Polyamino acid, polyamino acid nanoparticles, polyamino acid engineered bacterial vaccine and preparation method
Engineered vaccines prepared by grafting polyamino acid nanoparticles onto bacterial surfaces have solved the problem of balancing safety and therapeutic efficacy in tumor treatment, achieving immune activation and tumor suppression effects without toxic side effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHANGCHUN INSTITUTE OF APPLIED CHEMISTRY CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2026-01-13
- Publication Date
- 2026-04-10
AI Technical Summary
Existing bacterial vaccines have issues with toxic side effects and immunogenicity in tumor treatment, making it difficult to achieve a balance between safety and therapeutic efficacy. Furthermore, the chemical engineering modification of bacteria faces challenges in clinical translation.
Polyamino acid nanoparticles were grafted onto the surface of bacteria via amide condensation to prepare a polyamino acid engineered bacterial vaccine. Utilizing their good monodispersity and positive charge properties, the vaccine electrostatically adsorbs and loads antigens, thereby activating the immune response.
It achieves immune activation without toxic side effects, promotes the maturation of antigen-presenting cells, triggers a strong adaptive immune response, and effectively inhibits tumor growth.
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Figure CN121824677A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a polyamino acid, a polyamino acid nanoparticle, a polyamino acid engineered bacterial vaccine and a preparation method. BACKGROUND
[0002] As an important means of tumor immunotherapy, tumor vaccines can activate the patient's own immune system to specifically kill tumor cells. The composition of the vaccine includes an antigen component, a delivery carrier and an immune adjuvant. The delivery system not only protects the antigen from degradation, but also targets the lymph node or antigen-presenting cells (APC), and the adjuvant enhances antigen presentation and T cell activation by activating the natural immune signal (such as the TLR4 / MyD88 pathway or the cGAS-STING pathway). The tumor vaccine can simulate the natural immune process of pathogen infection, induce T cell response, and activate CD8 + or CD4 + T cells. Among them, CD8 + T cells differentiate into cytotoxic T lymphocytes (CTLs) to directly kill tumor cells expressing corresponding antigens, and CD4 + T cells differentiate into helper T cells to enhance anti-tumor response by secreting cytokines. In addition, the immune memory (memory T cells and memory B cells) induced by the vaccine can provide long-term protection against tumor recurrence.
[0003] Bacteria can actively chemotaxis and colonize in tumor tissues by taking advantage of the hypoxia, immunosuppressive properties and specific metabolic products of the tumor microenvironment, achieving precise delivery. In addition, the immune genetic material of the bacteria itself can act as a natural adjuvant to activate the innate immune response and reshape the immunosuppressive properties of the tumor microenvironment. The introduction of bacterial components into the tumor vaccine integrates the properties of microbiology and immunology. Compared with traditional vaccines relying on external delivery systems, the natural chemotaxis of bacterial carriers can significantly improve the enrichment efficiency of antigens in the tumor site, forming a sustained antigen release pool. However, the therapeutic effect and safety of natural bacteria are often difficult to balance, and the toxic side effects of bacteria and their surface pathogenicity and immunogenicity limit the development of bacteria as therapeutic drugs. Proper bacterial engineering can take full advantage of the innate advantages of bacterial metabolic regulation, improve safety, and expand the response rate.
[0004] Currently, significant progress has been made in chemical engineering modification of bacteria, but clinical translation is still difficult, and new strategies need to be developed to make bacterial surface modification more controllable and perfect. SUMMARY
[0005] Therefore, the present application aims to provide a polyamino acid, a polyamino acid nanoparticle, a polyamino acid engineered bacterial vaccine and a preparation method, the polyamino acid provided by the present application can be prepared into a nanoparticle and further prepared into a bacterial vaccine; the nanoparticle prepared from the polyamino acid provided by the present application can have good monodispersity, and the nanoparticle is simultaneously provided with an end amino group and a rich positive charge on the surface, can be grafted on the surface of bacteria through amide condensation, and can efficiently load different antigens through electrostatic adsorption for use as a vaccine; the polyamino acid engineered bacterial vaccine prepared from the nanoparticle has no toxic side effects on the human body and has good immune activation effect.
