WO2023060686A1 - 双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其应用 - Google Patents
双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其应用 Download PDFInfo
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/48—Polymers modified by chemical after-treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/542—Mucosal route oral/gastrointestinal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
Definitions
- the invention belongs to the field of biomedical materials, and in particular relates to amphiphilic imiquimod grafted with gamma-polylauryl glutamate and applications thereof.
- Vaccination is considered to be one of the most economical, convenient and effective ways to prevent infectious diseases in today's society.
- Antigen delivery is a crucial step in the immunization process.
- Toll-like receptors are a type of pattern recognition receptors (Pattern recognition receptors, PRRs). When activated, it not only induces an innate immune response, but also activates the acquired immune system, making it ideal for theoretical adjuvants.
- PRRs pattern recognition receptors
- artificially synthesized imiquimod (R837) as a TLR 7 agonist, is a small molecule immunomodulator with excellent antiviral and antitumor capabilities. Because of its relatively small molecular weight, it can enter the body through various channels, increase the activity of antigen-presenting cells, gather more dendritic cells, macrophages, B cells, and T cells to the vaccination site, and enhance the local immune response.
- imiquimod also has the following defects: (1) it has low solubility in water and common organic solvents, so it is not easy to prepare injections and has certain toxicity to cells; (2) single use of R837 will produce some adverse reactions, For example, erythema, erosion, exfoliation/peeling and edema are common adverse reactions; (3) The pharmacokinetics of imiquimod has the characteristics of rapid diffusion from local (such as subcutaneous or intramuscular) to the whole body, so that it can spread rapidly in multiple remote areas. End tissue causes unwanted intrinsic immune activation. Due to the existence of these shortcomings, its application is limited.
- the purpose of the present invention is to provide amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate, which not only has good water solubility, but also has good biocompatibility, reduces toxic and side effects, and can improve the specific immune response of the body.
- Ideal adjuvant is to provide amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate, which not only has good water solubility, but also has good biocompatibility, reduces toxic and side effects, and can improve the specific immune response of the body. Ideal adjuvant.
- Another object of the present invention is to provide the use of amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate in vaccine adjuvants.
- Amphiphilic imiquimod is grafted with ⁇ -polylauryl glutamate, which is prepared by a method comprising the following steps:
- the molecular weight of ⁇ -polyglutamic acid is 1-2 million, preferably 30-700,000;
- the chlorinating agent is thionyl chloride, oxalyl chloride or phosphorus pentachloride, preferably thionyl chloride and oxalyl chloride;
- fat-soluble alcohols are C8-C24 alcohols, alicyclic alcohols or sterols, preferably n-lauryl alcohol or cholesterol;
- acid-binding agents are triethylamine, 4-N,N-dimethylaminopyridine, pyridine, anhydrous carbonic acid One of cesium, anhydrous potassium carbonate, anhydrous sodium carbonate, sodium hydroxide and potassium hydroxide.
- the aprotic solvent is one of dichloromethane, chloroform, acetonitrile, dimethylsulfoxide, N,N-dimethylformamide, tetrahydrofuran, 1,4-dioxane and toluene;
- the aprotic solvent is preferably dichloromethane or acetonitrile.
- the molar ratio of ⁇ -polyglutamic acid, imiquimod, fat-soluble alcohol and acid-binding agent is 10:0.5-1.5:1-3:10-15;
- the ratio of acid to chlorinating agent is 1:(1-2.5), preferably 1:1, and the reaction time is 20-25 hours.
- reaction temperatures of steps (1) and (2) are both 10-40°C; preferably 20-25°C.
- step (1) every gram of ⁇ -polyglutamic acid is dispersed in 5-70 mL of aprotic solvent.
- the preparation method further includes the step of labeling with fluorescein;
- the fluorescein is aminofluorescein or aminorhodamine, preferably 5-aminofluorescein.
- the purification steps are as follows: remove the solvent, soak the residual solid with anhydrous acetone, methanol, ethanol or acetonitrile, filter, wash and vacuum dry.
- the present invention also provides the use of the amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate as a vaccine adjuvant.
- the vaccine is hand, foot and mouth disease, avian influenza, Newcastle disease, pseudorabies, porcine parvovirus, swine fever and porcine blue ear vaccine;
- the mass ratio to antigen is (0.01 ⁇ 1):(0.5 ⁇ 1).
- the present invention utilizes ⁇ -polyglutamic acid as a hydrophilic polymer backbone, and couples 5-aminofluorescein and R837 through an amide covalent bond. , through the coupling of hydrophobic fat-soluble alcohols (such as n-lauryl alcohol) through ester bonds to form amphiphilic polymer FIP (FL- ⁇ -PGA-R837-LA).
- hydrophobic fat-soluble alcohols such as n-lauryl alcohol
- ester bonds to form amphiphilic polymer FIP (FL- ⁇ -PGA-R837-LA).
- the modification of ⁇ -PGA is due to the fact that its carboxyl group forms esters and amides under EDC/NHS activation. Since the amino group of imiquimod is very inactive, it is difficult to use this method for amidation coupling.
- the carboxyl group of ⁇ -PGA is first made into a highly active acid chloride with thionyl chloride, oxalyl chloride or phosphorus pentachloride under the catalysis of N,N dimethylformamide, and then reacted with the amino group of imiquimod, Coupling via an amide bond was successful.
- the method of esterification or amidation after the carboxylic acid group of ⁇ -PGA is made into acid chloride has not been reported. The reason may be that the conditions of chlorination are not easy to control, and the reaction system is easily carbonized and blackened.
- the present invention successfully realizes the generation of acid chlorides by controlling the reaction temperature and the adding speed of the chlorinating agent. Therefore, the preparation method of FIP and ⁇ -PGA-R837-LA of the present invention is simple and ingenious. Due to the rich sources of raw materials, good biological safety and low price, the cost of FIP and ⁇ -PGA-R837-LA in the present invention is relatively low.
