Fluorine-nitrogen modified polymer immunoadjuvant material, preparation method and application thereof

Abstract

The application discloses a fluorine-nitrogen modified high-molecular immunoadjuvant material, a preparation method and application thereof, and belongs to the technical field of high-molecular materials and immunology, wherein the high-molecular immunoadjuvant material comprises a biodegradable polycarbonate main chain, and polyethylene glycol segments, perfluoroalkyl side chains and nitrogen-containing seven-membered ring side chains connected to the polycarbonate main chain. The perfluoroalkyl side chains are introduced to promote antigen delivery and improve the antigen presenting cell uptake efficiency; the nitrogen-containing seven-membered ring side chains are introduced to the side chains to make the material have intracellular environment response characteristics, so that the antigen intracellular release process is improved and the antigen utilization efficiency is improved. In addition, the polycarbonate which can be completely degraded by enzymes in the body is used as the main chain framework, so that the material has good delivery performance and biological safety.

Description

Technical Field

[0001] This invention relates to the fields of polymer materials and immunology, specifically to a fluorine-nitrogen modified polymeric immune adjuvant material, its preparation method, and its application. Background Technology

[0002] Rabies is a highly lethal zoonotic infectious disease caused by the rabies virus. Once typical clinical symptoms appear, the mortality rate is extremely high, and vaccination remains the most important and effective preventive measure. Currently, the rabies vaccines widely used in clinical and veterinary fields are mainly inactivated vaccines. These vaccines generally have good safety profiles, but because inactivated antigens lack the ability to actively infect and sustain expression, their immunogenicity still has room for improvement. For rabies vaccines, virus-neutralizing antibodies are considered an important indicator of protective immunity; international standards consider a neutralizing antibody level ≥0.5 IU / mL to provide effective protection. Therefore, while ensuring safety, how to further improve the efficiency of inactivated rabies vaccines in inducing a protective immune response remains an important issue in the optimization of related vaccines.

[0003] Aluminum adjuvants are among the most widely used traditional adjuvants. They primarily enhance vaccine-induced humoral immune responses by adsorbing antigens and forming a relatively sustained local antigen library, thus prolonging antigen exposure time in vivo. However, aluminum adjuvants have relatively limited ability to stimulate cellular immunity, and for vaccine systems requiring further improvements in antigen delivery efficiency and more comprehensive immune regulation, aluminum adjuvants still have certain limitations. With the development of subunit vaccines, recombinant vaccines, and novel inactivated vaccines, developing novel adjuvant materials that combine good safety, delivery capability, and immune-enhancing effects has become an important research direction in the vaccine field.

[0004] Co-delivering antigens and adjuvants through a rationally designed delivery system to improve antigen delivery efficiency, enhance immune cell targeting, and reduce systemic exposure is considered a feasible strategy for improving vaccine safety and efficacy. In recent years, polymer delivery systems have received widespread attention for the co-delivery of antigens and immunostimulatory molecules. Due to their tunable molecular weight, diverse structures, and easily modifiable functions, polymers can self-assemble into nanoscale structures, demonstrating promising application potential in antigen encapsulation, protection, and delivery. Further research has shown that some polymers, in addition to serving as delivery carriers, can also regulate the intensity and type of the immune response by influencing antigen uptake, intracellular transport, and immune cell recognition processes, thus being considered important candidate materials for constructing novel vaccine adjuvant systems.

[0005] Existing inactivated virus vaccine delivery and adjuvant systems typically have the following shortcomings in application:

[0006] 1. The problem of poor stability of nano-adjuvants in complex biological environments: Nanocarriers have limited stability in complex biological environments and are prone to aggregation, dissociation or non-specific adsorption, which affects antigen loading and delivery efficiency, and some systems are difficult to be efficiently taken up by antigen-presenting cells.

[0007] 2. The contradiction between the immune-activating properties and biosafety of highly effective chemical adjuvants: Some materials with strong immune-enhancing effects have problems such as high cytotoxicity or tissue irritation, making it difficult to balance immune efficacy and biosafety.