[0006] The present application provides a polyamino acid, which has the structure of formula (I):
[0007] Formula (I);
[0008] In formula (I), R1 is , , , or ; n and m are the degree of polymerization.
[0009] The present application provides a preparation method of a polyamino acid, comprising the following steps:
[0010] A) mixing and reacting an initiator with the structure of formula (i) and an amino acid-N-cyclic anhydride with the structure of formula (ii) in an organic solvent to obtain a polymer with the structure of formula (iii);
[0011] B) removing the protective group of the polymer with the structure of formula (iii) to obtain the polyamino acid with the structure of formula (I);
[0012] Formula (i);
[0013] Formula (ii);
[0014] Formula (iii);
[0015] Formula (I);
[0016] In formula (I), R1 is , , , or ; n and m are the degree of polymerization.
[0017] Preferably, in step A), the molar ratio of the initiator to the amino acid-N-cyclic anhydride is 1: (5-15).
[0018] Preferably, in step A), the temperature of the mixed reaction is 15~50 ℃, and the time is 24~96 h.
[0019] Preferably, in step B), the process of removing the protecting group specifically includes:
[0020] The polymer of formula (iii) is mixed with trifluoroacetic acid in an organic solvent to remove the tert-butyloxycarbonyl protecting group.
[0021] Preferably, the temperature of the removal is 10~40 ℃, and the time is 1~3 h.
[0022] The present application provides a polyamino acid nanoparticle, the components of which include the polyamino acid of the above technical solution or the polyamino acid prepared by the preparation method of the above technical solution.
[0023] The present application provides a preparation method of a polyamino acid nanoparticle, which includes the following steps:
[0024] The polyamino acid is self-assembled in an aqueous medium to obtain a polymer nanoparticle;
[0025] The polyamino acid is the polyamino acid of the above technical solution or the polyamino acid prepared by the preparation method of the above technical solution.
[0026] The present application provides a polyamino acid engineered bacterial vaccine, the components of which include a nanoparticle, an engineered bacterium, and a model antigen.
[0027] The nanoparticle is the polyamino acid nanoparticle of the above technical solution or the polyamino acid nanoparticle prepared by the preparation method of the above technical solution.
[0028] The engineered bacterium includes one or more of attenuated Salmonella, attenuated Escherichia coli, and attenuated Staphylococcus.
[0029] The present application provides a preparation method of a polyamino acid engineered bacterial vaccine, which includes the following steps:
[0030] The engineered bacterium suspension, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide, and N-hydroxysuccinimide are mixed, mixed into a polymer nanoparticle solution for co-incubation, centrifuged and washed, mixed into a model antigen solution for co-incubation, centrifuged and washed, to obtain a polyamino acid engineered bacterial vaccine.
[0031] The nanoparticle in the polymer nanoparticle solution is the polyamino acid nanoparticle of the above technical solution or the polyamino acid nanoparticle prepared by the preparation method of the above technical solution.
[0032] The engineered bacteria include one or more of attenuated Salmonella, attenuated Escherichia coli, and attenuated Staphylococcus.
[0033] Compared with the prior art, the application provides a polyamino acid, a polyamino acid nanoparticle, a polyamino acid engineered bacteria vaccine and a preparation method. The polyamino acid provided by the application has a structure of formula (I). The polyamino acid provided by the application can be prepared into a nanoparticle and further prepared into a bacteria vaccine. The nanoparticle prepared from the polyamino acid provided by the application can have good monodispersity, and the nanoparticle simultaneously has an end amino group and a surface with abundant positive charges, can be grafted on the surface of bacteria through amide condensation, and can efficiently load different antigens through electrostatic adsorption for use as a vaccine; the polyamino acid engineered bacteria vaccine prepared from the nanoparticle has no toxic side effects on the human body, has the function of promoting the activation of antigen presenting cells, can promote the maturation of DC cells in the body, further triggers a strong adaptive immune response, and has a good immune activation effect. BRIEF DESCRIPTION OF DRAWINGS
[0034] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.