- the FIP and ⁇ -PGA-R837-LA prepared by the present invention not only have good water solubility, good biocompatibility, reduced toxic and side effects, but also can effectively stimulate
- the immune response of the body can increase the secretion level of IgG, which can be used in fields such as vaccines, drug delivery, and probes.
- Animal experiments showed that no lesions were found in the skin, liver, spleen and kidney of the injection site of the mice inoculated with the vaccine containing FIP, which proved that the mice in the experimental group had normal signs and no adverse side effects. After the mice were vaccinated with different doses of vaccines containing FIP, the titer level increased significantly.
- FIP is a good water vaccine.
- Dosage form adjuvant better safety, is an ideal multi-dosage form adjuvant.
- FIP contains fluorescent groups, which can be used for in vivo/external fluorescence tracking and quantification, and intuitively determine the connection between humoral immunity and cellular immunity.
- the FIP of the present invention can be made into vaccines in W, O/W, W/O and W/O/W dosage forms, all of which can significantly improve the immune efficacy.
- FIP is an amphiphilic polymer, it can self-assemble into nanoparticles (micelle or vesicle) in water or oil phase medium through intermolecular force, which provides a new method for the preparation of drug and vaccine carriers. Methods. FIP (that is, FL- ⁇ -PGA-R837-LA) can be physically mixed with various antigens to make a vaccine for mucosal administration, which can be administered nasally and orally.
- Fig. 1 is the synthesis route of amphiphilic imiquimod grafted gamma-polylauryl glutamate (gamma-PGA-R837-LA);
- Fig. 2 is the synthetic route of fluorescein-labeled amphiphilic imiquimod grafted ⁇ -polylauryl glutamate (FIP, FL- ⁇ -PGA-R837-LA) and the chemical structural formula of 5-aminofluorescein;
- Fig. 3 is the 1 H NMR diagram of fluorescein-labeled amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate (FL- ⁇ -PGA-R837-LA);
- Figure 4 is a UV-vis characterization diagram of fluorescein-labeled amphiphilic imiquimod grafted gamma-polylauryl glutamate (FL-gamma-PGA-R837-LA);
- Fig. 5 is the fluorescence spectrum representation graph of FIP, imiquimod, fluorescein, wherein a is the fluorescence spectrum representation graph of FIP and imiquimod, b is the fluorescence spectrum representation graph of FIP and fluorescein;
- Figure 6 is a Zeta potential diagram of fluorescein-labeled amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate (FL- ⁇ -PGA-R837-LA) and ⁇ -polyglutamic acid ( ⁇ -PGA);
- Fig. 7 is the experimental result of the survival rate of RAW 264.7 cells with different concentrations of fluorescein-labeled amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate (FL- ⁇ -PGA-R837-LA), and the abscissa is the concentration , the ordinate is the cell survival rate (%), FIP is the FL- ⁇ -PGA-R837-LA solution of different concentrations; IMQ is the R837 solution of different concentrations; HAc is the acetic acid aqueous solution of pH 6.0; PBS is the PBS buffer solution;
- Figure 8 is the fluorescence flow diagram of RAW 264.7 cells engulfing fluorescein-labeled amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate (FL- ⁇ -PGA-R837-LA); a is the concentration of 50 ⁇ g/mL The FSC-SSC scatter diagram of the FIP-intervened cells, the abscissa is the forward angle scattered light intensity, and the ordinate is the side-scattered light intensity; b is the single-parameter histogram of the FIP-intervened cells with a concentration of 50 ⁇ g/mL, the abscissa is the value of the fluorescence signal; c is the average fluorescence map of cells intervened with different concentrations of FIP, the abscissa is the FIP concentration, and the ordinate is the fluorescence intensity;
- Fig. 9 is the photograph of the skin condition of mice 5 days after immunization, and the area where the inoculation site is located is in the dotted line frame; Wherein the vaccine containing imiquimod (R837, IMQ) is subcutaneously injected into the mouse in the figure a, and the mouse contains the vaccine subcutaneously in the b figure Vaccines for FIP;
- Figure 10 is a comparison chart of the immune effect of FIP and imiquimod (R837, IMQ) as an adjuvant;
- Figure 11 is the histological detection of the skin and viscera (such as liver, kidney, spleen) at the injection site of the inoculated mice, wherein the first row is the mice inoculated with PBS buffer solution, the second row is the mice inoculated with vaccine 1, a is the injection site skin, b is the liver, c is the spleen and d is the kidney;
- viscera such as liver, kidney, spleen
- Figure 12 is the serum IgG antibody level of mice inoculated with different formulations of FIP/OVA vaccines, wherein Figure A is the antibody titer two weeks after inoculation, and Figure B is the serum antibody titer after six weeks of inoculation.
- FIP/OVA is each vaccine (contains both FIP and OVA), and OVA is each positive control vaccine (contains OVA only).
- the preparation method of ⁇ -PGA-R837-LA comprises the following steps:
- the preparation method of FIP comprises the steps:
- FL- ⁇ -PGA-R837-LA was dissolved in DMSO-d 6 for characterization by hydrogen nuclear magnetic resonance ( 1 H NMR). Meanwhile, ⁇ -polyglutamic acid (dissolved in D 2 O) and R837 (dissolved in DMSO-d 6 ) were used as controls to evaluate the grafting and quantification of R837.
- the NMR detection results are shown in Figure 3.
- UV-vis detection sensitivity is high. Because the content of grafted fluorescein is very low, it is difficult to detect by hydrogen nuclear magnetic resonance ( 1 H NMR).
- 1 H NMR hydrogen nuclear magnetic resonance
- FL- ⁇ -PGA-R837-LA was dissolved in ultrapure water, and FL- ⁇ -PGA-R837 was detected by UV-vis (ultraviolet-visible light spectrophotometer) -The main component in LA; R837 was dissolved in hydrochloric acid acidified ultrapure water (pH 6) as control 1; 5-aminofluorescein aqueous solution was used as control 2.
- the absorbance was detected at a wavelength of 200-600nm, and the detection results were normalized and compared.
