Preparation method and application of QS-21-polyamino acid nano vaccine

Abstract

The invention discloses a preparation method and application of a QS-21-polyamino acid nano vaccine, and belongs to the field of biomedical nano materials. The preparation method comprises the following steps: modifying gamma-PGA with phenylalanine ethyl ester (F) to obtain a polymer PF40 of which the F grafting rate is 40%; aiming at the characteristic that QS-21 is strongly combined with cholesterol, PF40 (PFC) grafted with cholesterol (C) is developed, and PFC wraps OVA and QS-21 through hydrophobic force to construct a nano vaccine OQPFC. The cholesterol on the PFC can reduce the combination of the QS-21 and the cholesterol on a cell membrane so as to reduce the biotoxicity of the QS-21, and the QS-21 loading efficiency of the nano vaccine is improved, so that a better treatment effect is achieved. The OQPFC can escape from lysosome through carboxyl protonation, and a large amount of QS-21 and OVA are released in cytoplasm, so that the synergistic cellular immune activation effect of the carrier adjuvant PFC and the saponin adjuvant QS-21 is realized.

Description

Technical Field

[0001] The invention belongs to the field of biomedical nanomaterials and preparation thereof, and more specifically, relates to a preparation method and application of a QS-21-polyamino acid nanovaccine. Background Art

[0002] Vaccination is one of the most successful public health interventions in history. The protective ability of vaccines mainly comes from their ability to mobilize the immune system and induce long-term immune memory. After antigen exposure in the host, the vaccine stimulates the host's immune recognition system, destroys and remembers the microorganism, and provides protection for the human body when fighting the same microbial infection again (Pharmaceutics, 2021, 13, 455). With the development of vaccines, the types of vaccines are constantly enriched, but each vaccine has its advantages and disadvantages (Infection Control and Hospital Epidemiology, 2007, 28, 702). Traditional vaccines are mainly pathogen attenuated live vaccines, inactivated whole organisms or inactivated toxins (toxoids). Such vaccines often have excellent immune effects, but there is a risk of transmitting pathogens in the vaccine to immunocompromised patients or caregivers. In contrast, newly developed recombinant DNA vaccines, subunit vaccines, and conjugate vaccines are relatively safe and convenient to prepare in large quantities. However, since only specific parts of the entire pathogen are used, the immunogenicity of these vaccines is generally weak, and it is difficult to stimulate sufficient immune responses in the recipients. The use of vaccine adjuvants and delivery systems is often necessary.

[0003] QS-21 is a saponin, an active ingredient of the bark extract of Quillaja-saponaria Molina in South America. As a powerful saponin immune adjuvant, it can induce a strong cellular immune response, but its high biological toxicity limits its development as a human vaccine adjuvant. The use of polymer nanoparticles to encapsulate QS-21 can reduce the biological toxicity of QS-21 (Planta Medica, 2016, 82, 1496). At the same time, some polymer nanoparticles themselves have adjuvant functions (Nature Protocols, 2022, 17, 2240). Loading QS-21 and antigens on nanocarrier adjuvants can not only exert the immune activation properties of the two adjuvants at the same time, but also achieve co-localization of antigens and adjuvants, achieving a stronger immune activation effect.

[0004] This project is dedicated to designing a nano-delivery system based on γ-polyglutamic acid (γ-PGA) to encapsulate the model antigen ovalbumin (OVA) and the saponin adjuvant QS-21 to construct a new nano-vaccine. Through the encapsulation of nano-carrier adjuvants, the immune activation effects of the two adjuvants are exerted while reducing the toxic side effects of QS-21, providing new development ideas for the research of new nano-adjuvants.

[0005] The present invention first modifies γ-PGA with phenylalanine ethyl ester (F) to obtain a polymer PF40 with a F grafting rate of 40%. In view of the characteristic that QS-21 strongly binds to cholesterol, we developed PF40 (PFC) grafted with cholesterol (C). PFC encapsulates OVA and QS-21 through hydrophobic forces to construct a nanovaccine OQPFC. The cholesterol on PFC can reduce the binding of QS-21 with cholesterol on the cell membrane, thereby reducing its biological toxicity, and improve the QS-21 loading efficiency of the nanovaccine to achieve better therapeutic effects. OQPFC can escape from lysosomes through carboxyl protonation, and release QS-21 and OVA in large quantities in the cytoplasm, achieving a synergistic cellular immune activation effect of the carrier adjuvant PFC and the saponin adjuvant QS-21. Summary of the invention

[0006] The purpose of the present invention is to provide a nano delivery system with γ-polyglutamic acid (γ-PGA) as the main body. That is, using γ-PGA grafted polymer PFC as a carrier, encapsulating the model antigen ovalbumin (OVA) and the saponin adjuvant QS-21, constructing a new nano vaccine, while reducing the toxic and side effects of QS-21, while exerting the immune activation effects of the two adjuvants.

