Liposome nanoadjuvant targeting tlr7 / 8 design and evaluation of its immunopotency

Abstract

The application discloses a liposome nano-adjuvant targeting TLR7 / 8 and an immune efficacy evaluation method thereof, and belongs to the technical field of biological medicine. A core-shell type liposome nano-adjuvant PPGA / R@LNPs is constructed, in which hydrophobic polyglutamic acid is used as a drug loading core, and a cationic liposome is used as an outer shell, and a small molecule TLR7 / 8 agonist R848 is loaded; the physical and chemical properties, in-vitro immune regulation effect and in-vivo vaccine immune enhancement effect of the nano-adjuvant are further systematically evaluated. The results show that the nano-adjuvant has excellent particle size stability, encapsulation efficiency and biocompatibility, can significantly promote the activation and maturation of dendritic cells, induce Th1-biased immune response, effectively enhance the specific humoral immunity and cellular immunity of the foot-and-mouth disease virus inactivated vaccine, and simultaneously avoid local tissue damage of traditional oil adjuvants. The application effectively solves the problems of poor stability, easy degradation and local toxicity of the small molecule TLR7 / 8 agonist, and provides new technical support for safe and efficient delivery of the TLR agonist and development of a universal nano-adjuvant platform.

Description

Technical Field

[0001] This invention relates to the design of a liposomal nanoadjuvant targeting TLR7 / 8 and its immunogenicity evaluation, belonging to the field of life science technology. Background Technology

[0002] Adjuvants are an indispensable functional component in modern vaccine formulations. They can significantly enhance vaccine immunogenicity and reduce the required antigen dosage by improving antigen presentation, regulating the cytokine environment, and promoting synergistic responses of humoral and cellular immunity. In recent years, small molecule agonists targeting innate immune receptors have gradually become a research hotspot. Among them, Toll-like receptor (TLR) agonists have shown excellent potential in the development of infectious disease and tumor vaccines due to their ability to precisely regulate dendritic cell activation and Th1 immune responses. R848, as a classic TLR7 / 8 agonist, can potently induce the expression of pro-inflammatory factors such as type I interferon and IL-12. However, its poor stability and strong hydrophobicity easily lead to rapid diffusion in vivo and non-specific systemic exposure, causing significant toxic side effects, which is the main bottleneck limiting its clinical application.

[0003] To address the aforementioned issues, nanodelivery systems are considered a key approach to improving the safety and efficacy of TLR7 / 8 agonists due to their ability to regulate the in vivo distribution of small molecule agonists and prolong local retention time. Liposomes, with their cell membrane-like phospholipid bilayer structure, excellent biocompatibility, and multimodal encapsulation capabilities for proteins and small molecules, are widely used in various adjuvant and vaccine systems. Furthermore, polymers can be chemically modified to construct hydrophobic water domains, providing a stable encapsulation microenvironment for small molecule drugs. However, existing single liposome or polymer carriers still suffer from limitations when loaded with R848, such as insufficient drug loading stability, significant burst release effects, or high in vitro and in vivo toxicity, making it difficult to simultaneously achieve the synergistic goals of efficient encapsulation, controlled release with reduced toxicity, and immune enhancement.

[0004] Further developments in nanotechnology hold promise for overcoming these limitations. Hydrophobic γ-polyglutamic acid (PPGA) can self-assemble with R848 via hydrophobic interactions to form a drug-loaded core particle, which can then be introduced as a shell using cationic liposomes through electrostatic interactions, constructing a core-shell liposome adjuvant. This structure provides a stable hydrophobic encapsulation microenvironment for R848 and utilizes the liposome shell to buffer interference from complex physiological media, improving the stability and biocompatibility of the delivery system. However, there are currently no definitive research reports on the use of hydrophobic γ-polyglutamic acid to load R848 and combine it with a cationic liposome shell to construct a core-shell liposome adjuvant (PPGA / R@LNPs) for the safe and efficient delivery of TLR7 / 8 agonists and synergistic enhancement of vaccine immunogenicity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to construct a liposomal nanoadjuvant design targeting TLR7 / 8 and a method for evaluating its immunogenicity, thereby contributing to the effective prevention and control of infectious diseases. The specific technical solution is as follows:

[0006] (1) Preparation of hydrophobic polyglutamic acid.