- the results are shown in Figure 4, the characteristic peaks of R837 at 200-250nm, the characteristic peaks of ⁇ -PGA at 250-300nm and the The characteristic peaks of 5-FL proved that both R837 and fluorescein were grafted onto the ⁇ -PGA backbone, and verified the structure of FL- ⁇ -PGA-R837-LA.
- FL- ⁇ -PGA-R837-LA was dissolved in ultrapure water and detected by a fluorescence spectrophotometer. Dissolve R837 in ultrapure water (pH 6) solution acidified with hydrochloric acid as control 1; use 5-aminofluorescein aqueous solution as control 2.
- the positive and negative values of the Zeta potential correspond to the stability of the material structure and the positive and negative charges. It can be seen from Figure 6 that the Zeta potential of FL- ⁇ -PGA-R837-LA is -3.3mV, indicating that FL- ⁇ -PGA-R837-LA can coagulate rapidly, and if the concentration is too high, a gel-like substance will be formed.
- aqueous phase for example, ultrapure water, 0.1M PBS buffer with pH 7.4, 0.9% normal saline, 5% glucose In aqueous solution and acidic aqueous solution with pH 1-6, SBF simulated body fluid
- solvent phase ethanol, DMSO, DMF
- ⁇ -PGA-R837-LA and FL- ⁇ -PGA-R837-LA were dissolved in 5 mL of ultrapure water, 0.1 M, PBS buffer at pH 7.4, 0.9% normal saline, 5% glucose aqueous solution, All dissolved in acidic aqueous solution with pH 1-6 and SBF simulated body fluid; 20 mg of ⁇ -PGA-R837-LA and FL- ⁇ -PGA-R837-LA were all dissolved in 3 mL of DMSO, ethanol, DMF and acetone. Therefore, ⁇ -PGA-R837-LA and FL- ⁇ -PGA-R837-LA are amphiphilic materials, which are expected to be used in the preparation of water adjuvants.
- R837 cannot be dissolved in ultrapure water, 0.1M, PBS buffer solution with pH 7.4, 0.9% normal saline, 5% glucose aqueous solution and SBF simulated body fluid, and only dissolves in acidic aqueous solution with pH ⁇ 6; R837 is slightly soluble in in hot DMSO and DMF solutions. Therefore, its application in water adjuvants is limited.
- a microplate reader (BioTek microplate reader) was used to detect the degree of cell apoptosis. Results: As shown in Figure 7, when the concentration of FIP solution was 1000 ⁇ g/mL (the concentration of R837 contained in it was 100 ⁇ g/mL), the cells still maintained a survival rate of 88%. When the concentration of FIP solution was less than 1000 ⁇ g/mL, the cells had Apoptosis phenomenon, cell survival rate was similar to that of cells treated with PBS buffer; on the contrary, R837 solution did more damage to cells, and when the concentration was 100 ⁇ g/mL, a large number of RAW 264.7 cells underwent apoptosis. Therefore, FIP has good biocompatibility and low toxicity, and can be used in vaccines, drug loading, probes and other fields.
- OVA 500 ⁇ g/mL OVA solution
- FIP 100 ⁇ g/mL FIP solution
- mice Female Balb/c mice (20-25g) aged 6-8 weeks were randomly divided into 5 groups, with 6 mice in each group, and were inoculated with vaccines containing FIP, vaccines containing R837, 500 ⁇ g/mL OVA solution, 100 ⁇ g/mL FIP solution and PBS buffer solution with pH 7.4, the inoculation dose was 200 ⁇ L vaccine per mouse. After inoculation, observe the skin condition of the injection site within one week. 28 days after inoculation, blood was collected from the orbit, and the serum was separated to detect the antibody titer.
- the antibody titer was detected by the following method: a 10 ⁇ g/mL OVA protein solution was prepared with a pH 9.6, 0.05 mol/L Na 2 CO 3 -NaHCO 3 buffer as a solvent. Take 50 ⁇ L of OVA protein solution to coat a 96-well plate, absorb overnight at 4°C; discard the solution, and wash the plate with PBST buffer (500 ⁇ L Tween-20 added to 1 L, 0.1 M PBS buffer solution with pH 7.4) 2 Place on clean absorbent paper and pat dry; add 100 ⁇ L of blocking solution (obtained after adding 10 g of BSA to 1 L, 0.1 M pH 7.4 PBS buffer) to each well, seal the membrane, and incubate at 37 ° C for 1 hour , then wash the plate twice with PBST buffer, put it on clean absorbent paper and pat dry; add 100 ⁇ L of mouse serum diluted with PBST buffer, incubate at 37°C in the dark for 1.5 hours, discard the solution, add PBST buffer to wash
- the imiquimod component in the vaccine containing FIP is only 10 ⁇ g/mL, which is only 10% of the vaccine containing R837, FIP, as a water adjuvant, not only has good water solubility, reduces toxic and side effects, but also significantly reduces When the dosage of imiquimod is used, it can effectively stimulate the immune response of the body and increase the secretion level of IgG.
- Example 1 illustrates the application of the fluorescein-labeled amphiphilic imiquimod grafted with ⁇ -polylauryl glutamate (FL- ⁇ -PGA-R837-LA, FIP for short) prepared in Example 1 as a vaccine immune adjuvant.