[0035] Figure 1 is a nuclear magnetic resonance hydrogen spectrum of the 1-methyl-D-tryptophan polymer with a tert-butyloxycarbonyl protecting group provided in Embodiment 2 of the application;
[0036] Figure 2 is a nuclear magnetic resonance hydrogen spectrum of the 1-methyl-D-tryptophan polymer provided in Embodiment 3 of the application;
[0037] Figure 3 is a particle size distribution diagram of the polyamino acid nanoparticle provided in Embodiment 4 of the application;
[0038] Figure 4 is a transmission electron microscope diagram provided in Embodiment 5 of the application, wherein (a) is a transmission electron microscope diagram of an unmodified nanoparticle, and (b) is a transmission electron microscope diagram of the polyamino acid engineered bacteria vaccine obtained after modification;
[0039] Figure 5is a graph of in vivo lymph node (LN) analysis test results provided by the embodiment 6 of the present application, wherein (a) is the change of cell number in LN at 3, 5, 7 and 14 days after subcutaneous injection of each group of reagents; (b) is the change of CD40, a maturation marker of dendritic cells, in LN at 3, 5, 7 and 14 days after subcutaneous injection of each group of reagents; (c) is the change of CD80, a maturation marker of dendritic cells, in LN at 3, 5, 7 and 14 days after subcutaneous injection of each group of reagents; (d) is the change of MHC II, a maturation marker of dendritic cells, in LN at 3, 5, 7 and 14 days after subcutaneous injection of each group of reagents; (e) is the change of CD8 + T cells in LN at 3, 5, 7 and 14 days after subcutaneous injection of each group of reagents; (f) is the change of CD4 + T cells in LN at 3, 5, 7 and 14 days after subcutaneous injection of each group of reagents.
[0040] Figure 6 is a graph of in vivo tumor treatment experiment results provided by the embodiment 7 of the present application, wherein (a) is the tumor inhibition curve of B16F10-OVA model; (b) is the body weight change curve of mice during treatment. DETAILED DESCRIPTION
[0041] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.
[0042] The present application provides a polyamino acid with the structure of formula (I):
[0043] Formula (I);
[0044] In formula (I), R1 is , , , or ;
[0045] In formula (I), n and m are the polymerization degree; n is preferably 100-150, more preferably 100-130, and more preferably 114-120; m is preferably 5-15, more preferably 5-10, and more preferably 5-8.
[0046] The present application provides a preparation method of a polyamino acid, comprising the following steps:
[0047] A) reacting an initiator of structure (i) with an amino acid-N- cyclic anhydride of structure (ii) in an organic solvent to obtain a polymer of structure (iii);
[0048] B) removing the protecting group of the polymer of structure (iii) to obtain a polyamino acid of structure (I);
[0049] structure (i);
[0050] structure (ii);
[0051] structure (iii);
[0052] structure (I);
[0053] wherein R1 is , , , or ; n and m are the degree of polymerization.
[0054] In the preparation method provided by the present application, in step A), the amino acid-N-cyclic anhydride can be specifically selected as 1-methyl-D-tryptophan-N-cyclic anhydride, having structure (ii-1):
[0055] structure (ii-1).
[0056] In the preparation method provided by the present application, in step A), the source of the 1-methyl-D-tryptophan-N-cyclic anhydride is not particularly limited, and can be a commercially available product or prepared according to the following method:
[0057] reacting 1-methyl-D-tryptophan with diphosgene to obtain 1-methyl-D-tryptophan-N-cyclic anhydride.