[0008] 3. Solve the problem of exogenous antigens being easily retained in endosomes / lysosomes and degraded during intracellular delivery, making it difficult to achieve effective intracellular release: After being taken up by cells, existing delivery systems often lack effective intracellular transport and release capabilities, causing exogenous antigens to be easily retained in the endosome / lysosome pathway and degraded after endocytosis, resulting in insufficient effective intracellular release, which in turn affects antigen processing, presentation and the initiation of immune response.

[0009] Furthermore, among numerous polymer systems, polycarbonate materials have gradually attracted attention due to their good biocompatibility, degradability, and strong structural designability. Existing research has shown that degradable polycarbonate platforms, while maintaining pH-responsive characteristics, can be used for antigen delivery and immune activation. Their backbone, upon hydrolysis, forms small-molecule alcohols and carbon dioxide, providing a good safety basis for their use as biomedical materials. Functional modification of the polycarbonate backbone or side chains can further regulate the material's hydrophilicity / hydrophobicity, ionization characteristics, and interactions with cell membranes and immune cells, thereby improving its performance in vaccine delivery and immune enhancement. For example, introducing nitrogen-containing functional groups into the polycarbonate side chains helps enhance the material's interaction with the cell surface and improves its response behavior in weakly acidic intracellular environments. In other biomolecule delivery studies, fluorine modification has been shown to improve interfacial properties, enhance cellular uptake, increase serum tolerance, and, in some systems, help improve endosome escape efficiency. However, existing research on fluorine-containing modifications is mostly focused on nucleic acid or protein delivery, and research combining them with biodegradable polycarbonate backbones and immune adjuvant design remains relatively limited.

[0010] Current research on polycarbonate-based adjuvants or delivery materials largely focuses on the regulation of single functions. However, for the specific application scenario of inactivated rabies vaccines, how to further integrate hydrophilic segments, nitrogen-containing ring structures, and fluorine-containing regulatory units on a biodegradable polycarbonate backbone to simultaneously ensure delivery stability, intracellular delivery efficiency, immune enhancement, and preliminary safety remains a technical problem to be solved in this field. Therefore, developing a functionalized polycarbonate polymeric adjuvant material suitable for inactivated rabies vaccines has clear research value and application prospects. Summary of the Invention

[0011] The purpose of this invention is to provide a fluorine-nitrogen modified polymeric immune adjuvant material, its preparation method, and its application, so as to solve the problems mentioned in the background art.

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] A fluorine-nitrogen modified polymeric immunoadjuvant material comprises a biodegradable polycarbonate backbone, and polyethylene glycol segments, perfluoroalkyl side chains, and nitrogen-containing seven-membered ring side chains connected to the polycarbonate backbone.

[0014] Furthermore, the general structural formula of the polymeric immune adjuvant material is:

[0015] ;

[0016] In the formula, n, m, and p represent the degree of aggregation; 1 ≤ q ≤ 15.

[0017] Furthermore, q is 8.

[0018] Another object of the present invention is to provide a method for preparing the above-mentioned polymeric immune adjuvant material, comprising the following steps:

[0019] With the aid of a catalyst and a co-catalyst, polyethylene glycol was used as a chain transfer agent to induce a ring-opening copolymerization reaction between the monomer allyl glycidyl ether and carbon dioxide, resulting in PAGEC-PEG-PAGEC segments.

[0020] The PAGEC-PEG-PAGEC segment was reacted with protonated 2-(aza-1-yl)ethane-1-thiol under ultraviolet light irradiation in the presence of a photoinitiator to obtain the PAGEC-PEG-PC7A intermediate.

[0021] PAGEC-PEG-PC7A intermediate was reacted with perfluoroalkyl thiols under ultraviolet light in the presence of a photoinitiator to obtain a polymeric immune adjuvant material.

[0022] Furthermore, the catalyst is Salen-Co-TFA; the co-catalyst is PPN-TFA.

[0023] Furthermore, the polyethylene glycol is polyethylene glycol 2000, and its equivalent ratio to the monomer allyl glycidyl ether is (0.01-0.05):1.

[0024] Furthermore, the protonation method for 2-(aza-1-yl)ethane-1-thiol is as follows: 2-(aza-1-yl)ethane-1-thiol is reacted with hydrogen chloride; the equivalent ratio of PAGEC-PEG-PAGEC segment to protonated 2-(aza-1-yl)ethane-1-thiol is 1:(10-30).