- OVA ovalbumin
- Vaccine number or composition OVA concentration FIP concentration dosage form Injection dosage 0.1M PBS buffer, pH 7.4 0 0 W 200 ⁇ L Positive Control Vaccine 1 500 ⁇ g/mL 0 W 200 ⁇ L Positive Control Vaccine 2 500 ⁇ g/mL 0 O/W 200 ⁇ L Positive Control Vaccine 3 500 ⁇ g/mL 0 W/O 200 ⁇ L Positive Control Vaccine 4 500 ⁇ g/mL 0 W/O/W 200 ⁇ L
- Vaccine 1 500 ⁇ g/mL 100 ⁇ g/mL W 200 ⁇ L Vaccine 2 500 ⁇ g/mL 100 ⁇ g/mL O/W 200 ⁇ L Vaccine 3 500 ⁇ g/mL 100 ⁇ g/mL W/O 200 ⁇ L Vaccine 4 500 ⁇ g/mL 100 ⁇ g/mL W/O/W 200 ⁇ L
- FIP/OVA aqueous vaccine (referred to as vaccine 1): use 0.1M PBS (pH 7.4) buffer as solvent, prepare 1000 ⁇ g/mL of OVA solution and 200 ⁇ g/mL of FIP (i.e. FL- ⁇ -PGA- R837-LA) solution, and then the two solutions were mixed in equal volumes to prepare a FIP/OVA aqueous vaccine (referred to as vaccine 1).
- FIP FL- ⁇ -PGA- R837-LA
- vaccine 1 the FIP concentration was 100 ⁇ g/mL and the OVA concentration was 500 ⁇ g/mL.
- Positive control vaccine 1 was prepared according to the preparation method of vaccine 1, except that water was used instead of FIP solution.
- FIP/OVA oil-in-water formulation vaccine (referred to as vaccine 2): use formula 4 in Chinese patent ZL201310021011.3 as oil phase; use 0.1M PBS (pH 7.4) buffer as solvent to prepare water containing OVA and FIP phase; the water phase and the oil phase were mixed at a volume ratio of 1:1, and homogenized under high pressure to obtain a FIP/OVA oil-in-water formulation vaccine (referred to as vaccine 2).
- the concentration of FIP in the vaccine is 100 ⁇ g/mL, and the concentration of OVA is 500 ⁇ g/mL.
- Positive control vaccine 2 was prepared according to the preparation method of FIP/OVA oil-in-water formulation vaccine, except that the vaccine did not contain FIP.
- FIP/OVA water-in-oil vaccine (referred to as vaccine 3): use 0.1M PBS (pH 7.4) buffer as solvent to prepare an aqueous phase containing OVA and FIP; the aqueous phase and white oil in a volume ratio of 1 : 3 were mixed and emulsified to obtain the FIP/OVA water-in-oil formulation vaccine (referred to as vaccine 3).
- the concentration of OVA in vaccine 3 was 500 ⁇ g/mL and the concentration of FIP was 100 ⁇ g/mL.
- Positive control vaccine 3 was prepared according to the preparation method of vaccine 3, except that the aqueous phase did not contain FIP.
- W/O/W dosage form vaccine use 0.1M PBS (pH 7.4) buffer as a solvent to prepare an aqueous phase containing FIP and OVA; mix the aqueous phase and ISA201 in a volume ratio of 1: 1 mixed and emulsified to obtain the FIP/OVA water-in-oil-in-water formulation vaccine (referred to as vaccine 4), the concentration of FIP in this vaccine is 100 ⁇ g/mL, and the concentration of OVA is 500 ⁇ g/mL.
- Positive control vaccine 4 was prepared according to the preparation method of vaccine 4, except that the aqueous phase did not contain FIP.
- mice Female Balb/c mice (20-25g) at the age of 6-8 weeks were randomly divided into 9 groups, each group of mice had 6, and 8 groups of mice were respectively immune to positive control vaccine 1-4, vaccine 1-4 ( Table 1), the remaining group of mice were inoculated with 0.1M, pH 7.4 PBS buffer as a negative control.
- the inoculation method of each vaccine is as follows: a total of two inoculations, on the 1st day and 14th day, the inoculation dose is 200 ⁇ L of vaccine per mouse.
- mice inoculated with PBS buffer and vaccine 1 were sliced at the inoculation site, liver, kidney, and spleen tissue 28 days after the first immunization to observe whether there were any damages and lesions.
- the specific method is as follows: 3 mice in each group were euthanized, and the skin and viscera (such as liver, kidney, and spleen) at the injection site were surgically separated, and treated with 4% paraformaldehyde (purchased from Legen Bio, product number DF0135) Fixed and soaked, embedded in paraffin, sliced and processed for immunohistochemical staining. The results are shown in Figure 11.
- mice inoculated with PBS buffer Compared with the mice inoculated with PBS buffer, no lesions were found in the skin, liver, spleen, and kidney of the injection site of the mice inoculated with vaccine 1, which proved that the mice in the experimental group had normal signs and no adverse side effects. reaction.
- Orbital blood was collected every two weeks after the first immunization, the serum was separated, and the specific antibody (IgG) level in the serum of each mouse was determined according to the method in Example 2.
- IgG specific antibody
- the applicant used the amide bond to covalently couple imiquimod to ⁇ -polyglutamic acid for the first time, and grafted FL- ⁇ -PGA-R837 modified with liposoluble groups and fluorescent chromophores - LA polymer.
- FL- ⁇ -PGA-R837-LA has good biocompatibility and amphipathic solubility.