[0058] In the preparation step of the above 1-methyl-D-tryptophan-N-cyclic anhydride provided by the present application, the molar ratio of 1-methyl-D-tryptophan to diphosgene is preferably 1: (1-6), more preferably 1: (1-3), and most preferably 1:1.5; the reaction is preferably carried out in a protective gas atmosphere, and the protective gas is preferably nitrogen; the reaction is preferably carried out in an organic solvent, and the organic solvent is preferably tetrahydrofuran; the reaction is preferably carried out under stirring; the temperature of the reaction is preferably 40-60°C, and more preferably 50°C; and the time of the reaction is preferably 1-2 h, and more preferably 1 h.
[0059] In the step of preparing 1-methyl-D-tryptophan-N-lactam provided by the present application, after the reaction, the reaction product is treated, and the treatment process preferably comprises: sequentially performing ice-precipitated n-hexane settlement, ice-precipitated ethyl acetate re-dissolution, ice-precipitated saturated sodium chloride solution washing, drying water removal and solvent suction, to obtain 1-methyl-D-tryptophan-N-lactam.
[0060] In the preparation method provided by the present application, in step A), the molar ratio of the initiator to the amino acid-N-lactam is preferably 1: (5-15), more preferably 1: (5-10), and most preferably 1:5.
[0061] In the preparation method provided by the present application, in step A), the organic solvent is preferably N,N-dimethylformamide or chloroform, and more preferably N,N-dimethylformamide; and the mass of 1-methyl-D-tryptophan-N-lactam to the volume of the organic solvent is preferably 1.0 g: (10.0-60.0) mL, more preferably 1.0 g: (20.0-50.0) mL, and most preferably 1.0 g: (35.0-50.0) mL.
[0062] In the preparation method provided by the present application, in step A), the temperature of the mixed reaction is preferably 15-50 ℃, more preferably 20-40 ℃, and most preferably 20-25 ℃; and the time of the mixed reaction is preferably 24-96 h, more preferably 48-72 h, and most preferably 72 h.
[0063] In the preparation method provided by the present application, in step A), after the mixed reaction, the reaction product is treated, and the treatment process preferably comprises: precipitating the reaction mixture in excess ethyl ether, separating, dialyzing, and drying, to obtain the polymer with the structure of formula (iii).
[0064] In the preparation method provided by the present application, in step B), the process of removing the protecting group specifically comprises:
[0065] The polymer with the structure of formula (iii) is mixed with trifluoroacetic acid in an organic solvent to remove the tert-butyloxycarbonyl protecting group.
[0066] In the specific process of removing the protecting group provided in the present application, the ratio of the polymer to trifluoroacetic acid is preferably 0.5 g: (2-10) mL, more preferably 0.5 g: 5.0 mL; the organic solvent is preferably one or more of dichloromethane, trichloromethane and N,N-dimethylformamide; the ratio of the polymer to the organic solvent is preferably 0.5 g: (2-10) mL, more preferably 0.5 g: 5.0 mL; the temperature of the removal is preferably 10-40 ℃, more preferably 20-30 ℃, and most preferably 25 ℃; and the time of the removal is preferably 1-3 h, more preferably 1-2 h, and most preferably 1.5 h.
[0067] In the specific process of removing the protecting group provided in the present application, after the removal of the protecting group is completed, the reaction product is subjected to post-treatment, and the post-treatment process preferably includes: settling the reaction mixture in excess ethyl ether, separating, dialyzing, drying, and obtaining the polyamino acid of the structure of formula (I).
[0068] The present application also provides a polyamino acid nanoparticle, which comprises the polyamino acid provided in the above technical solution or prepared by the preparation method provided in the above technical solution.
[0069] In the polyamino acid nanoparticle provided in the present application, the average particle size of the polyamino acid nanoparticle is preferably 50-150 nm, more preferably 60-100 nm, and more preferably 70-80 nm.
[0070] The present application also provides a preparation method of a polyamino acid nanoparticle, which comprises the following steps:
[0071] The polyamino acid is self-assembled in an aqueous medium to obtain a polymer nanoparticle;
[0072] The polyamino acid is the polyamino acid provided in the above technical solution or prepared by the preparation method provided in the above technical solution.