[0025] Furthermore, the photoinitiator is benzoin dimethyl ether.

[0026] Furthermore, the perfluoroalkyl thiol has the general structural formula C0. a H5F 2a-3 S, where 3≤a≤20; the equivalent ratio of the PAGEC-PEG-PC7A intermediate to the perfluoroalkyl thiol is 1:(10-30).

[0027] Another object of the present invention is to provide the application of the above-mentioned polymeric immune adjuvant material in the preparation of rabies vaccine.

[0028] This invention provides a fluorine-nitrogen-modified polymeric immunoadjuvant material. The introduction of perfluoroalkyl functional units into the polymer structure enhances the self-assembly driving force of the polymer, improves the structural stability of the nanodelivery system, and improves its interaction with the antigen-presenting cell membrane, thereby promoting the uptake of the antigen / adjuvant complex by antigen-presenting cells and improving antigen delivery efficiency. Furthermore, this invention uses biodegradable polycarbonate as the main chain backbone and combines it with hydrophilic polyethylene glycol segments to construct the polymer adjuvant system. The polycarbonate backbone can be gradually degraded in vivo, helping to reduce the safety risks associated with long-term material accumulation; the polyethylene glycol segments help improve the dispersibility and biocompatibility of the material, reducing non-specific interactions, thus enabling the system to achieve both delivery capability and good biocompatibility. Moreover, this invention introduces nitrogen-containing seven-membered ring functional groups with acid-responsive properties into the polymer structure. These groups can undergo protonation in a weakly acidic intracellular environment, thereby enhancing the carrier's responsiveness to the endosomal environment, promoting antigen release from the endosomal environment, reducing antigen degradation, and improving intracellular antigen delivery efficiency and subsequent antigen presentation effects. Attached Figure Description

[0029] Figure 1 For PC 10 F 17 -PEG 2000 -Schematic diagram of the synthesis route for PC7A.

[0030] Figure 2 PAGEC-PEG 2000 -PAGEC's hydrogen NMR spectrum.

[0031] Figure 3 PAGEC-PEG2000 -H NMR spectrum of PC7A.

[0032] Figure 4 This is the hydrogen NMR spectrum of C7ASH.

[0033] Figure 5 For PC 10 F 17 -PEG 2000 -H NMR spectrum of PC7A.

[0034] Figure 6 For PC 10 F 17 -PEG 2000 -PC7A-induced mouse dendritic cells; in the figure, A represents CD11c. + MHC II + Cell proportion; B is CD11c + MHC I + Cell percentage; C is CD11c + CD86 + The proportion of cells; D represents CD11c. + CD80 + The proportion of cells.

[0035] Figure 7 For PC 10 F 17 -PEG 2000 -PC7A induces neutralizing antibody levels in mice; in the figure, A is a schematic diagram of the experimental procedure for inducing humoral immune responses in mice; B is a comparison of neutralizing antibody levels in each group.

[0036] Figure 8 For PC 10 F 17 -PEG 2000 -PC7A-induced mouse TFH cells; In the figure, A represents the proportion of TFH cells; B represents the results of flow cytometry.

[0037] Figure 9 For PC 10 F 17 -PEG 2000 -PC7A-induced mouse GCB cells; In the figure, A represents the proportion of GCB cells; B represents the results of flow cytometry.

[0038] Figure 10 For PC 10 F 17 -PEG 2000 PC7A induces CD19 in mice + CD69 + B cell status; in the figure, A represents CD19.+ CD69 + B cell proportion; B represents the results of flow cytometry analysis.

[0039] Figure 11 For PC 10 F 17 -PEG 2000 PC7A induces CD19 in mice + CD40 + B cell status; in the figure, A represents CD19. + CD40 + B cell proportion; B represents the results of flow cytometry analysis.

[0040] Figure 12 For PC 10 F 17 -PEG 2000 PC7A induces CD4 in mice + CD69 + T cell status; in the figure, A represents CD4. + CD69 + T cell percentage; B is the result of flow cytometry analysis.