[0007] A method for preparing a QS-21-polyamino acid nano-vaccine, characterized in that an ultrapure aqueous solution of QS-21 and a model antigen ovalbumin (OVA) is prepared and added to a centrifuge tube; phenylalanine ethyl ester is grafted onto the side chain of γ-polyglutamic acid to obtain a polymer (PF40) with a grafting rate of 40%, and then cholesterol amine (Chol-NH2) is grafted onto the side chain of PF40 to obtain PFC, a dimethyl sulfoxide (DMSO) solution of PFC is added to a centrifuge tube, and aspirated and mixed; the centrifuge tube after aspirated and mixed is centrifuged to remove the supernatant, and repeatedly washed with ultrapure water to obtain a nano-vaccine OQPFC coated with OVA and QS-21;

[0008] The preparation method of PF40 comprises the following steps: 1) preparing a NaHCO3 solution of γ-polyglutamic acid (γ-PGA); 2) pre-ice-bathing the γ-PGA solution, adding N-(3-dimethylaminopropyl)-N'-ethylcarbonyldiimide hydrochloride (EDC) under ice-bathing conditions, reacting in an ice-bath for 10 minutes, and moving the magnetic stirrer to an atmospheric environment for reaction for 16 hours; 4) purifying the reaction system by dialyzing in deionized water using a dialysis bag with a molecular weight cutoff of 15 kDa, changing the water every 3 hours, and dialyzing for 48 hours; 5) after the dialysis is completed, freezing the sample with liquid nitrogen, and drying it in a freeze dryer for 48 hours to obtain a γ-PGA-F (PF40) powder with a grafting rate of 40%; the concentration of the NaHCO3 solution is 50 mM, and the molar ratio of γ-PGA:EDC:F is 4:3:3;

[0009] The preparation method of Chol-NH2 comprises the following steps: 1) preparing a toluene solution of cholesterol chloroformate (Chol-Cl) and placing it in ice water for standby use; 2) adding ethylenediamine (EDA) to toluene and stirring at 600 rpm until completely dissolved; 3) adding the toluene solution of Chol-Cl dropwise to the toluene solution of EDA and reacting for 16 hours under atmospheric environment; the molar ratio is Chol-Cl:EDA=20:1; the system is divided into centrifuge tubes, and equal volumes of deionized water are added respectively, and anhydrous Magnesium sulfate (MgSO4), shake thoroughly to mix, then stand to separate; 5) extract the upper organic phase after separation, and rotary evaporate at 50°C and vacuum degree 0.9Mpa; 6) add dichloromethane to the rotary evaporator, then add methanol to the dichloromethane solution, extract the supernatant after centrifugation, and rotary evaporate the supernatant at 50°C and vacuum degree 0.9Mpa; the molar ratio is anhydrous magnesium sulfate: dichloromethane: methanol = 1:2:3; take out the product from the rotary evaporator to obtain cholesterol modified with amino group (Chol-NH2);

[0010] The preparation method of PFC comprises the following steps: dissolving PF40 in DMSO, preheating at 40°C for 10 minutes, adding N,N'-carbonyldiimidazole (CDI); preparing a tetrahydrofuran (THF) solution of Chol-NH2, adding the THF solution of Chol-NH2 to the PF40 solution, rotary evaporating, and dialyzing the remaining solution; adding toluene to the dialyzed reaction system, shaking well and then standing; retaining the aqueous phase after separation, rotary evaporating the aqueous phase to obtain PFC; the molar ratio is PF40:CDI:Chol-NH2=3:2:1.

[0011] The centrifugal speed was 10000 rpm, and the centrifugal time was 5 min. The concentration of QS-21 solution was 1 mg / mL, and the concentration of OVA solution was 4 mg / mL.

[0012] The QS-21-polyamino acid nano vaccine prepared by the method.