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Abstract
双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其应用,属于生物医用材料领域。双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,采用包括如下步骤的方法制备:(1)在无水氛围下,将γ-聚谷氨酸分散在非质子溶剂中,然后加入催化剂N,N-二甲基甲酰胺,在搅拌状态下,滴入氯化剂,反应10-40小时;(2)将咪喹莫特、脂溶性醇和缚酸剂加入步骤(1)反应后所得溶液中,反应42-54小时;(3)纯化后,得到双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯材料。双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其荧光素标记物,不仅水溶性好,具良好的生物相容性,降低了毒副作用,而且能够提高机体特异性免疫应答,是理想的佐剂。
Description
本发明属于生物医用材料领域,具体涉及双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其应用。
接种疫苗被认为是当今社会预防传染病最经济、最方便和最有效的方法之一。在免疫过程中,抗原递送是至关重要的一步。在疫苗的实际使用过程中,由于许多抗原单独施用时不足以赋予免疫性抗体应答,这就需要设计能固定抗原并刺激免疫应答的佐剂。
Toll样受体(Toll-like receptor,TLR)是一类模式识别受体(Pattern recognition receptor,PRR),这种受体在受到微生物特殊的保守产物-病原体相关分子模式(Pathogenassociated molecular patterns,PAMPs)激活时,不仅会诱导天然免疫应答,同时还能激活获得性免疫系统,是理论上佐剂的理想选择。近年来,人工合成的咪喹莫特(R837)作为TLR 7激动剂,是小分子免疫调节剂,具有优异的抗病毒、抗肿瘤能力。因为相对分子量小,可以通过多种途径进入体内,提高抗原呈递的细胞活性,聚集较多的树突状细胞、巨噬细胞、B细胞和T细胞等到接种位点,增强局部免疫反应。可是,咪喹莫特也存在以下缺陷:(1)在水中以及常见有机溶剂中溶解度较小,因此不易制备成注射剂,对细胞有一定的毒性;(2)单一使用R837会产生一些不良反应,例如红斑、糜烂、剥脱/剥落和水肿等属于常见不良反应;(3)咪喹莫特的药代动力学具有从局部(例如皮下或肌肉内)迅速向全身扩散等特点,从而在多个远端组织引起不必要的内在免疫激活。由于这些缺点的存在,使其应用受到了限制。
发明内容
本发明的目的是提供双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,不仅水溶性好,具良好的生物相容性,降低了毒副作用,而且能够提高机体特异性免疫应答,是理想的佐剂。
本发明的再一目的是提供双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯在疫苗佐剂方面的用途。
本发明的目的采用如下技术方案实现:
双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,采用包括如下步骤的方法制备:
(1)在无水氛围下,将γ-聚谷氨酸分散在非质子溶剂中,然后加入催化剂N,N-二甲基甲酰胺,在搅拌状态下,滴入氯化剂,反应10-40小时;
(2)将咪喹莫特、脂溶性醇和缚酸剂加入步骤(1)反应后所得溶液中,反应42-54小时;
(3)纯化后,得到双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯材料。
在本发明中,γ-聚谷氨酸的分子量为1-200万,优选为30-70万;所述氯化剂为氯化亚砜、草酰氯或五氯化磷,优选氯化亚砜和草酰氯;脂溶性醇为C8-C24醇、脂环醇或者甾醇,优选正月桂醇或胆固醇;缚酸剂为三乙胺、4-N,N-二甲氨基吡啶、吡啶、无水碳酸铯、无水碳酸钾、无水碳酸钠、氢氧化钠和氢氧化钾中的一种。
在本发明中,非质子溶剂中为二氯甲烷、氯仿、乙腈、二甲亚砜、N,N-二甲基甲酰胺、四氢呋喃、1,4-二氧六环和甲苯中的一种;所述非质子溶剂优选为二氯甲烷或乙腈。
在本发明中,γ-聚谷氨酸、咪喹莫特、脂溶性醇和缚酸剂的摩尔比为10:0.5-1.5:1-3:10-15;步骤(1)中γ-聚谷氨酸与氯化剂的物质的量之比为1:(1~2.5),优选为1:1,反应时间为20-25小时。
在本发明中,步骤(1)、(2)的反应温度均为10-40℃;优选20-25℃。
在本发明中,步骤(1)中每克γ-聚谷氨酸分散在5~70mL的非质子溶剂中。
在本发明中,所述制备方法还包括采用荧光素进行标记的步骤;所述荧光素为氨基荧光素或氨基罗丹明,优选5-氨基荧光素。
在本发明中,纯化步骤如下:除去溶剂,残留固体用无水丙酮、甲醇、乙醇或乙腈浸泡,过滤、洗涤和真空干燥。
本发明还提供所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯作为疫苗佐剂方面的用途。
在本发明中,所述疫苗为手足口病、禽流感、新城疫、伪狂犬、猪细小、猪瘟和猪蓝耳疫苗;所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯与抗原的质量比为(0.01~1):(0.5~1)。
为了克服现有的R837免疫佐剂水溶性差和毒副作用较大的缺陷,本发明利用γ-聚谷氨酸作为亲水性聚合物骨架,通过酰胺共价键偶联5-氨基荧光素、R837,通过酯键偶联憎水性脂溶性醇(如正月桂醇),形成双亲性聚合物FIP(FL-γ-PGA-R837-LA)。通常对γ-PGA进行修饰是鉴于其羧基在EDC/NHS活化下形成酯和酰胺。由于咪喹莫特的氨基很不活泼,难以用该方法进行酰胺化偶联。因此,先将γ-PGA的羧基用氯化亚砜、草酰氯或五氯化磷在N,N二甲基甲酰胺催化下制成高活性的酰氯,然后与咪喹莫特的氨基反应,成功地通过酰胺键进行偶联。通过Scifinder和Web of Science文献搜索,发现将γ-PGA的羧酸基制成酰氯后再进行酯化或酰胺化的方法也未曾报导。其原因可能是氯化的条件不易控制,反应体系很容易碳化发黑。本发明通过控制反应温度和氯化剂加入的速度,成功地实现酰氯的产生。因此,本发明FIP和γ-PGA-R837-LA的制备方法简单,巧妙。由于原料来源丰富,生物安全性良好,价格低,因此本发明中的FIP和γ-PGA-R837-LA成本较低。