[0041] Figure 13 For PC 10 F 17 -PEG 2000 PC7A induces CD4 in mice + CD44 + CD62L + T cell status; in the figure, A represents CD4. + CD44 + CD62L + T cell percentage; B is the result of flow cytometry analysis.

[0042] Figure 14 For PC 10 F 17 -PEG 2000 -PC7A immune protection effect; In the figure, A represents the survival rate of mice after challenge; B represents the change in body weight of mice after challenge.

[0043] Figure 15 For PC 10 F 17 -PEG 2000 -PC7A Biosafety Assessment. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0045] Existing rabies vaccine adjuvants primarily rely on adsorption and sustained release, offering limited active regulation of the antigen delivery process and failing to effectively promote antigen uptake by antigen-presenting cells, thus affecting immune enhancement. This invention, by introducing a perfluoroalkyl side chain, provides an adjuvant delivery system that promotes antigen delivery and improves antigen uptake efficiency by antigen-presenting cells.

[0046] Existing polymeric delivery systems often suffer from antigen retention and degradation in endosomes / lysosomes after endocytosis, resulting in insufficient intracellular release efficiency and consequently affecting antigen presentation and effective initiation of the immune response. This invention introduces a nitrogen-containing seven-membered ring (PC7A) into the side chain, providing a polymeric immunoadjuvant material with intracellular responsive properties to improve the intracellular release process and enhance antigen utilization efficiency.

[0047] Existing polymeric materials with strong delivery capabilities often suffer from insufficient degradability or limited safety, making it difficult to balance delivery efficiency and application safety. This invention utilizes polycarbonate, which can be completely degraded by in vivo enzymes, as the main chain backbone, providing a degradable polymeric immunoadjuvant material that combines excellent delivery performance with biosafety.

[0048] Specifically, in one embodiment of the present invention, a fluorine-nitrogen modified polymeric immunoadjuvant material is provided, comprising a biodegradable polycarbonate backbone, and polyethylene glycol segments, perfluoroalkyl side chains and nitrogen-containing seven-membered ring side chains connected to the polycarbonate backbone.

[0049] The general structural formula of polymeric immune adjuvant materials is as follows:

[0050] ;

[0051] In the formula, n, m, and p represent the degree of polymerization; 10 ≤ m ≤ 113, 20 ≤ n + p ≤ 50, 0.2 ≤ p / n ≤ 0.8, and 1 ≤ q ≤ 15. Preferably, q = 8.

[0052] In another embodiment of the present invention, a method for preparing the above-mentioned polymeric immune adjuvant material is also provided, specifically including the following steps:

[0053] S1. Synthesis of PAGEC-PEG-PAGEC segments: The catalyst Salen-Co-TFA (0.001-0.00 eq, structure as shown in Formula I) and the co-catalyst PPN-TFA (0.001-0.003 eq, structure as shown in Formula II) were added to the reactor, followed by the chain transfer agent polyethylene glycol (PEG, 0.01-0.05 eq) and the monomer allyl glycidyl ether (AGE, 1 eq). A small amount of dichloromethane (DCM) was added to dissolve the reactants. The reactor was then purged with 2-4 MPa CO2 and reacted at room temperature for 36-60 h. The polymer obtained from the reaction was then dissolved in DCM, precipitated in diethyl ether to remove byproducts and catalyst, and dried under vacuum to obtain PAGEC-PEG-PAGEC segments.

[0054] ;

[0055] Formula I

[0056] .

[0057] Formula II

[0058] S2. The synthesized PAGEC-PEG-PAGEC segment was modified with a nitrogen-containing seven-membered ring to obtain the PAGEC-PEG-PC7A intermediate: 2-(aza-1-yl)ethane-1-thiol (1 eq) was protonated by reacting with hydrogen chloride (1 eq); the protonated 2-(aza-1-yl)ethane-1-thiol (10-30 eq) and the above PAGEC-PEG-PAGEC segment (1 eq) were added sequentially to a reaction flask, and benzoin dimethyl ether (DMPA, 1-3 eq) was added. The reaction was carried out at room temperature under ultraviolet light for 2-6 h; then the polymer obtained from the reaction was dissolved with DCM, and the byproducts were removed by precipitating into ether. The product was dissolved in PBS solution with pH=8-10, stirred for 3-5 h to remove protonation, dialyzed, and vacuum dried to obtain the PAGEC-PEG-PC7A intermediate. The dialysis time for this step is 36-60 hours. During the first 6-10 hours, the deionized water is changed every 1-3 hours. During the middle 12-20 hours, the deionized water is changed every 3-5 hours. During the last 18-30 hours, the deionized water is changed every 9-15 hours.