[0013] The obtained nanoparticles have the following characteristics:

[0014] (1) It has a uniform size, with a particle size of about 200 nm;

[0015] (2) Good biocompatibility, reducing the toxic and side effects of QS-21;

[0016] (3) It can enhance humoral immunity and has the potential to serve as a humoral immune adjuvant;

[0017] (4) High drug loading rate and good in vitro cell therapy effect; BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : The nuclear magnetic hydrogen spectrum analysis spectrum of PF40, Chol-NH2 and PFC of the present invention.

[0019] Figure 2 : Particle size diagram of OQPFC of the present invention.

[0020] Figure 3 : Transmission electron microscope image of the nanospheres of the present invention.

[0021] Figure 4 : Drug loading rate test diagram of the nano-microspheres of the present invention.

[0022] Figure 5 : The cell compatibility test diagram of the nano-microspheres of the present invention.

[0023] Figure 6 : Cytokine test diagram of the nanoparticles of the present invention.

[0024] Figure 7 : Flow cytometry CD80 and CD86 expression test diagram of the nanospheres of the present invention.

[0025] Figure 8 : Flow cytometry MHC-I and MHC-II expression test diagram of the nanospheres of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the following examples, but the present invention is not limited to the following examples. For example, the concentration and volume of the solution used can be adjusted as required.

[0027] Example 1

[0028] Preparation of γ-PGA-F

[0029] (1) 51.6 mg of γ-polyglutamic acid (γ-PGA) was completely dissolved in 50 mM NaHCO 3 solution.

[0030] (2) Pre-cooling the γ-PGA solution, and adding 57.6 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) under ice-cooling conditions, and reacting in the ice-cooling condition for 10 min to fully activate the carboxyl groups on the γ-PGA. The speed of the magnetic stirrer was 400 rpm.

[0031] (3) Immediately add 69 mg of phenylalanine ethyl ester hydrochloride (F) to the reaction bottle, react in an ice bath for 1 h, and then move the reaction system to the atmosphere for 16 h.

[0032] (4) The reaction system was then dialyzed and purified in deionized water using a dialysis bag with a molecular weight cutoff of 15 kDa, with the water changed every 3 hours for 48 hours.

[0033] (5) After dialysis, the reaction system was divided into 3 mL portions, rapidly cooled in liquid nitrogen, and then dried in a freeze dryer for 48 h to obtain γ-PGA-F (PF) powder. 3 mg of freeze-dried PF was weighed and dissolved in deuterated DMSO solution. The results were analyzed by H NMR ( Figure 1 ) verified the successful synthesis of γ-PGA-F and calculated that the grafting rate of F was 40%.

[0034] Preparation of Chol-NH2

[0035] (1) Dissolve 225 mg of cholesterol chloroformate (Chol-Cl) completely in 5 mL of toluene and place in ice water for later use.

[0036] (2) 1.67 mL of ethylenediamine (EDA) was added to 25 mL of toluene and completely dissolved at 600 rpm.

[0037] (3) The toluene solution of Chol-Cl was added dropwise to the toluene solution of EDA and stirred at 600 rpm for 16 h.

[0038] (4) The reaction system was divided into two 50 mL centrifuge tubes, and equal volumes of deionized water were added to each of the two tubes. 4.5 g of anhydrous magnesium sulfate (MgSO4) was added to the centrifuge tubes. The mixture was shaken thoroughly and allowed to stand to separate into layers.

[0039] (5) After separation, the upper organic phase was extracted and toluene was removed by rotary evaporation at 50° C. and a vacuum degree of 0.9 MPa.

[0040] (6) Add 5 mL of dichloromethane to the rotary evaporator to completely dissolve the dry product in the rotary evaporator. Add 5 mL of methanol to the dichloromethane solution to completely precipitate the residual MgSO4.

[0041] (7) The solution was centrifuged at 10,000 rpm for 5 min, the supernatant was extracted, and the organic solution was removed by rotary evaporation at 50°C and a vacuum degree of 0.9 MPa. The product was taken out from the rotary evaporator to obtain cholesterol modified with amino groups (Chol-NH2). Chol-NH2 was characterized by hydrogen nuclear magnetic resonance spectroscopy ( Figure 1 ) confirmed the successful synthesis and calculated the purity of Chol-NH2 to be 93%.

[0042] Preparation of PFC

[0043] (1) 29.9 mg of PF40 was dissolved in 1 mL of dimethyl sulfoxide (DMSO), heated at 40 °C for 10 min, and 14.6 mg of N,N'-carbonyldiimidazole (CDI) was added to pre-activate the carboxyl groups on PF for 12 h.

