymer shell on the surface of bacteria at different monomer ratios of the present invention, wherein the figure shows the effect of the monomer acrylamide (Aam) on the activity of bacteria;

[0045] Fig.19 The figure shows the formation of a thin polymer shell on the bacterial surface at different monomer ratios of the present invention, wherein the figure shows the effect of the monomer N-(3-aminopropyl) methacrylamide hydrochloride (Apm) on the bacterial activity;

[0046] Fig. 20 The figure shows the formation of a thin polymer shell on the surface of bacteria at different monomer ratios of the present invention, wherein the figure shows the effect of the monomer N,N'-methylenebisacrylamide (Bis) on the activity of bacteria;

[0047] Fig.21 The thin polymer shell formed on the bacterial surface under different monomer ratios of the present invention, wherein the composition of various monomers is shown in the figure;

[0048] Fig. 22 The surface potential changes of the bacteria prepared in Example 3 of the present invention before and after in-situ polymerization modification;

[0049] Fig.23 The figure shows the formation of a thin polymer shell on the bacterial surface by the monomer (acrylamide) of the present invention at different ratios; the scale bar in the figure is 500 nm. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0052] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0053] The Escherichia coli described below is pKillerRed-B plasmid bacterial solution purchased from Shanghai Kelei Biotechnology Co., Ltd.

[0054] Example 1

[0055] A method for surface modification of bacteria, comprising the following steps:

[0056] (1) Preparation of E. coli culture liquid: Use a burned inoculation loop to dip the E. coli culture liquid and quickly apply it in a zigzag shape on the bacterial culture dish, and then place the bacterial culture dish in a humidified constant temperature incubator at 37°C for incubation for 10 hours. After that, select the E. coli colony and put it into 10mL LB culture medium, add 10μL (100mg / mL) of ampicillin solution, and culture it in a constant temperature shaker at 37°C and 200rpm for 12 hours. Dilute the culture liquid after 12 hours of culture at a ratio of 1:10 (v / v) into fresh LB liquid culture medium with the above-mentioned ampicillin solution (100mg / mL), wherein the volume ratio of LB liquid culture medium to ampicillin solution is 1mL:1μL, and culture it in a constant temperature shaker at 37°C and 200rpm until the logarithmic phase of growth. Take the logarithmic phase bacterial liquid, centrifuge it at 3397g for 5 minutes, wash it with sterile ice-cold PBS, and repeat 3 times. Use sterile PBS as diluent and adjust the OD600 to about 1.0 (i.e. dilute to about 10 colonies) using a UV spectrophotometer. 8 CFU / mL) for further use in coating process or viability assay.

[0057] (2) Preparation of E. coli-NAS: For the acrylation of bacterial membrane surface, 1 mg of N-hydroxysuccinimidyl acrylate (NAS) was first dissolved in 100 μL of DMSO to obtain a solution, and then 10 μL of the above solution was added to 10 mL of bacterial solution (10 mL) diluted with sterile PBS. 8CFU / mL). The reaction was vortexed at 4-8°C for 2 h. Then, the acryloylated bacteria were collected by centrifugation, washed with sterile ice-cold PBS, repeated 3 times, and resuspended in 10 mL of sterile ice-cold PBS to obtain E. coli-NAS with acryloylated bacterial membrane surface.

[0058] (3) Preparation of thin polymer shell on bacterial surface: 1 mL of E. coli-NAS obtained in step (2) was added to a 1.5 mL sterile EP tube and centrifuged at 3397 g for 5 min at 4°C. The supernatant was removed and the tube was resuspended in 790 μL of fresh sterile ice-cold PBS. Then, various monomer solutions were added in sequence: 180 μL of acrylamide Aam (70 mg / mL) aqueous solution, 10 μL of N-(3-aminopropyl)methacrylamide hydrochloride Apm (50 mg / mL) aqueous solution and 10 μL of N,N'-methylenebisacrylamide Bis (50 mg / mL) DMSO solution was incubated for 10 min to obtain a suspension, and encapsulation was initiated by adding an initiator / catalyst, including adding 5 μL of ammonium persulfate Aps (100 mg / mL) aqueous solution and 5 μL of tetramethylethylenediamine TMEDA (775 mg / mL) aqueous solution to the above suspension, and the temperature was maintained at 4-8°C for 8 h under shaking, and the encapsulated bacteria were collected by centrifugation and washed with sterile ice-cold PBS to obtain bacteria nEcN wrapped in a thin polymer shell;