本发明制备的FIP和γ-PGA-R837-LA,不仅水溶性好,具良好的生物相容性,降低了毒副作用,而且在显著减少了咪喹莫特用量的情况下,可以有效地刺激机体的免疫应答, 提高IgG的分泌水平,可用于疫苗、载药、探针等领域。动物实验显示,接种含有FIP的疫苗的小鼠的注射部位皮肤、肝、脾和肾均未发现病变问题,证明实验组小鼠体征指标正常,无不良副反应。含有FIP的不同剂型的疫苗接种小鼠后,效价水平均有明显的上升,在首免后6周,效价约达到仅含有OVA的疫苗的2倍,说明FIP是一种很好的水剂型佐剂,安全性更好,是一种较为理想多剂型佐剂。FIP中含有荧光基团,可用于生物体内/外荧光追踪和定量,直观的确定体液免疫和细胞免疫之间的联系。本发明FIP可以制成W、O/W、W/O、W/O/W剂型的疫苗,均能够显著提高免疫效力。由于FIP是两亲性高分子聚合物,因此能在水或者油相介质中通过分子间作用力自组装形成纳米微粒(胶束或囊泡),为药物和疫苗载体的制备提供了一种新的方法。FIP(即FL-γ-PGA-R837-LA)能与多种抗原物理混合,制成粘膜给药疫苗,可进行滴鼻和口服给药。
图1为双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(γ-PGA-R837-LA)合成路线;
图2为荧光素标记的双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FIP,FL-γ-PGA-R837-LA)合成路线及5-氨基荧光素的化学结构式;
图3为荧光素标记的双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FL-γ-PGA-R837-LA)
1H NMR图;
图4为荧光素标记的双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FL-γ-PGA-R837-LA)的UV-vis表征图;
图5为FIP、咪喹莫特、荧光素的荧光光谱表征图,其中a是FIP与咪喹莫特的荧光光谱表征图,b是FIP和荧光素的荧光光谱表征图;
图6为荧光素标记的双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FL-γ-PGA-R837-LA)与γ-聚谷氨酸(γ-PGA)的Zeta电位图;
图7为不同浓度的荧光素标记的双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FL-γ-PGA-R837-LA)对RAW 264.7细胞的存活率实验结果,横坐标为浓度,纵坐标为细胞存活率(%),FIP为不同浓度FL-γ-PGA-R837-LA溶液;IMQ是不同浓度R837溶液;HAc为pH 6.0的醋酸水溶液;PBS为PBS缓冲液;
图8为RAW 264.7细胞吞噬荧光素标记的双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FL-γ-PGA-R837-LA)的荧光流式图;a是浓度为50μg/mL的FIP干预细胞的FSC-SSC散点图,横坐标是前向角度散射光强度,纵坐标是侧向散射光强度;b是浓度为50μg/mL的FIP干预细胞的单参数直方图,横坐标是荧光信号的值;c是不同浓度的FIP干预细胞的平均荧光图,横坐标为FIP浓度,纵坐标为荧光强度;
图9为小鼠免疫后5天皮肤情况的照片,虚线框内为接种部位所在区域;其中a图中 小鼠皮下注射含有咪喹莫特(R837,IMQ)的疫苗,b图中小鼠皮下注射含有FIP的疫苗;
图10为FIP和咪喹莫特(R837,IMQ)作为佐剂的免疫效果对比图;
图11为接种小鼠的注射部位皮肤及内脏(如肝、肾、脾)的组织学检测,其中第一行是接种PBS缓冲液的小鼠,第二行是接种疫苗1的小鼠,a为注射位点皮肤,b为肝,c为脾和d为肾;
图12为接种FIP/OVA不同剂型疫苗的小鼠血清IgG抗体水平,其中图A是接种两周后抗体效价,图B是接种六周后血清抗体效价。FIP/OVA是各疫苗(同时含有FIP和OVA),OVA是各阳性对照疫苗(仅含有OVA)。
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其荧光素标记物的制备及鉴定
1.双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(缩写为γ-PGA-R837-LA)的制备
γ-PGA-R837-LA的制备方法,包括如下步骤:
(1)将0.1mol(13g)的γ-聚谷氨酸(γ-PGA,MW=700000,轩凯生物科技有限公司),分散在200mL无水二氯甲烷(DCM)中,然后加入0.5mL的N,N-二甲基甲酰胺(DMF)作为催化剂,在室温(20-25℃)搅拌状态下,滴入0.1mol(7.3mL)的氯化亚砜(SOCl
2),滴速为2秒/滴,体系为无水操作;滴加结束后,反应24小时,反应过程中产生的尾气用10%NaOH水溶液吸收。
(2)将0.01mol(2.4g)的咪喹莫特(R837)、0.02mol(3.72g)正月桂醇(LA)和0.12mol(12g)缚酸剂三乙胺(NEt
3)加入到步骤(1)反应后所得溶液中,室温(20-25℃)反应48小时。
(3)旋转蒸发除去溶剂,残留固体用无水甲醇浸泡,过滤取滤渣、用水洗涤、真空干燥,得到双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯。
反应原理见图1。