[0059] S3, Preparation of PC a F 2a-3 -PEG-PC7A compound, namely a polymeric immunoadjuvant material: a perfluoroalkyl thiol C a H5F 2a-3S (10-30 eq, 3≤a≤20) and the PAGEC-PEG-PC7A intermediate (1 eq) obtained above were added sequentially to a reaction flask, followed by the addition of benzoin dimethyl ether (DMPA, 1-3 eq). The reaction was carried out under UV irradiation at room temperature for 2-6 h. The polymer obtained from the reaction was dissolved in DCM, precipitated in diethyl ether to remove byproducts, dialyzed, and then vacuum dried to obtain PC. a F 2a-3 -PEG-PC7A compound. The dialysis time for this step is 36-60 hours. For the first 6-10 hours, the deionized water is changed every 1-3 hours. For the middle 12-20 hours, the deionized water is changed every 3-5 hours. For the last 18-30 hours, the deionized water is changed every 9-15 hours.

[0060] In another embodiment of the present invention, a vaccine composition (preferably a rabies vaccine) for human or veterinary use is also provided, comprising the above-mentioned polymeric adjuvant material and antigen components.

[0061] Unless otherwise specified, all raw materials used in the following embodiments are commercially available products and can be purchased through commercial channels. The invention will be described in detail below through specific embodiments in practical applications.

[0062] Example 1: This example provides a polymeric immune adjuvant material PC. 10 F 17 -PEG 2000 -PC7A, the preparation method of which specifically includes the following steps:

[0063] S1, Synthesis of PAGEC-PEG 2000 -PAGEC segment: The catalyst Salen-Co-TFA (0.002 eq, structure as shown in Formula I) and the co-catalyst PPN-TFA (0.002 eq, structure as shown in Formula II) were added to the reactor, followed by the chain transfer agent polyethylene glycol 2000 (PEG). 2000 PAGEC-PEG was prepared by dissolving allyl glycidyl ether (AGE, 1 eq) and allyl glycidyl ether (AGE, 1 eq) in diethyl ether with a small amount of dichloromethane (DCM). The reaction mixture was then purged with 3 MPa CO2 and reacted at room temperature for 48 h. The resulting polymer was then dissolved in DCM, precipitated into diethyl ether to remove byproducts and catalyst, and dried under vacuum to obtain PAGEC-PEG. 2000 -PAGEC segment, see detailed synthesis steps. Figure 1 , 1 The H-NMR spectrum is shown below. Figure 2 .

[0064] ;

[0065] Formula I

[0066] .

[0067] Formula II

[0068] S2, the above-synthesized PAGEC-PEG 2000 - PAGEC segments were modified with nitrogen-containing seven-membered rings to obtain PAGEC-PEG. 2000 -PC7A intermediate: 2-(aza-1-yl)ethane-1-thiol (1 eq) is protonated by reacting it with hydrogen chloride (1 eq); the protonated 2-(aza-1-yl)ethane-1-thiol (20 eq) is then reacted with the above-mentioned PAGEC-PEG. 2000 PAGEC segments (1 eq) were added sequentially to a reaction flask, followed by benzoin dimethyl ether (DMPA, 2 eq). The reaction was carried out under UV irradiation at room temperature for 4 h. Subsequently, the polymer obtained from the reaction was dissolved in DCM, and the precipitated polymer was placed in diethyl ether to remove byproducts. The product was then dissolved in PBS solution at pH 9, stirred for 4 h to remove protonation, dialyzed, and dried under vacuum to obtain PAGEC-PEG. 2000 -PC7A intermediate, see detailed synthesis steps. Figure 1 , 1 The H-NMR spectrum is shown below. Figure 3 The dialysis time for this step is 48 hours. For the first 8 hours, the deionized water is changed every 2 hours. For the middle 16 hours, the deionized water is changed every 4 hours. For the last 24 hours, the deionized water is changed every 12 hours.