[0059] (4) Preparation of thin polymer shell surface modified tumor targeting polypeptide: The nEcN obtained in step (3) was added to a sterile EP tube, and an aqueous solution of tumor targeting polypeptide (NHS-PEG3400-RGD) was added (n(Apm):n(NHS-PEG3400-RGD)=1:5). After reacting at room temperature for 1 h under shaking, the mixture was centrifuged and washed with sterile ice-cold PBS to obtain bacteria nEcN@RGD with a thin polymer shell surface modified with tumor targeting polypeptide.

[0060] Example 2

[0061] A method for surface modification of bacteria, comprising the following steps:

[0062] (1) Preparation of E. coli culture liquid: Use a burned inoculation loop to dip the E. coli culture liquid and quickly apply it in a zigzag shape on the bacterial culture dish, and then place the bacterial culture dish in a humidified constant temperature incubator at 37°C for incubation for 10 hours. After that, select the E. coli colony and put it into 10mL LB culture medium, add 10μL (100mg / mL) of ampicillin solution, and culture it in a constant temperature shaker at 37°C and 200rpm for 12 hours. Dilute the culture liquid after 12 hours of culture at a ratio of 1:10 (v / v) into fresh LB liquid culture medium with the above-mentioned ampicillin solution (100mg / mL), wherein the volume ratio of LB liquid culture medium to ampicillin solution is 1mL:1μL, and culture it in a constant temperature shaker at 37°C and 200rpm until the logarithmic phase of growth. Take the logarithmic phase bacterial liquid, centrifuge it at 3397g for 5 minutes, wash it with sterile ice-cold PBS, and repeat 3 times. Use sterile PBS as diluent and adjust the OD600 to about 1.0 using a UV spectrophotometer (i.e. dilute with sterile PBS until the colony count is about 10 8 CFU / mL) for further use in coating process or viability assay.

[0063] (2) Preparation of E. coli-NAS: For the acrylation of bacterial membrane surface, 10 mg of N-hydroxysuccinimide acrylate (NAS) was first dissolved in 100 μL of DMSO to obtain a solution. Then, 10 μL of the above solution was added to 10 mL of bacterial solution (10 mL) diluted with sterile PBS in (1). 8 CFU / mL). The reaction was vortexed at 4-8°C for 4 h. Then, the acryloylated bacteria were collected by centrifugation, washed with sterile ice-cold PBS, repeated 3 times, and resuspended in 10 mL of sterile ice-cold PBS to obtain E. coli-NAS with acryloylated bacterial membrane surface.

[0064] (3) Preparation of thin polymer shell on bacterial surface: 1 mL of E. coli-NAS obtained in step (2) was added to a 1.5 mL sterile EP tube, centrifuged at 3397 g for 5 min at 4°C, the supernatant was removed, and 750 μL of fresh sterile ice-cold PBS was added for resuspending, and then various monomer solutions were added in sequence: 220 μL of Aam (70 mg / mL) aqueous solution, 10 μL of Apm (50 mg / mL) aqueous solution and 10 μL of Bis (50 mg / mL) DMSO solution, incubated for 10 min to obtain a suspension, and encapsulation was initiated by adding an initiator / catalyst, including adding 5 μL of Aps (100 mg / mL) aqueous solution and 5 μL of TMEDA (775 mg / mL) aqueous solution to the above suspension, and the temperature was maintained at 4-8°C under shaking for 12 h. After that, the encapsulated bacteria were collected by centrifugation and washed with sterile ice-cold PBS to obtain bacterial nEcN wrapped with a thin polymer shell;