[0007] L-phenylalanine ethyl ester was grafted onto the γ-PGA molecular chain using the carbodiimide method. First, 297 mg of γ-PGA was dissolved in 40 mL of 0.3 M NaHCO3 aqueous solution and magnetically stirred at a constant temperature until completely dissolved. Then, 441 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was dissolved in 10 mL of 0.3 M NaHCO3 aqueous solution and slowly added dropwise to the above γ-PGA solution system under ice bath conditions, with continuous stirring for 15 min. After activation, the ice bath was removed, and 528 mg of L-phenylalanine ethyl ester was weighed and added to the reaction system. The reaction was continuously stirred at room temperature for 24 h. The reaction product was transferred to a dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed against pure water for 3 days to remove unreacted small molecule impurities, with the dialysate changed every 4 h. Finally, the dialyzed product was freeze-dried, and the obtained hydrophobic polyglutamic acid (PPGA) was dried and stored at 4 °C for later use.

[0008] (2) Preparation of R848 / polyglutamic acid nanoparticles.

[0009] Drug-loaded particles were prepared using a thin-film hydration method. 50 mg of PPGA and 1 mg of R848 were weighed and dissolved together in 10 mL of methanol. The mixture was vortexed for 10 s and incubated at room temperature for 20 min. The organic solvent was removed by rotary evaporation to form a uniform film. After complete solvent evaporation and drying, 10 mL of PBS buffer (10 mmol / L, pH 7.4) was added for hydration. Self-assembly was promoted by vortexing and sonication for 30 min. The particles were then centrifuged at 5000 rpm for 5 min using a 3 kDa ultrafiltration centrifuge tube to remove unencapsulated free R848. The remaining particles were resuspended in 10 mL of PBS buffer to obtain 5.1 mg / mL drug-loaded core particles (PPGA / R).

[0010] (3) Preparation of cationic blank liposomes.

[0011] Cationic blank liposomes were prepared using a thin-film dispersion method. 41.91 mg of DOTAP and 47.16 mg of DOPE were dissolved in 44.55 mL of chloroform. The chloroform was removed by rotary evaporation under reduced pressure, allowing the lipids to form a uniform film on the bottle wall. Subsequently, 44.55 mL of PBS (10 mmol / L, pH 7.4) solution was added for hydration, and the mixture was sonicated for 30 min. A blank cationic liposome (LNP) solution with a final concentration of 2 mg / mL was prepared.

[0012] (4) Preparation of R848 / polyglutamic acid liposome nanoadjuvant.

[0013] PPGA / R cores were encapsulated within liposome shells using electrostatic adsorption. Under continuous vortexing, 1.6 mL of a 5.1 mg / mL PPGA / R solution was rapidly added to 32 mL of a 2 mg / mL LNPs solution. After vortexing for 10 s, the mixture was incubated at room temperature for 2 h to allow for complete binding. The resulting nano-adjuvant (PPGA / R@LNPs) was then freeze-dried and stored at 4 °C for later use.

[0014] (5) Preparation of vaccines.

[0015] Take 72 mg of PPGA / R@LNPs adjuvant, dissolve it in 5 mL of PBS buffer, and then mix it with 5 mL of 50 μg / mL foot-and-mouth disease virus antigen solution (to make the final concentration of foot-and-mouth disease antigen 25 μg / mL). Then vortex for 10 min to mix it evenly to obtain the foot-and-mouth disease vaccine.

[0016] (6) Immunization grouping and procedure.

[0017] Female SPF Balb / c mice aged 6-8 weeks and in good health were randomly divided into 5 groups (n=8) to establish a vaccine immunization evaluation model. The candidate vaccine formulations for each group are as follows: ① Control group: sterile PBS; ② Antigen group (FMDV): FMDV antigen; ③ ISA 201 adjuvant group (ISA 201+FMDV): ISA 201 adjuvant and FMDV antigen were mixed at a 1:1 (w / w) ratio; ④ Blank vector group (LNPs+FMDV): LNPs solution was mixed with FMDV antigen, ensuring that its lipid component concentration was consistent with that of the nano-adjuvant group; ⑤ Nano-adjuvant group (PPGA / R@LNPs+FMDV): PPGA / R@LNPs were mixed with FMDV antigen, and the final concentration of R848 in the system was controlled at 16 μg / mL. In addition, except for group ①, each group of mice was immunized with 5 μg of FMDV antigen, and the single vaccination dose was 200 μL. All mice were subcutaneously vaccinated in the back of the neck, receiving a total of two immunizations, with each immunization spaced 14 days apart.

[0018] Figure 1 This is a schematic diagram showing the particle size variation of PPGA / R@LNPs in different media.

[0019] Figure 2 This is a schematic diagram showing the changes in mouse body weight after immunization.

[0020] Figure 3 This is a schematic diagram showing the changes in specific antibodies in mouse serum after immunization. Detailed Implementation

[0021] Example 1: Stability evaluation of PPGA / R@LNPs nanoadjuvant.