[0069] In addition, the synthesis method of 2-(aza-1-yl)ethane-1-thiol (C7ASH) is as follows:

[0070] Cyclothione (2 eq) and cyclohexylimine (1 eq) were dissolved separately in DCM and transferred sequentially to flasks. The mixtures were stirred at room temperature for 3 hours, and excess solvent and unreacted reactants were removed under reduced pressure to obtain C7ASH. The reaction equation is as follows:

[0071] ;

[0072] C7ASH 1 The H-NMR spectrum is shown below. Figure 4 .

[0073] S3, Preparation of PC 10 F 17 -PEG 2000 -PC7A compound, namely a high molecular weight immunoadjuvant material: a perfluoroalkyl thiol C 10 H5F 17 S(20eq) and the PAGEC-PEG obtained above 2000PC7A intermediate (1 eq) was added sequentially to a reaction flask, followed by benzoin dimethyl ether (DMPA, 2 eq). The reaction was carried out under UV irradiation at room temperature for 4 h. The resulting polymer was dissolved in DCM, precipitated in diethyl ether to remove byproducts, dialyzed, and then dried under vacuum to obtain PC. 10 F 17 -PEG 2000 -PC7A compound, detailed synthesis steps are shown in [link to details]. Figure 1 , 1 The H-NMR spectrum is shown below. Figure 5 Other polymers with similar structures can also be synthesized and characterized using the same method, and will not be described in detail here. The dialysis time for this step is 48 hours. For the first 8 hours, the deionized water is changed every 2 hours; for the middle 16 hours, it is changed every 4 hours; and for the last 24 hours, it is changed every 12 hours.

[0074] Example 2: This example examines PC 10 F 17 -PEG 2000 The effects of PC7A on the activation of dendritic cells (DCs) in mice are as follows:

[0075] To evaluate PC 10 F 17 -PEG 2000 The ability of PC7A to induce dendritic cell (DC) activation in vivo was evaluated in this embodiment using BALB / c mice. Mice were randomly divided into three groups: inactivated CVS11 + PC7A. 10 F 17 -PEG 2000 -PC7A immunity (CVS11+PC) 10 F 17 -PEG 2000 Mice were immunized with three groups: PC7A group, inactivated CVS11+Alum group, and inactivated CVS11 group. Seven days after the first immunization, mice were euthanized, and inguinal lymph nodes were collected to prepare single-cell suspensions. CD11c was detected by flow cytometry. + The expression levels of co-stimulatory molecules CD80 and CD86, as well as antigen presentation-related molecules MHC II and MHC I, were analyzed in cells. Results showed that, compared to the control groups (i.e., the CVS11+A1 group and the CVS11 group), inactivation of CVS11+PC... 10 F 17 -PEG 2000 CD11c in the inguinal lymph nodes of PC7A group mice + CD80 + CD11c + CD86 + CD11c+ MHC I + and CD11c + MHC II + The proportion of cells was significantly increased ( Figure 6 The results show that PC 10 F 17 -PEG 2000 -PC7A can effectively promote the activation of dendritic cells in lymph nodes and enhance their antigen presentation ability, thereby facilitating the initiation of subsequent humoral and cellular immune responses.

[0076] Example 3: This example examines PC 10 F 17 -PEG 2000 The effects of PC7A on inducing humoral immune responses in mice are as follows:

[0077] To evaluate PC 10 F 17 -PEG 2000 The ability of PC7A to induce a specific humoral immune response in vivo was evaluated in this embodiment using BALB / c mice. Mice were randomly divided into three groups: inactivated CVS11 + PC7A. 10 F 17 -PEG 2000 -PC7A immunity (CVS11+PC) 10 F 17 -PEG 2000 Mice were immunized with three doses of CVS11 (PC7A group), inactivated CVS11+Alum (CVS11+A1 group), and inactivated CVS11 (CVS11 group). A three-dose immunization program was used. Mice were euthanized on day 14 after the last immunization, and inguinal lymph nodes were collected to prepare single-cell suspensions. CD4 counts were detected by flow cytometry. + The proportion of TFH cells in T cells and B220 + The proportion of GCB cells in B cells; simultaneously, CD19 was detected. + CD69 + B cells and CD19 + CD40 + The proportion of B cells was used to assess B cell activation-related status, and serum was collected to determine neutralizing antibody levels. Results showed that, compared to the control groups (i.e., the CVS11+A1 group and the CVS11 group), the CVS11+PC... 10 F 17 -PEG 2000 -PC7A group of mouse CD19+CD69+ B cells ( Figure 10 ) and CD19+CD40+B ( Figure 11The proportion of Tfh cells in the lymph nodes was elevated, suggesting that B cells were in a more active immune state; at the same time, the proportion of Tfh cells in the lymph nodes was also elevated. Figure 8 ) and GCB cell ratio ( Figure 9 The levels of both antibodies were elevated, suggesting that the immunized group induced a stronger germinal center-associated immune response. Serum neutralizing antibody titers were also higher than those in the control group. Figure 7 This trend is consistent with the changing trend of the proportion of cells related to the germinal center. These results indicate that PC... 10 F 17 -PEG 2000 -PC7A can promote Tfh cell and germinal center B cell-related responses, thereby increasing the level of specific neutralizing antibodies and inducing humoral immune responses.

[0078] Example 4: This example examines the PC 10 F 17 -PEG 2000 The evaluation of PC7A on mouse T cell immune activation is as follows:

[0079] This embodiment evaluated PC by detecting the activation phenotype of T lymphocytes in the spleen. 10 F 17 -PEG 2000 - The promoting effect of PC7A on cellular immunity. BALB / c mice were immunized three times (two weeks apart). On day 14 after the last immunization, the spleens of each group of mice were aseptically dissected and single-cell suspensions were prepared. Flow cytometry was used to detect CD4+ in the spleen. + The study also examined the expression levels of T cell populations and activation markers CD44 and CD62L within these cells. Results showed that, compared to the control groups (i.e., the CVS11+A1 group and the CVS11 group), PC... 10 F 17 -PEG 2000 -PC7A group can induce the body to produce more CD4. + CD69 + T( Figure 12 ), at the same time, CD4 + / CD44 + CD62L + The activation rate of T cells also increased significantly. Figure 13 The results show that PC 10 F 17 -PEG 2000 -PC7A can enhance antigen-induced cellular immune responses.

[0080] Example 5: This example is an immune protection experiment, as detailed below:

[0081] Healthy female BALB / c mice aged 6-8 weeks were randomly divided into three groups (n=10): PBS group (inoculated with an equal volume of PBS), Al group (inoculated with an aluminum adjuvant-containing inactivated CVS11 vaccine), and PC group. 10 F 17 -PEG 2000 -PC7A group (vaccinated with PC) 10 F 17 -PEG 2000 -PC7A inactivated CVS11 vaccine). Mice in all groups received the same immunization dose and number of doses. On day 14 after the last immunization, mice in all groups were intramuscularly injected with a 10 LD50 dose of virulent rabies virus strain CVS11. Clinical symptoms and weight changes in mice were observed and recorded daily after challenge. Results showed that mice in the PBS group began to exhibit typical neurological symptoms and progressive weight loss from day 4 after challenge, and all died within 14 days; PC7A inactivated CVS11 vaccine. 10 F 17 -PEG 2000 The PC7A group mice maintained stable physiological condition and showed no signs of disease throughout the observation period, with a final survival rate of 100%. The experimental results indicate that ( Figure 14 ), PC 10 F 17 -PEG 2000 -PC7A, as an adjuvant, can significantly enhance the immune protection level of inactivated rabies virus vaccines, enabling test animals to obtain complete immune protection against lethal doses of viral challenge, with efficacy comparable to clinical standard aluminum adjuvants.

[0082] Example 5: This example is a safety evaluation experiment, as detailed below:

[0083] At the endpoint of the humoral immune response experiment, orbital venous blood was collected from mice to detect complete blood count and blood biochemical indicators. It was found that compared with the PBS group, PC... 10 F 17 -PEG 2000 -No significant difference was found in the PC7A group, indicating that PC 10 F 17 -PEG 2000 -PC7A has good biocompatibility.