[0065] (4) Preparation of thin polymer shell surface modified tumor targeting polypeptide: The nEcN obtained in step (3) was added to a sterile EP tube, and an aqueous solution of tumor targeting polypeptide (NHS-PEG3400-RGD) was added (n(Apm):n(NHS-PEG3400-RGD)=1:20). After reacting at room temperature for 4 h under shaking, the thin polymer shell surface modified tumor targeting polypeptide bacterial nEcN@RGD was obtained by centrifugation and washing with sterile ice-cold PBS.

[0066] Example 3

[0067] A method for surface modification of bacteria, comprising the following steps:

[0068] (1) Preparation of E. coli culture liquid: Use a burned inoculation loop to dip the E. coli culture liquid and quickly apply it in a zigzag shape on the bacterial culture dish, and then place the bacterial culture dish in a humidified constant temperature incubator at 37°C for incubation for 10 hours. After that, select the E. coli colony and put it into 10mL LB culture medium, add 10μL (100mg / mL) of ampicillin solution, and culture it in a constant temperature shaker at 37°C and 200rpm for 12 hours. Dilute the culture liquid after 12 hours of culture at a ratio of 1:10 (v / v) into fresh LB liquid culture medium with the above-mentioned ampicillin solution (100mg / mL), wherein the volume ratio of LB liquid culture medium to ampicillin solution is 1mL:1μL, and culture it in a constant temperature shaker at 37°C and 200rpm until the logarithmic phase of growth. Take the logarithmic phase bacterial liquid, centrifuge it at 3397g for 5 minutes, wash it with sterile ice-cold PBS, and repeat 3 times to obtain free bacteria. Use sterile PBS as diluent and adjust the OD600 to about 1.0 using a UV spectrophotometer (i.e. dilute with sterile PBS until the colony count is about 10 8 CFU / mL) for further use in coating process or viability assay.

[0069] (2) Preparation of E. coli-NAS: For the acrylation of bacterial membrane surface, 5 mg of N-hydroxysuccinimide acrylate (NAS) was first dissolved in 100 μL of DMSO to obtain a solution. Then, 10 μL of the above solution was added to 10 mL of bacterial solution (10 mL) diluted with sterile PBS in (1). 8 CFU / mL). The reaction was vortexed at 4-8°C for 3 h. Then, the acryloylated bacteria were collected by centrifugation, washed with sterile ice-cold PBS, repeated 3 times, and resuspended in 10 mL of sterile ice-cold PBS to obtain E. coli-NAS with acryloylated bacterial membrane surface.

[0070] (3) Preparation of thin polymer shell on bacterial surface: 1 mL of E. coli-NAS obtained in step (2) was added to a 1.5 mL sterile EP tube, centrifuged at 3397 g for 5 min at 4°C, the supernatant was removed, and 770 uL of fresh sterile ice-cold PBS was added for resuspending, and then various monomer solutions were added in sequence: 200 μL of Aam (70 mg / mL) aqueous solution, 10 μL of Apm (50 mg / mL) aqueous solution and 10 μL of Bis (50 mg / mL) DMSO solution, incubated for 10 min to obtain a suspension, and encapsulation was initiated by adding an initiator / catalyst, including adding 5 μL of Aps (100 mg / mL) aqueous solution and 5 μL of TMEDA (775 mg / mL) aqueous solution to the above suspension, and the temperature was maintained at 4-8°C under shaking for 10 h. After the reaction, the encapsulated bacteria were collected by centrifugation and washed with sterile ice-cold PBS to obtain bacterial nEcN wrapped with a thin polymer shell;

[0071] (4) Preparation of thin polymer shell surface modified tumor targeting polypeptide: The nEcN obtained in step (3) was added to a sterile EP tube, and an aqueous solution of tumor targeting polypeptide (NHS-PEG3400-RGD) was added (n(Apm):n(NHS-PEG3400-RGD)=1:10). After reacting at room temperature for 2 h under shaking, the thin polymer shell surface modified tumor targeting polypeptide bacterial nEcN@RGD was obtained by centrifugation and washing with sterile ice-cold PBS.