2.荧光素标记双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(缩写为FL-γ-PGA-R837-LA,缩写为FIP)的制备
FIP的制备方法,包括如下步骤:
(1)将0.1mol(13g)γ-聚谷氨酸(γ-PGA,MW=700000,轩凯生物科技有限公司),分散在200mL无水二氯甲烷(DCM)中,然后加入0.5mL的N,N-二甲基甲酰胺(DMF)作为催化剂,在室温(20-25℃)搅拌状态下,滴入0.1mol(7.3mL)的氯化亚砜(SOCl
2),滴速为2秒/滴,体系为无水操作;滴加结束后,反应24小时,反应过程中产生的尾气用 10%NaOH水溶液吸收;
(2)将0.01mol(2.4g)的咪喹莫特(R837)、0.02mol(3.72g)正月桂醇(LA)、0.5mmol(0.17g)5-氨基荧光素(缩写为5-NH
2-FL)和0.12mol(12g)缚酸剂三乙胺(NEt
3)加入到步骤(1)反应后所得溶液中,室温(20-25℃)反应48小时。
(3)旋转蒸发除去溶剂,残留固体用无水甲醇浸泡,过滤取滤渣,用水洗涤、真空干燥,得到荧光素标记的双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FL-γ-PGA-R837-LA)。
反应原理见图2。
3.物质鉴定
(1)核磁检测
将FL-γ-PGA-R837-LA溶解于DMSO-d
6中,用于核磁共振氢谱(
1H NMR)的表征。同时,γ-聚谷氨酸(溶于D
2O)和R837(溶于DMSO-d
6)作为对照,评价R837的嫁接与定量。核磁检测结果见图3,FL-γ-PGA-R837-LA的
1H NMR谱图中,7-9ppm为R837芳环区氢的化学位移,说明R837已经嫁接到γ-聚谷氨酸骨架上,同时与γ-聚谷氨酸积分面积比表明嫁接率(R837在FL-γ-PGA-R837-LA中的质量百分含量)为10%左右;高场区出现的峰表明,正月桂醇也嫁接到γ-聚谷氨酸骨架上。由于FL-γ-PGA-R837-LA中荧光素含量很低,在
1H NMR谱上几乎被噪音淹没。
(2)紫外-可见(UV-vis)检测
UV-vis检测灵敏度高。由于嫁接的荧光素含量很低,核磁共振氢谱(
1H NMR)难以检测出。为了确定R837和5-氨基荧光素是否被嫁接上,将FL-γ-PGA-R837-LA溶于超纯水,用UV-vis(紫外-可见光分光光度计)检测FL-γ-PGA-R837-LA中的主要成分;将R837溶于盐酸酸化的超纯水(pH 6),作为对照1;以5-氨基荧光素水溶液作为对照2。在200-600nm的波长下检测吸光度,将检测结果归一化后比较。结果如图4,结合FL-γ-PGA-R837-LA和其他单一成分的紫外图中出现200-250nm处的R837特征峰、250-300nm处的γ-PGA的特征峰和475-525nm处的5-FL的特征峰,证明了R837、荧光素均嫁接到γ-PGA骨架上,验证了FL-γ-PGA-R837-LA的结构。
(3)荧光(FL)检测
为了进一步确证R837和5-氨基荧光素是否被嫁接到γ-PGA侧链上,将FL-γ-PGA-R837-LA溶于超纯水,用荧光分光光度计检测。将R837溶于盐酸酸化的超纯水(pH 6)溶液,作为对照1;以5-氨基荧光素水溶液,作为对照2。结果如图5,当使用激发波长(λ
Ex)为280nm的光激发R837发色团时,归一化后,发现FL-γ-PGA-R837-LA和R837的R837发射峰(λ
Em=340nm)峰形几乎形同,仅有3nm的位移,这表明R837已经被嫁接到γ-PGA侧链上;当使用激发波长(λ
Ex)为455nm的光激发荧光素发色团时, FL-γ-PGA-R837-LA出现荧光素发射峰(λ
Em=519nm),归一化后发现,FL-γ-PGA-R837-LA和5-氨基荧光素的最大发射峰仅有4nm的位移,但两者峰形几乎相同,因此,5-氨基荧光素已经被嫁接到γ-PGA侧链上。
(4)Zeta电位检测
Zeta电位值的正负和大小对应着着物质结构的稳定性和正负电荷。从图6可见,FL-γ-PGA-R837-LA的Zeta电位为-3.3mV,说明FL-γ-PGA-R837-LA能快速凝结,如果浓度过大,则形成凝胶状物质。
(5)溶解性检测
将R837、γ-PGA-R837-LA和FL-γ-PGA-R837-LA分别溶于水相(例如,超纯水,0.1M pH为7.4的PBS缓冲液、0.9%生理盐水,5%葡萄糖水溶液和pH 1~6的酸性水溶液,SBF模拟体液)和溶剂相(乙醇、DMSO、DMF)中,对比观察其溶解度,评价其水佐剂的可行性及注射缓冲液的选择。
结果:20毫克的γ-PGA-R837-LA和FL-γ-PGA-R837-LA在5mL的超纯水、0.1M、pH为7.4的PBS缓冲液、0.9%生理盐水、5%葡萄糖水溶液、pH 1~6的酸性水溶液和SBF模拟体液中均全部溶解;20毫克的γ-PGA-R837-LA和FL-γ-PGA-R837-LA在3mL的DMSO、乙醇、DMF和丙酮均全部溶解。因此γ-PGA-R837-LA和FL-γ-PGA-R837-LA是双亲性材料,有望于制备水佐剂。
R837不能溶解在超纯水、0.1M、pH为7.4的PBS缓冲液、0.9%生理盐水、5%葡萄糖水溶液和SBF模拟体液中,仅溶解在pH≤6以下的酸性水溶液中;R837微溶于热的DMSO和DMF溶液中。因此,限制了它在水佐剂类的应用。
实施例2 FIP的体外实验、FIP与R837免疫效果比较
(1)FIP的体外细胞毒性测试
以pH 7.4的PBS缓冲液为溶剂,配制不同浓度的FIP溶液。以pH 6的醋酸水溶液为溶剂,配制不同浓度的R837溶液。考察FIP溶液、R837溶液对细胞的毒性,以pH 6.0的醋酸水溶液为阴性对照,以pH 7.4的PBS缓冲液为空白对照,采用小鼠巨噬细胞RAW 264.7作为模式源,利用CCK-8法进行毒性检测,具体方法如下:在96孔板上铺上RAW 264.7细胞,在37℃、5%CO
2/95%O
2的生物环境下孵育24小时,显微镜观察,当细胞计数达到2×10