[0073] In the preparation method of the nanoparticle provided in the present application, it more specifically comprises the following steps:
[0074] The polyamino acid is mixed with an organic solvent and then dropped into water, stirred, dialyzed, and a polymer nanoparticle is obtained.
[0075] In the specific preparation steps of the nanoparticles provided in the present application, the organic solvent is preferably N,N-dimethylformamide and / or dimethyl sulfoxide, more preferably N,N-dimethylformamide; the ratio of the amount of the organic solvent to the polyamino acid is preferably (0.5-2) mL:10.0 mg, more preferably 1.0 mL:10.0 mg; the water is preferably ultrapure water; the ratio of the amount of the water to the polyamino acid is preferably (0.5-2) mL:10.0 mg, more preferably 1.0 mL:10.0 mg; the stirring time is preferably 1-24 h, more preferably 8-12 h, and most preferably 12 h; the dialysis temperature is preferably 4-30 ℃, more preferably 10-25 ℃, and most preferably 25 ℃; and the dialysis time is preferably 4-12 h, more preferably 4-8 h, and most preferably 6 h.
[0076] The present application also provides a polyamino acid engineered bacterial vaccine, which comprises nanoparticles, engineered bacteria, and model antigens.
[0077] The nanoparticles are the polyamino acid nanoparticles described in the above technical solution or prepared by the preparation method described in the above technical solution.
[0078] The engineered bacteria comprise one or more of attenuated Salmonella, attenuated Escherichia coli, and attenuated Staphylococcus.
[0079] In the polyamino acid engineered bacterial vaccine provided in the present application, the engineered bacteria are preferably attenuated Salmonella; and the model antigens are preferably tumor antigens and / or viral antigens, and specifically can be ovalbumin (OVA).
[0080] The present application also provides a preparation method of a polyamino acid engineered bacterial vaccine, comprising the following steps:
[0081] The engineered bacterial suspension, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and N-hydroxysuccinimide are mixed, and then mixed into a polymeric nanoparticle solution for co-incubation, centrifuged and washed, mixed into a model antigen solution for co-incubation, centrifuged and washed, to obtain a polyamino acid engineered bacterial vaccine.
[0082] The nanoparticles in the polymeric nanoparticle solution are the polyamino acid nanoparticles described in the above technical solution or prepared by the preparation method described in the above technical solution.
[0083] The engineered bacteria comprise one or more of attenuated Salmonella, attenuated Escherichia coli, and attenuated Staphylococcus.
[0084] In the method for preparing the bacterial vaccine provided by the application, the engineered bacteria in the engineered bacteria suspension are preferably attenuated Salmonella; the concentration of the engineered bacteria suspension is preferably 1×10 5 CFU, more preferably 1×10 9 CFU, more preferably 1×10 7 CFU, more preferably 1×10 9 CFU, and most preferably 1×10 8 CFU.
[0085] In the method for preparing the bacterial vaccine provided by the application, the ratio of the use amounts of the engineered bacteria suspension, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is preferably 1.0 mL:(0.7-1.5) mg:(1-1.7) mg, more preferably 1.0 mL:1.15 mg:1.3 mg.
[0086] In the method for preparing the bacterial vaccine provided by the application, the concentration of the polymer nanoparticle solution is preferably 0.2-2 mg mL -1 , more preferably 0.5-1 mg mL -1 , and most preferably 1.0 mg mL -1 ; and the volume ratio of the polymer nanoparticle solution to the engineered bacteria suspension is preferably 1:(0.5-2), more preferably 1:1.
[0087] In the method for preparing the bacterial vaccine provided by the application, the time for co-incubation with the mixed polymer nanoparticle solution is preferably 0.5-6 h, more preferably 0.5-2 h, and most preferably 1 h.
[0088] In the method for preparing the bacterial vaccine provided by the application, the antigen in the model antigen solution is preferably a tumor antigen and / or a viral antigen, and can be specifically OVA; the concentration of the model antigen solution is preferably 20-200.0 μg mL -1 , more preferably 50-100.0 μg mL -1 , and most preferably 100.0 μg mL -1 .