[0072] Example 4

[0073] Observe the resistance of surface-modified bacteria to environmental threats and improve their bioavailability or drug availability at disease sites

[0074] First, a free bacterial solution (i.e., the bacterial solution prepared in Example 3 (1)), a bacterial solution wrapped in a thin polymer shell prepared in Example 3, and a bacterial solution whose surface was modified with a tumor-targeting polypeptide (the density of the bacterial solution was 10 8 CFU / mL, the solvent is sterile PBS buffer) and lysozyme (10 mg / mL) in PBS solution were mixed in equal volumes and incubated at 37°C for 120 min. After the incubation, 100 μL of bacterial solution was taken to a 96-well plate, and three replicate wells were set for each group. 10 μL of CCK8 solution was added simultaneously and mixed, and incubated in a 37°C incubator in the dark for 30 min; according to the depth of the orange-yellow formazan salt produced by the free bacterial group, it was detected with an enzyme reader, and the excitation wavelength was set to 450 nm. The results are as follows Figure 8 As shown, PBS is a blank control, that is, the test results of the free bacterial solution and the PBS solution are mixed in equal volumes and then subjected to subsequent treatment;

[0075] like Figure 3-4 As shown in Figures 8 and 22, compared with free bacteria, the surface of bacteria modified by surface in situ free radical polymerization has a clearly visible thin polymer shell and the surface potential is improved. After incubation with lysozyme, the free bacterial cell wall is obviously damaged, while the bacteria wrapped in the thin polymer shell still maintain the morphology of the bacteria themselves and maintain about 80% of the activity, which is consistent with the expected function of protecting bacteria from enzyme degradation.

[0076] Example 5

[0077] Observe the application of reducing the immunogenicity and pathogenicity of bacteria and improving the safety of bacterial therapy

[0078] The bacteria coated with thin polymer shells and the bacteria with tumor-targeting peptides modified on the surface of thin polymer shells prepared in Example 3 were used to establish a mouse subcutaneous tumor model by subcutaneously injecting tumor cells into mice. When the tumor volume reached about 300 mm 3 When the bacteria wrapped by thin polymer shells and the bacteria with tumor-targeting peptides modified on the surface of thin polymer shells were added, 1×10 8 100 μL of the solution was injected into the tail vein of mice at a concentration of CFU / mL, and free bacteria were injected as a control group. At various time points after the injection, one mouse in each group was killed, its organs were removed and the tissue was broken using a homogenizer, followed by centrifugation at a speed of (500 rpm, centrifugation for 5 min), the supernatant was taken, diluted to different concentrations, and then plated using the coating plate method, placed in a humidified incubator at 37°C for culture, and then the enrichment of free bacteria and bacteria wrapped in thin polymer shells in various organs was analyzed.

[0079] The experimental results are as follows Figure 9-11 As shown: compared with the control group, the bacteria wrapped in thin polymer shells and the bacteria with tumor targeting peptides modified on the surface of thin polymer shells can more stably control their growth, thereby improving the biosafety of bacterial therapy, wherein Pre in the figure represents the result after the mice without bacterial injection were killed, 1 represents the supernatant stock solution, 1 / 10 and 1 / 100 represent the supernatant stock solution diluted with sterile PBS to 10 times and 100 times the original volume, respectively.