[0022] In this embodiment, the preparation method of the nano-adjuvant is the same as (1)-(4). The prepared PPGA / R@LNPs were dispersed in ddH2O, PBS (10 mmol / L, pH 7.4) buffer, DMEM medium and physiological saline buffer, respectively, and the particle size was detected at specific time points (1, 7, 14, 30, 60 and 90 days). Figure 1 As shown, PPGA / R@LNPs maintained a relatively stable particle size after 90 days of continuous storage in different media, demonstrating good physical stability.

[0023] Example 2: Vaccine preparation and immunization process.

[0024] In this embodiment, the vaccine preparation and immunization process are the same as (1)-(6). During the first 10 days after the initial immunization, the mouse weight was measured and recorded daily, and a weight change curve was plotted. For example... Figure 2 As shown, during the 10-day observation period after primary immunization, all mice exhibited normal clinical signs and no abnormalities such as lethargy, decreased appetite, or limited spontaneous activity. The weight gain trend of the PPGA / R@LNPs+FMDV group was basically consistent with that of the Control and FMDV groups, with no significant differences among the groups, preliminarily indicating that this nanoadjuvant has no negative impact on the growth and development of mice.

[0025] Example 3: Vaccine preparation and immunization process.

[0026] In this embodiment, the preparation and immunization process of the vaccine are the same as in (1)-(6). Blood was collected from the orbital cavity at 4, 6, 8 and 12 weeks after the initial immunization of mice. The blood was placed at 4°C for 3 h, centrifuged at 3000 rpm for 10 min, and the serum was separated to detect the level of specific antibody IgG. ① The antigen was diluted to a final concentration of 1 μg / mL using antigen coating, 100 μL per well, coated overnight at 4°C, and washed 5 times with 1×PBST. ② The 96-well plate was blocked using blocking buffer, 350 μL per well, incubated overnight at 4°C, and washed 5 times with 1×PBST. ③ The sample was diluted to a suitable concentration using diluent, 50 μL per well, incubated at 37°C for 1 h, and washed 5 times with 1×PBST. ④ The HRP-labeled anti-mouse IgG was diluted to 1:15000 using diluent, 50 μL per well, incubated at 37°C for 1 h, and washed 5 times with 1×PBST. ⑤ Add 50 μL of TMB developing solution to each well and incubate at 37°C in the dark for 30 min. ⑥ Add 50 μL of stop solution to each well and measure the OD450 nm value. Figure 3 As shown, the vaccine significantly increased FMDV-specific IgG levels by week 4 post-immunization, outperforming the traditional ISA201 adjuvant; it peaked at week 6 and remained at a high level until week 12. These results indicate that the vaccine not only rapidly induces high levels of FMDV-specific IgG but also maintains strong humoral immune memory for a longer period, with superior response strength and persistence compared to traditional commercial adjuvants.

Claims

1. Design and Immunopotency Evaluation of a Liposome Nanoadjuvant Targeting TLR7 / 8. A core-shell liposome nanoadjuvant, PPGA / R@LNPs, was constructed with a hydrophobic polyglutamic acid core and a cationic liposome shell to load the small molecule TLR7 / 8 agonist R848. The physicochemical properties, in vitro immunomodulatory effects, and in vivo vaccine-enhancing effects of this nanoadjuvant were systematically evaluated. Results showed that the nanoadjuvant possessed excellent particle size stability, encapsulation efficiency, and biocompatibility. It significantly promoted dendritic cell activation and maturation, induced Th1-biased immune responses, and effectively enhanced specific humoral and cellular immunity in foot-and-mouth disease virus inactivated vaccines, while avoiding local tissue damage associated with traditional oil adjuvants. This invention effectively solves the problems of poor stability, easy degradation, and local toxicity of small molecule TLR7 / 8 agonists, providing new technical support for the safe and efficient delivery of TLR agonists and the development of a universal nanoadjuvant platform.

2. The design and immunopotency evaluation of a liposomal nanoadjuvant targeting TLR7 / 8 according to claim 1, wherein the preparation steps of the hydrophobic polyglutamic acid are as follows: L-phenylalanine ethyl ester is grafted onto the γ-PGA molecular chain using the carbodiimide method. First, 297 mg of γ-PGA is dissolved in 40 mL of 0.3 M NaHCO3 aqueous solution and magnetically stirred at a constant temperature until completely dissolved. Subsequently, 441 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is dissolved in 10 mL of 0.3 M NaHCO3 aqueous solution and slowly added dropwise to the above γ-PGA solution system under ice bath conditions, with continuous stirring for 15 min for activation. After activation, the ice bath is removed, and 528 mg of L-phenylalanine ethyl ester is weighed and added to the reaction system, and the reaction is continuously stirred at room temperature for 24 h. The reaction product is transferred to a dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed in pure water for 3 days to remove unreacted small molecule impurities, with the dialysate being changed every 4 h. Finally, the dialysis product was freeze-dried, and the obtained hydrophobic polyglutamic acid (PPGA) was dried and stored at 4°C for later use.