[0044] (2) Dissolve 21.3 mg of Chol-NH2 completely in 1 mL of tetrahydrofuran (THF), add the THF solution of Chol-NH2 into the reaction bottle in (1), and react for 12 h.

[0045] (3) The reaction system was subjected to rotary evaporation at 45° C. and a vacuum degree of 0.9 MPa to remove THF in the reaction system, and the remaining DMSO solution was dialyzed in deionized water using a 15,000 Da dialysis bag to remove DMSO and hydrolyze CDI.

[0046] (4) Add 6 mL of toluene to the dialyzed reaction system, shake well and let stand, and extract the unreacted Chol-NH2 in the aqueous phase with toluene. After separation, retain the aqueous phase, and evaporate the aqueous phase at 50° C. and vacuum degree 0.9 MPa to remove the small amount of toluene remaining in the aqueous phase to obtain PFC. PFC is characterized by hydrogen nuclear magnetic resonance spectroscopy ( Figure 1 )Confirm that the synthesis is successful

[0047] Preparation of OQPFC

[0048] (1) Dissolve QS-21 in ultrapure water to prepare a 1 mg / mL QS-21 solution. Dissolve OVA in ultrapure water to prepare a 4 mg / mL OVA solution. Mix the two in a 3:1 volume ratio in a 1.5 mL centrifuge tube to prepare 100 μL of ultrapure aqueous solution.

[0049] (2) Prepare a 15 mg / mL DMSO solution of PFC.

[0050] (3) Take 100 μL of the solution prepared in (2), add it to the centrifuge tube prepared in (1), and mix by pipetting.

[0051] (4) The centrifuge tube was centrifuged at 10,000 rpm for 5 min and washed three times with ultrapure water to obtain the nano-vaccine OQPFC encapsulated with OVA and QS-21. The particle size of OQPFC was tested. The results are as follows: Figure 2 shown.

[0052] (5) OQPFC powder was obtained by freeze drying and weighed. The synthesized OQPFC was measured using Mastersize3000 dynamic light scattering (DLS) (Malvern Instruments, UK) to obtain a hydrated particle size of 200 nm ( Figure 3 ).

[0053] Drug loading rate test: In order to compare the performance of adjuvants, OVA and QS-21 in OQPFC were quantified. The drug loading rate of OVA was determined using the Lowry protein detection kit, and the drug loading rate of QS-21 was determined by external standard HPLC using pure QS-21 as the QS-21 standard. Figure 4 The data showed that the OVA loading rate of OQPFC was 7.87%, and the OVA loading rate of QS-21 was 9.49%.

[0054] Biosafety evaluation: In order to verify whether the nanovaccine has good biocompatibility, the MTT experiment was used to detect the effect of the nanovaccine on the cell proliferation and activity of macrophage RAW264.7. The corresponding molar concentration of free QS-21 was used as the control group, and the difference in biocompatibility between NPs and free QS-21 was compared to verify the effect of polymer carrier encapsulation on reducing the biological toxicity of QS-21. Figure 5 The data showed that when the concentration of free QS-21 exceeded 6μg / mL, it would significantly inhibit the proliferation and activity of macrophages. After 24 hours of co-incubation, the survival rate of macrophages dropped to less than 80% of the negative control group. However, after QS-21 was encapsulated with a polymer carrier, the nanoparticles could maintain a survival rate of more than 90% at a concentration of 200μg / mL.

[0055] Cytokine detection: The secretion of inflammation-related cytokines is an important indicator for evaluating the immunostimulatory ability of drug pairs. In order to detect the immune activation effect of the constructed nanovaccine, the secretion of inflammation-related cytokines was tested. In order to detect the immune activation effect of each constructed nanovaccine, the secretion of each related cytokine was tested after different systems were incubated with RAW264.7 cells for 24 hours. The secretion of each related cytokine was tested after 24 hours of incubation with RAW264.7 cells. Increased secretion of TNF-α and IL-1β is an important sign of cellular immune activation, and increased secretion of IL-6 is an important sign of humoral immune activation. Figure 6The data showed that OQPFC could significantly enhance the secretion of TNF-α and IL-1β compared with free OVA. The ability of OQPFC to enhance humoral immunity was comparable to that of aluminum adjuvant, proving that OQPFC had the potential to serve as a humoral immune adjuvant.