[0080] Example 6

[0081] Observe the application of endowing bacteria with exogenous functions to effectively and precisely control bacterial activity and biological behavior, thereby improving therapeutic effects

[0082] The CCK8 method can be used to detect the activity of bacteria before and after modification. First, the free bacteria in Example 3, the bacteria wrapped in the thin polymer shell, and the bacteria with the tumor-targeting polypeptide modified on the surface of the thin polymer shell are diluted with sterile PBS, and OD600 is adjusted to 1 using a UV spectrophotometer; then 100 μL of the bacterial solution is taken into a 96-well plate, and three replicate wells are set for each group. 10 μL of CCK8 solution is added simultaneously to mix, and incubated in a 37°C incubator in the dark for 30 minutes; according to the depth of the orange-yellow formazan salt produced by the free bacterial group, the microplate reader is used for detection, and the excitation wavelength is set to 450 nm. Similarly, the activity of the bacteria before and after modification can also be detected using a bacterial activity detection kit. First, take 1mL of the above bacterial solution with OD600=1 in a sterile 1.5mL EP tube, prepare 3 samples for each group, and then prepare the staining working solution: take 1μL of NucGreen and 2μL of EthD-III, mix thoroughly, and add 8μL of 0.85% NaCl solution by mass volume. Add 10μL of the staining working solution to each EP tube containing 1mL of the above bacterial solution. Mix thoroughly and incubate at room temperature and away from light for 15min. The optimal incubation time for different bacteria is different. The staining time can be appropriately adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect. After the staining is completed, take 10μL of the bacterial suspension and drop it on a slide with a square cover glass (product size: 18×18×0.13-0.17 mm), take pictures as soon as possible to avoid dry slides. Use a laser scanning confocal fluorescence microscope (CLSM) for imaging. Green fluorescence can be observed using a FITC-compatible filter, and red fluorescence can be observed using a PI-compatible filter. In the above experiment, the free bacteria group was used as a control. Fig.14 As shown in the figure, NucGreen is a green nucleic acid dye that can stain both live and dead bacteria; EthD-III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When NucGreen and EthD-III are properly mixed, bacteria with intact cell membranes appear green, while bacteria with damaged cell membranes appear green and red, respectively, under different channels. Fig.14 It can be seen that there is no significant decrease in the activity of bacteria before and after modification, based on the performance of nucleic acid dyes under laser confocal microscopy.

[0083] The growth of bacteria in each group at different time periods was observed by ultraviolet spectrophotometer, and the same amount of free bacteria, bacteria wrapped in thin polymer shells, and bacteria with tumor-targeting peptides modified on the surface of thin polymer shells were compared.

[0084] The experimental results are as follows Figure 12-13As shown: Compared with the control group, the activity of bacteria wrapped in thin polymer shells and bacteria with tumor-targeting peptides modified on the surface of thin polymer shells is retained at about 80%, and the growth of bacteria is restricted within 8 hours, which can effectively control their biological behavior.

[0085] Example 7

[0086] Observation of the application of bacteria encapsulated by thin polymer shells for enrichment at tumor sites

[0087] First, Cy-7 (near-infrared anthocyanin fluorescent dye) was used to label the free bacteria in Example 3, and the bacteria encapsulated by thin polymer shells and the bacteria with tumor-targeting peptides modified on the surface of thin polymer shells were prepared according to Example 3. Subsequently, a subcutaneous tumor model was established in mice by subcutaneously injecting tumor cells into mice. When the tumor volume reached about 300 mm 3 When the bacteria wrapped in thin polymer shells and the bacteria modified with tumor targeting peptides on the surface of thin polymer shells were added, the concentration of 1×10 8 100 μL of the solution was injected into the tail vein of mice at a concentration of 100 μL CFU / mL, and free bacteria modified with Cy-7 were injected as a control group. The mice were killed 72 hours after the injection, and their organs were removed and analyzed using a small animal in vivo imaging device to analyze the enrichment of free bacteria, bacteria wrapped in thin polymer shells, and bacteria with tumor-targeting peptides modified on the surface of thin polymer shells in various organs.

[0088] The experimental results are as follows Figure 15-17 As shown by Figure 15-17 It can be seen that the Cy-7-labeled bacteria wrapped in the thin polymer shell and the Cy-7-labeled bacteria with tumor-targeting peptides modified on the surface of the thin polymer shell can be enriched in the tumor site faster and more efficiently than the control group.