[0084] In summary, the embodiments of this invention construct a functionalized polymeric adjuvant system based on a polycarbonate backbone. By introducing polyethylene glycol segments, nitrogen-containing seven-membered ring functional units, and fluorinated side chains, the synergistic design of material composition and properties is achieved. This material system exhibits good structural designability and parameter tunability. The dispersibility, interfacial properties, and application performance of the material can be optimized by adjusting the segment composition, the degree of functional group grafting, and hydrophilic / hydrophobic characteristics.

[0085] In this invention, polycarbonate is used as the main framework, and its structural design possesses biodegradability potential. Animal experiments show that after the material was used as a vaccine adjuvant, no significant abnormalities were observed in blood routine and serum liver function indicators, suggesting that it has good preliminary biosafety and biocompatibility, providing a basis for its further development and application.

[0086] Furthermore, the polymeric immune adjuvant material provided in this invention can be combined with inactivated rabies virus antigen to form an immune preparation, and has shown good adjuvant effects in animal experiments. It can improve the level of neutralizing antibodies and the protective effect against challenge, and promote the growth of dendritic cells, B cells, and CD4+. + T-cell activation indicates that the material has a synergistic effect of antigen delivery and immune enhancement, and has application value in the development of adjuvants for inactivated rabies vaccines.

[0087] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A fluorine-nitrogen-modified polymeric immunoadjuvant material, characterized in that, It comprises a biodegradable polycarbonate backbone, and polyethylene glycol segments, perfluoroalkyl side chains and nitrogen-containing seven-membered ring side chains connected to the polycarbonate backbone.

2. The fluorine-nitrogen-modified polymeric immunoadjuvant material according to claim 1, characterized in that, The general structural formula of the polymeric immune adjuvant material is: ； In the formula, n, m, and p represent the degree of aggregation; 1 ≤ q ≤ 15.

3. The fluorine-nitrogen-modified polymeric immunoadjuvant material according to claim 2, characterized in that, The value of q is 8.

4. A method for preparing a polymeric immune adjuvant material as described in any one of claims 1-3, characterized in that, Includes the following steps: With the aid of a catalyst and a co-catalyst, polyethylene glycol was used as a chain transfer agent to induce a ring-opening copolymerization reaction between the monomer allyl glycidyl ether and carbon dioxide, resulting in PAGEC-PEG-PAGEC segments. The PAGEC-PEG-PAGEC segment was reacted with protonated 2-(aza-1-yl)ethane-1-thiol under ultraviolet light irradiation in the presence of a photoinitiator to obtain the PAGEC-PEG-PC7A intermediate. PAGEC-PEG-PC7A intermediate was reacted with perfluoroalkyl thiols under ultraviolet light in the presence of a photoinitiator to obtain a polymeric immune adjuvant material.

5. The method for preparing the polymeric immune adjuvant material according to claim 4, characterized in that, The catalyst is Salen-Co-TFA; the co-catalyst is PPN-TFA.

6. The method for preparing the polymeric immune adjuvant material according to claim 4, characterized in that, The polyethylene glycol is polyethylene glycol 2000, and its equivalent ratio to the monomer allyl glycidyl ether is (0.01-0.05):

1.

7. The method for preparing the polymeric immune adjuvant material according to claim 4, characterized in that, The protonation method for 2-(aza-1-yl)ethane-1-thiol is as follows: 2-(aza-1-yl)ethane-1-thiol is reacted with hydrogen chloride; the equivalent ratio of PAGEC-PEG-PAGEC segment to protonated 2-(aza-1-yl)ethane-1-thiol is 1:(10-30).

8. The method for preparing the polymeric immune adjuvant material according to claim 4, characterized in that, The photoinitiator is benzoin dimethyl ether.

9. The method for preparing the polymeric immune adjuvant material according to claim 4, characterized in that, The perfluoroalkyl thiol has the general structural formula C0 a H5F 2a-3 S, where 3≤a≤20; the equivalent ratio of the PAGEC-PEG-PC7A intermediate to the perfluoroalkyl thiol is 1:(10-30).

10. The use of a polymeric immune adjuvant material as described in any one of claims 1-3 in the preparation of a rabies vaccine.

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