[0089] In the method for preparing the bacterial vaccine provided by the application, the time for co-incubation with the mixed antigen solution is preferably 0.5-6 h, more preferably 0.5-2 h, and most preferably 1 h.
[0090] For a clearer understanding, the following examples are provided in detail.
[0091] In the following examples of the application, unless otherwise specified, the relevant operations are carried out at room temperature (25°C) and under normal pressure (1 atm).
[0092] Example 1
[0093] 1-methyl-D-tryptophan-N-cyclic anhydride was prepared as follows:
[0094] 60.0 mL of tetrahydrofuran, 2.0 g (9.16 mmol) of 1-methyl-D-tryptophan and 2.8 g (14.15 mmol) of diphosgene were stirred and mixed to obtain a mixture, and the reaction was carried out under nitrogen (50 °C, 1 h) while stirring. After the reaction was completed, ice n-hexane was precipitated, ice ethyl acetate was re-dissolved, ice saturated sodium chloride solution was washed, and water was removed by drying and solvent was extracted to obtain 1-methyl-D-tryptophan-N-cyclic anhydride.
[0095] Example 2
[0096] 1-methyl-D-tryptophan polymer with a tert-butyloxycarbonyl protecting group was prepared as follows:
[0097] 0.6 g (2.5 mmol) of 1-methyl-D-tryptophan-N-cyclic anhydride prepared in Example 1 was added to a dry round bottom flask, and 25.0 mL of anhydrous N,N-dimethylformamide was added to be stirred and dissolved. 2.6 g (0.5 mmol) of a polyethylene glycol initiator with a tert-butyloxycarbonyl protecting group and an amino group, BOC-PEG 5K -NH2, was reacted at 25 °C for 72 h, and after the reaction was completed, the reaction mixture was precipitated in excess ethyl ether, separated, dialyzed, dried to obtain a 1-methyl-D-tryptophan polymer with a tert-butyloxycarbonyl protecting group.
[0098] In this example, the chemical structure of the initiator used is shown in formula (i), and the chemical structure of the 1-methyl-D-tryptophan polymer with a tert-butyloxycarbonyl protecting group prepared is shown in formula (iii-1):
[0099] Formula (i);
[0100] Formula (iii-1);
[0101] wherein n = 114, m = 5.
[0102] The 1-methyl-D-tryptophan polymer with a tert-butyloxycarbonyl protecting group prepared in this example was detected by nuclear magnetic resonance hydrogen spectrum, and the results are shown in Figure 1 .
[0103] Example 3
[0104] 1-methyl-D-tryptophan polymer was prepared as follows:
[0105] Weigh 0.5 g of 1-methyl-D-tryptophan polymer with tert-butyloxycarbonyl protecting group prepared in Example 2 into a clean flask, add 5.0 mL of dichloromethane to stir and dissolve, after complete dissolution, add 5.0 mL of trifluoroacetic acid to form a yellowish solution, and the reaction is carried out at 25 ℃ for 1.5 h. After the reaction is completed, the reaction mixture is precipitated in excess ethyl ether, separated, dialyzed, and dried to obtain 1-methyl-D-tryptophan polymer.
[0106] In this example, the chemical structure of the prepared 1-methyl-D-tryptophan polymer is shown in formula (I-1):
[0107] Formula (I-1);
[0108] Wherein, n = 114, m = 5.
[0109] The 1-methyl-D-tryptophan polymer prepared in this example is detected by nuclear magnetic resonance hydrogen spectrum, and the result is shown in Figure 2 .
[0110] Example 4
[0111] The preparation of 1-methyl-D-tryptophan polymer nanoparticles is as follows:
[0112] Dissolve 10.0 mg of 1-methyl-D-tryptophan polymer prepared in Example 3 in 1.0 mL of N,N-dimethylformamide to obtain a polymer organic solution. Slowly drop the polymer organic solution into 1.0 mL of ultrapure water, stir for 12 h, and dialyze at 25 ℃ for 6 h to obtain 1-methyl-D-tryptophan polymer nanoparticles.