[0089] Example 8

[0090] Comparison of the formation of thin polymer shells on the bacterial surface at different monomer ratios

[0091] Preparation of thin polymer shell on bacterial surface: 1 mL of E. coli-NAS obtained in step (2) of Example 1 was added to a 1.5 mL sterile EP tube, centrifuged at 3397 g for 5 min at 4°C, the supernatant was removed, and fresh sterile ice-cold PBS was added for resuspending, and then various monomer solutions were added in sequence: Aam (70 mg / mL) aqueous solution, Apm (50 mg / mL) aqueous solution and Bis (50 mg / mL) DMSO solution, wherein the amount of fresh sterile ice-cold PBS added was such that the system was maintained at 1 ml. The suspension was incubated for 10 min to obtain an encapsulation, and encapsulation was initiated by adding an initiator / catalyst, including adding 5 μL of Aps (100 mg / mL) aqueous solution and 5 μL of TMEDA (775 mg / mL) aqueous solution to the above suspension. After the reaction was maintained at 4-8°C under shaking for 8 h, the encapsulated bacteria were collected by centrifugation and washed with sterile ice-cold PBS.

[0092] The amount of each substance used, such as Fig.21 As shown, the activity of bacteria was detected by CCK8 method in Example 6. The experimental results are as follows Figure 18-20 As shown: Among them, Fig.18 In the medium, the volumes of Apm and Bis were fixed at 5 μL and 10 μL, respectively; Fig.19 In the medium, the volumes of fixed Aam and Bis were 210 μL and 10 μL, respectively; Fig. 20 In the experiment, the volumes of fixed Aam and Apm were 210 μL and 5 μL respectively. Compared with free bacteria (shown in the bar graph with ordinate 0 in the figure), the activity of bacteria decreased with the increase of the amount of various monomers added, and the system formed a gel phenomenon when various monomers were added to a certain proportion (for the volumes of 1 mL, 10 8 For a bacterial colony, when the volume of the monomer Aam (70 mg / ml) aqueous solution added exceeds 260 μL or the volume of the cross-linking agent Bis (50 mg / ml) aqueous solution added exceeds 20 μL, the entire system will form a gel instead of remaining in a solution due to the violent reaction. This is consistent with the atom transfer radical polymerization reaction. Ultimately, by controlling the amount of monomer added, the activity of the bacteria can be maintained to the maximum under the condition that no gel is formed and a thin polymer shell is formed on the surface of the bacteria.

[0093] Figure 1 Schematic diagram of bacterial surface modification in an embodiment of the present invention, Figure 1 It can be seen that the surface of bacteria is rich in amino groups. After modification with NAS, the amino groups on the surface of bacteria are conjugated with double bonds. Then, various monomers, cross-linkers, initiators and catalysts are added according to the preparation method of the embodiment to carry out polymerization reaction to form a dense thin polymer shell, and then tumor targeting peptides are added outside the polymer shell for modification.

[0094] Figure 2 Schematic diagram of the in vivo delivery mechanism of modified bacteria in the embodiment of the present invention, Figure 2 It can be seen that after the modified bacteria enter the mouse through the tail vein, they take advantage of the characteristic of Escherichia coli to tend to hypoxic environment and actively tend to the tumor site. The tumor targeting peptide can better target the bacteria to the tumor site. Under this dual targeting condition, the bacteria reach the tumor site.

[0095] The present invention measures the change in particle size before and after bacterial modification, such as Figure 5-7 As shown, the particle size of free bacteria is 955±19nm, the particle size of bacteria wrapped by thin polymer shell is 1080±32nm, and the particle size of bacteria modified with tumor targeting peptide on the surface of thin polymer shell is 1140±46nm. It can be seen that there is no significant change in the particle size of bacteria after in situ polymerization, proving that a thin polymer shell is formed on its surface.