3. The design and immunopotency evaluation of a liposomal nanoadjuvant targeting TLR7 / 8 according to claim 1, wherein the preparation steps of R848 / polyglutamic acid nanoparticles are as follows: Drug-loaded particles are prepared using a thin-film hydration method. 50 mg of PPGA and 1 mg of R848 are weighed and dissolved together in 10 mL of methanol. The mixture is vortexed for 10 s, incubated at room temperature for 20 min, and the organic solvent is removed by rotary evaporation to form a uniform film. After the solvent has completely evaporated and dried, 10 mL of PBS buffer (10 mmol / L, pH 7.4) is added for hydration. Self-assembly is promoted by vortexing and sonication for 30 min. The unencapsulated free R848 is removed by centrifugation at 5000 rpm for 5 min using a 3 kDa ultrafiltration centrifuge tube, and the mixture is resuspended in 10 mL of PBS buffer to obtain 5.1 mg / mL drug-loaded core particles (PPGA / R).

4. The design and immunopotency evaluation of a liposomal nanoadjuvant targeting TLR7 / 8 according to claim 1, wherein the preparation steps of the cationic blank liposomes are as follows: Cationic blank liposomes are prepared using a thin-film dispersion method. 41.91 mg DOTAP and 47.16 mg DOPE are dissolved in 44.55 mL of chloroform. The chloroform is removed by rotary evaporation under reduced pressure, allowing the lipids to form a uniform thin film on the bottle wall; subsequently, 44.55 mL of PBS (10 mmol / L, pH 7.4) solution is added for hydration, and the mixture is sonicated for 30 min. A blank cationic liposome (LNPs) solution with a final concentration of 2 mg / mL is prepared.

5. The design and immunopotency evaluation of a liposomal nanoadjuvant targeting TLR7 / 8 according to claim 1, wherein the preparation steps of the R848 / polyglutamic acid liposomal nanoadjuvant are as follows: A PPGA / R core is encapsulated in a liposome shell using electrostatic adsorption. Under continuous vortexing, 1.6 mL of a 5.1 mg / mL PPGA / R solution is rapidly added to 32 mL of a 2 mg / mL LNPs solution. After vortexing for 10 s, the mixture is incubated at room temperature for 2 h to allow for complete binding. The resulting nanoadjuvant (PPGA / R@LNPs) is then freeze-dried and stored at 4°C for later use.

6. The design of a liposomal nanoadjuvant targeting TLR7 / 8 and its immunogenicity evaluation according to claim 1, wherein the vaccine preparation steps are as follows: 72 mg of PPGA / R@LNPs adjuvant is dissolved in 5 mL of PBS buffer, and then mixed with 5 mL of 50 μg / mL foot-and-mouth disease virus antigen solution (to make the final concentration of foot-and-mouth disease antigen 25 μg / mL), and then vortexed for 10 min to mix evenly to obtain the foot-and-mouth disease vaccine.

7. The design and immunogenicity evaluation of a liposomal nanoadjuvant targeting TLR7 / 8 according to claim 1, wherein the steps of immunization grouping and procedure are as follows: Female SPF Balb / c mice aged 6-8 weeks and in good health are randomly divided into 5 groups (n=8) to establish a vaccine immunization evaluation model. The candidate vaccine formulations for each group are as follows: ① Control group: sterile PBS; ② Antigen group (FMDV): FMDV antigen; ③ ISA 201 adjuvant group (ISA 201+FMDV): ISA 201 adjuvant and FMDV antigen are mixed evenly at a ratio of 1:1 (w / w); ④ Blank vector group (LNPs+FMDV): LNPs solution is mixed with FMDV antigen, ensuring that its lipid component concentration is consistent with that of the nanoadjuvant group; ⑤ Nanoadjuvant group (PPGA / R@LNPs+FMDV): PPGA / R@LNPs is mixed with FMDV antigen, and the final concentration of R848 in the system is controlled to be 16 μg / mL. In addition, except for Group ①, each group of mice was immunized with 5 μg of FMDV antigen, with a single vaccination dose of 200 μL. All mice were subcutaneously vaccinated in the back of the neck, for a total of 2 immunizations, with an interval of 14 days between each immunization.

Images