[0113] The nanoparticles prepared in this example are subjected to particle size test experiment, and the result is shown in Figure 3 . It can be seen that the average particle size of the nanoparticles is 80 nm. Figure 3
[0114] Example 5
[0115] (1) Preparation of experimental group vaccine (polyamino acid engineered bacterial vaccine), the specific process is as follows:
[0116] Adjust the concentration of attenuated Salmonella suspension to 1×10 8 CFU, add 1.15 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and 1.3 mg of N-hydroxysuccinimide to 1.0 mL of bacterial suspension, mix uniformly, and then add 1 mL of 1 mg mL -1 nanoparticle solution (nanoparticles from Example 4) at a concentration of 1 mg mL-1, 1.0 mL of OVA solution (OVA purchased from Sigma-Aldrich) at a concentration of 0.1 mg mL-1was added, and stirring was performed at 25 °C for 1 h. After centrifugal washing, the polyamino acid engineered bacterial vaccine was obtained. -1 -1 -1
[0117] The above polyamino acid engineered bacterial vaccine prepared in this example was subjected to transmission electron microscopy characterization and compared with unmodified nanoparticles, and the results are shown in Figure 4 Figure 4 It can be seen that the polyamino acid nanoparticles are successfully loaded on the surface of the bacteria.
[0118] (2) Preparation of the control vaccine (polyamino acid nanoparticle vaccine complexed with OVA), the specific process is as follows:
[0119] In 1.0 mL of nanoparticle solution (nanoparticles from Example 4) at a concentration of 1 mg mL-1, 1.0 mL of OVA solution (OVA purchased from Sigma-Aldrich) at a concentration of 0.1 mg mL-1was added, and stirring was performed at 25 °C for 1 h. After centrifugal washing, the polyamino acid engineered bacterial vaccine was obtained.
[0120] Example 6
[0121] In vivo lymph node (LN) analysis test, the specific process is as follows:
[0122] PBS, OVA, Example 5 polyamino acid nanoparticle vaccine complexed with OVA (denoted as P(D-1-MT)-O) and Example 5 polyamino acid engineered bacterial vaccine (denoted as P(D-1-MT)@S-O) were subcutaneously injected (injection dose: OVA 4.0 μg + nanoparticles 100.0 μg per mouse) into mice on day 0, and the inguinal lymph nodes of the mice were dissected on days 3, 5, 7 and 14, and were treated by mechanical disruption at 37 °C. Then the sample was filtered through a 200 mesh nylon mesh filter to obtain a single cell suspension, and cell counting was performed by a cell counting plate. Then incubation was performed at 4 °C for 30 minutes with anti-CD11c, anti-CD80, anti-MHC-II, anti-CD40, anti-CD3, anti-CD4 and anti-CD8 to analyze the changes of DC and T cells in the lymph nodes. The sample was subjected to flow data acquisition on a flow cytometer (BD FACSCelesta) and analysis by FlowJo software. The results are shown in Figure 5 .
[0123] It can be seen that the polyamino acid nanoparticles are successfully loaded on the surface of the bacteria. Figure 5 The results showed that the polyamino acid-engineered bacterial vaccine increased the number of lymphocytes in the draining lymph nodes. Furthermore, the levels of dendritic cell activation markers CD80, MHC-II, and CD40 in the lymph nodes were elevated, indicating that the nanovaccine activated dendritic cells. In addition, CD80... + T and CD4 + The increased T-cell levels indicate that the nanovaccine enhanced the immune response. The strongest effect of activating dendritic cells was observed on day 7 after subcutaneous injection.