[0096] Various monomers will produce free radicals during polymerization, which have a certain impact on the vitality of the bacteria. On the one hand, if too much monomer is added, the bacterial vitality will be reduced and the reaction system may gel. On the other hand, if the amount of monomer added is small, it is not enough to form a dense shell on the surface of the bacteria. Fig.23 The figure shows the formation of a thin polymer shell on the surface of bacteria by the monomer (acrylamide) of the present invention at different ratios. The scale bar in the figure is 500 nm, wherein the leftmost is a TEM image of bacteria prepared by the method of Example 3, except that the Aam feed is 150 μL, i.e., 10.5 mg; the middle is a TEM image of bacteria prepared by the method of Example 3, except that the Aam feed is 210 μL, i.e., 14.7 mg; the rightmost is a TEM image of bacteria prepared by the method of Example 3, except that the Aam feed is 270 μL, i.e., 18.9 mg. Fig.23 It can be seen that when the amount of monomer Aam added is insufficient, a dense shell is not formed on the bacterial surface and the effect on the bacterial vitality can be ignored; when the amount added is within an appropriate range, a polymer shell can be clearly seen on the bacterial surface, and a certain bacterial vitality is maintained; when the amount of monomer added is too much, the polymerization reaction is too intense, the entire system will form a network-like gel structure, and the activity of the bacteria will decrease accordingly.

[0097] When the mass of Aam is less than 11.9 mg, the test results are Fig.23 Similar to the left figure, in the range of 11.9-17.5mg, Fig.23 The middle figure is similar. When the dosage is greater than 17.5 mg and less than or equal to 21 mg, Fig.23 The right picture is similar.

[0098] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bacterial surface modification method based on atom transfer radical polymerization, characterized in that: The following steps are involved: S1. acrylylating the surface of bacteria using N-hydroxysuccinimide acrylate to introduce double bonds on the surface to obtain acrylylated bacteria; S2. Using acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride and N,N'-methylenebisacrylamide as monomers, a polymerization reaction is carried out on the surface of acrylylated bacteria in the presence of an initiator and a catalyst to obtain bacteria wrapped in a thin polymer shell; wherein the mass ratio of acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride and N,N'-methylenebisacrylamide is 11.9-17.5:0.125-1:0.1-0.

5.

2. The bacterial surface modification method based on atom transfer radical polymerization according to claim 1, characterized in that: In S1, the bacteria is Escherichia coli.

3. The bacterial surface modification method based on atom transfer radical polymerization according to claim 1, characterized in that: In S2, the initiator is ammonium persulfate; and the catalyst is tetramethylethylenediamine.

4. The bacterial surface modification method based on atom transfer radical polymerization according to claim 1, characterized in that: In S2, the mass ratio of the initiator to N,N'-methylenebisacrylamide is 1:1; the mass ratio of the catalyst to N,N'-methylenebisacrylamide is 7.75:

1.

5. The bacterial surface modification method based on atom transfer radical polymerization according to claim 1, characterized in that: In S2, every 10 8 The mass of acrylamide used for each colony is 11.9-17.5 mg.

6. The bacterial surface modification method based on atom transfer radical polymerization according to any one of claims 1 to 5, characterized in that: The method also includes modifying the bacterial surface obtained after the polymerization reaction with a tumor targeting polypeptide.

7. The bacterial surface modification method based on atom transfer radical polymerization according to claim 6, characterized in that: The tumor targeting polypeptide is NHS-PEG3400-RGD.

8. The bacterial surface modification method based on atom transfer radical polymerization according to claim 6 or 7, characterized in that: The molar ratio of the tumor targeting polypeptide to the N-(3-aminopropyl)methacrylamide hydrochloride used is 5-20:

1.

9. A bacterium encapsulated by a thin polymer shell, characterized in that: The bacterial surface modification method based on atom transfer radical polymerization as described in any one of claims 1 to 8 is prepared.

10. Use of the bacteria encapsulated by the thin polymer shell as claimed in claim 9 as a drug delivery carrier.

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