[0056] Flow cytometry: Figure 7 , Figure 8 The test results proved that the vaccine construction strategy of using PFC to encapsulate antigens and adding QS-21 can significantly promote the maturation of macrophages and strongly activate cellular immune responses. The expression of macrophage surface proteins was detected by flow cytometry. The expression rates of CD80, 86, MHC-I, and II of OQPFC far exceeded those of commercial aluminum adjuvants. In the OQPFC group, not only a large amount of MHC-II expression on the surface of macrophages was observed, but also a significant increase in MHC-I expression was observed, which indicates that OQPFC significantly enhances the cellular immune response, showing the strong potential of OQPFC as an adjuvant system for cellular immune activation.

Claims

1. A method for preparing a QS-21-polyamino acid nanovaccine, characterized in that: An ultrapure aqueous solution of QS-21 and model antigen ovalbumin (OVA) is prepared and added to a centrifuge tube; phenylalanine ethyl ester is grafted onto the side chain of γ-polyglutamic acid to obtain a polymer (PF40) with a grafting rate of 40%, and then cholesterol amine (Chol-NH2) is grafted onto the side chain of PF40 to obtain PFC, and a dimethyl sulfoxide (DMSO) solution of PFC is added to a centrifuge tube and mixed by pipetting; the centrifuge tube after pipetting is centrifuged to remove the supernatant, and is repeatedly washed with ultrapure water to obtain a nano vaccine OQPFC coated with OVA and QS-21; The preparation method of PF40 comprises the following steps: 1) preparing a NaHCO3 solution of γ-polyglutamic acid (γ-PGA); 2) pre-ice-bathing the γ-PGA solution, adding N-(3-dimethylaminopropyl)-N'-ethylcarbonyldiimide hydrochloride (EDC) under ice-bathing conditions, reacting in an ice-bath for 10 minutes, and moving the magnetic stirrer to an atmospheric environment for reaction for 16 hours; 4) purifying the reaction system by dialyzing in deionized water using a dialysis bag with a molecular weight cutoff of 15 kDa, changing the water every 3 hours, and dialyzing for 48 hours; 5) after the dialysis is completed, freezing the sample with liquid nitrogen, and drying it in a freeze dryer for 48 hours to obtain a γ-PGA-F (PF40) powder with a grafting rate of 40%; the concentration of the NaHCO3 solution is 50 mM, and the molar ratio of γ-PGA:EDC:F is 4:3:3; The preparation method of Chol-NH2 comprises the following steps: 1) preparing a toluene solution of cholesterol chloroformate (Chol-Cl) and placing it in ice water for standby use; 2) adding ethylenediamine (EDA) to toluene and stirring at 600 rpm until completely dissolved; 3) adding the toluene solution of Chol-Cl dropwise to the toluene solution of EDA and reacting for 16 hours under atmospheric environment; the molar ratio is Chol-Cl:EDA=20:1; the system is divided into centrifuge tubes, and equal volumes of deionized water are added respectively, and anhydrous Magnesium sulfate (MgSO4), shake thoroughly to mix, then stand to separate; 5) extract the upper organic phase after separation, and rotary evaporate at 50°C and vacuum degree 0.9Mpa; 6) add dichloromethane to the rotary evaporator, then add methanol to the dichloromethane solution, extract the supernatant after centrifugation, and rotary evaporate the supernatant at 50°C and vacuum degree 0.9Mpa; the molar ratio is anhydrous magnesium sulfate: dichloromethane: methanol = 1:2:3; take out the product from the rotary evaporator to obtain cholesterol modified with amino group (Chol-NH2); The preparation method of PFC comprises the following steps: dissolving PF40 in DMSO, preheating at 40°C for 10 minutes, adding N,N'-carbonyldiimidazole (CDI); preparing a tetrahydrofuran (THF) solution of Chol-NH2, adding the THF solution of Chol-NH2 to the PF40 solution, rotary evaporating, and dialyzing the remaining solution; adding toluene to the dialyzed reaction system, shaking well and then standing; retaining the aqueous phase after separation, rotary evaporating the aqueous phase to obtain PFC; the molar ratio is PF40:CDI:Chol-NH2=3:2:

1.

2. The preparation method according to claim 1, characterized in that: The centrifugal speed was 10000 rpm, and the centrifugal time was 5 min. The concentration of QS-21 solution was 1 mg / mL, and the concentration of OVA solution was 4 mg / mL.

3. The QS-21-polyamino acid nanovaccine prepared by the preparation method according to claim 1.

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