[0124] Example 7
[0125] The in vivo tumor treatment experiment proceeded as follows:
[0126] On day 0, B16-F10-OVA cells (2.0 × 10⁶ cells) were introduced. 5 Cells were subcutaneously inoculated into the left back of 6-8 week old female C57BL / 6N mice. On days 4, 7, 11, and 14, PBS, OVA, the polyamino acid nanoparticle vaccine combined with OVA from Example 5 (denoted as P(D-1-MT)-O), and the polyamino acid engineered bacterial vaccine from Example 5 (denoted as P(D-1-MT)@SO) were subcutaneously injected into the right back of mice (injection dose of 4.0 μg OVA + 100.0 μg nanoparticles per mouse), respectively. The PBS group served as the control group. The weight of the mice and the volume of the tumor were then checked every other day. When the tumor volume reached 2000 mm, the tumor was classified as a tumor growth factor. 3 If the skin is severely ulcerated, the mouse is euthanized, and the results are shown in [the table below]. Figure 6 .
[0127] Depend on Figure 6 The results showed that the polyamino acid-engineered bacterial vaccine of Example 5 effectively inhibited tumor growth, demonstrating significant advantages and potential application value. Furthermore, there were no significant differences in mouse body weight among the groups, proving that the material had no toxic side effects.
[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A polyamino acid, characterized by, The polyamino acid has a structure of formula (I): Formula (I); In formula (I), R1is , , , or ; n and m are the degree of polymerization.
2. A polyamino acid nanoparticle, characterized in that, The polyamino acid nanoparticle comprises the polyamino acid of claim 1.
3. A method of preparing polyamino acid nanoparticles, characterized by, The method comprises the following steps: The polyamino acid is self-assembled in an aqueous medium to obtain polymer nanoparticles. The polyamino acid is the polyamino acid of claim 1.
4. A polyamino acid engineered bacterial vaccine, characterized in that, The polyamino acid engineered bacterial vaccine comprises nanoparticles, engineered bacteria, and model antigens. The nanoparticles are the polyamino acid nanoparticles of claim 2 or the polyamino acid nanoparticles prepared by the method of claim 3. The engineered bacteria comprise one or more of attenuated Salmonella, attenuated Escherichia coli, and attenuated Staphylococcus.
5. A method of producing a polyamino acid engineered bacterial vaccine, comprising, The method comprises the following steps: The engineered bacterial suspension, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and N-hydroxysuccinimide are mixed, and the mixture is mixed with a solution of polymer nanoparticles for co-incubation, centrifuged and washed, mixed with a solution of model antigens for co-incubation, centrifuged and washed to obtain a polyamino acid engineered bacterial vaccine. The nanoparticles in the solution of polymer nanoparticles are the polyamino acid nanoparticles of claim 2 or the polyamino acid nanoparticles prepared by the method of claim 3. The engineered bacteria comprise one or more of attenuated Salmonella, attenuated Escherichia coli, and attenuated Staphylococcus.
6. A process for the preparation of a polyamino acid, characterized by, The method comprises the following steps: A) an initiator having a structure of formula (i) is mixed with an amino acid-N-cyclic anhydride having a structure of formula (ii) in an organic solvent to obtain a polymer having a structure of formula (iii); B) the polymer having a structure of formula (iii) is deprotected to obtain a polyamino acid having a structure of formula (I); Equation (i); Formula (ii); Equation (iii); Formula (I); wherein R1is , , , or ; n and m are the degree of polymerization.
7. The production method according to claim 6, wherein In step A), the molar ratio of the initiator to the amino acid-N-cyclic anhydride is 1: (5-15).
8. The preparation method according to claim 6, characterized in that, In step A), the mixing reaction is carried out at a temperature of 15-50 ℃ for 24-96 h.
9. The preparation method according to claim 6, characterized in that, In step B), the deprotection process specifically comprises: The polymer having a structure of formula (iii) is mixed with trifluoroacetic acid in an organic solvent to remove the tert-butyloxycarbonyl protecting group.
10. The method of claim 9, wherein, The removal is carried out at a temperature of 10-40 ℃ for 1-3 h.