6cells/孔时,每孔加入100μL不同浓度的FIP溶液或R837溶液,继续孵育24小时,然后加入CCK-8试剂(细胞增殖试剂盒中试剂,购自百赛生物),孵育4小时,采用酶标仪(BioTek酶标仪)检测细胞凋亡程度。结果:如图7所示,当FIP溶液浓度为1000μg/mL(所含R837成分浓度为100μg/mL)时,细胞仍保持着88%的存活率,FIP溶液浓度 小于1000μg/mL时,细胞无凋亡现象,细胞存活率与PBS缓冲液处理的细胞类似;相反,R837溶液对细胞伤害较大,当浓度为100μg/mL时,大量的RAW 264.7细胞发生了凋亡。因此,FIP具有良好的生物相容性和低毒性,可用于疫苗、载药、探针等领域。
(2)FIP/OVA纯水剂型的荧光流式测定
以pH 7.4的PBS缓冲液为溶剂,配制50μg/mL的FIP溶液,其中R837的浓度为5μg/mL。
在24孔细胞板上铺上RAW 264.7细胞,孵育18小时,细胞数量达到2×10
6cells/孔,每孔加入100μL、50μg/mL的FIP溶液。继续孵育24小时后,吹打细胞,用PBS洗涤离心三次,最后用冰的PBS重悬后用流式细胞仪(BD FACSCalibur流式细胞仪)进行检测。另外,采用上述相同方法考察不同浓度FIP溶液对RAW 264.7细胞的影响。结果如图8所示,当FIP浓度为50μg/mL时,RAW 264.7细胞仍能看到明显的荧光峰,其未分化的CD
3+细胞展现较强的活性。随着FIP浓度的增加,其细胞的荧光强度逐渐增高。这些结果说明了FIP很容易被细胞摄入,能够在生物体内的组织器官中呈现荧光标记,通过不同的时间段进行为追踪免疫路径提供了帮助。
(3)FIP与R837免疫效果比较
以pH 7.4的PBS缓冲液为溶剂,分别配制1000μg/mL的OVA(卵清蛋白)溶液和200μg/mL的FIP溶液,然后将两溶液等体积混合,得到含有FIP的疫苗。
以pH 6的醋酸水溶液为溶剂,分别配制1000μg/mL的OVA溶液和200μg/mL的R837溶液,然后将两溶液等体积混合,得到含有R837的疫苗。
另外,以pH 7.4的PBS缓冲液为溶剂,配制500μg/mL的OVA溶液(缩写为OVA);以pH 7.4的PBS缓冲液为溶剂,配制100μg/mL的FIP溶液(缩写为FIP)。
将6-8周龄的雌性Balb/c小鼠(20-25g)随机分成5组,每组小鼠有6只,分别接种含有FIP的疫苗、含有R837的疫苗、500μg/mL的OVA溶液、100μg/mL的FIP溶液和pH 7.4的PBS缓冲液,接种剂量均为200μL疫苗/只。接种后,一周内观察注射部位皮肤状况。接种后28天,眼眶采血,分离血清,检测抗体效价。
抗体效价采用如下方法检测:以pH 9.6、0.05mol/L的Na
2CO
3-NaHCO
3缓冲液为溶剂,配制10μg/mL的OVA蛋白溶液。取50μL的OVA蛋白溶液包被96孔板,4℃过夜吸附;弃液,使用PBST缓冲液(在1L、0.1M的pH 7.4的PBS缓冲液中添加500μL的吐温-20所得)洗板2次,置于干净吸水纸上拍干;每孔加入100μL的封闭液(1L、0.1M的pH 7.4 PBS缓冲液中添加10g的BSA后所得)后封膜,置于37℃摇床孵育1小时,然后用PBST缓冲液洗板2次,置于干净吸水纸上拍干;加入100μL用PBST缓冲液稀释后的小鼠血清,37℃避光孵育1.5小时,弃去溶液,加入PBST缓冲液洗板5次,置于干净吸水纸上拍干; 每孔加入50μL HRP标记的山羊抗小鼠IgG二抗(购自碧云天,产品编号A0216),37℃避光孵育1小时,弃去溶液,加入PBST缓冲液洗板5次,置于干净吸水纸上拍干;每孔加入50μL的显色液(将购自英创生物的双组分TMB显色液中的A溶液和B溶液按照体积比为1:1混合)在37℃避光孵育30分钟,最后每孔加入50μL终止液(2mol/L的H
2SO
4水溶液),用酶标仪在450nm处检测吸光度。
结果:接种含有R837的疫苗的小鼠,出现了小面积的皮肤溃烂、红肿、化脓等不良反应,见图9;接种含有FIP的疫苗的小鼠,被毛柔顺,饮食稳定,生命体征正常,注射部位没有红肿,鼓包等炎症反应。由图10可见,虽然咪喹莫特作为佐剂时IgG效价最高(OD
450
nm=3.07),但是FIP作为水佐剂时仍展现较强的效价(OD
450nm=2.1)。由于含有FIP的疫苗中咪喹莫特成分仅有10μg/mL,仅是含有R837的疫苗中的10%,因此FIP作为水佐剂,不仅水溶性好,降低了毒副作用,而且在显著减少了咪喹莫特用量的情况下,可以有效地刺激机体的免疫应答,提高IgG的分泌水平。
由于上述免疫实验中咪喹莫特结构的浓度仅有10μg/mL,小鼠注射部位没有红肿,鼓包等炎症反应,为了验证高浓度下是否存在毒副作用,以pH 7.4的PBS缓冲液为溶剂,分别配制1000μg/mL的OVA溶液和1000μg/mL的FIP(即FL-γ-PGA-R837-LA)溶液,然后将两溶液等体积混合,得到含有1000μg/mL的FIP的疫苗。采用上述相同方法进行接种,一周内,小鼠被毛柔顺,饮食稳定,生命体征正常,注射部位没有红肿,鼓包等炎症反应。因此FIP无毒副作用。
实施例3免疫实验
本实施例说明实施例1制备的荧光素标记双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯(FL-γ-PGA-R837-LA,简称FIP),作为疫苗免疫佐剂的应用。
一、制备疫苗制剂
以卵清蛋白(OVA)为模式抗原,不同配方的疫苗按表1进行配制,然后对小鼠进行皮下注射。
表1.各疫苗组成及免疫剂量
疫苗编号或组成 | OVA浓度 | FIP浓度 | 剂型 | 注射用量 |
0.1M、pH 7.4的PBS缓冲液 | 0 | 0 | W | 200μL |
阳性对照疫苗1 | 500μg/mL | 0 | W | 200μL |
阳性对照疫苗2 | 500μg/mL | 0 | O/W | 200μL |
阳性对照疫苗3 | 500μg/mL | 0 | W/O | 200μL |
阳性对照疫苗4 | 500μg/mL | 0 | W/O/W | 200μL |
疫苗1 | 500μg/mL | 100μg/mL | W | 200μL |
疫苗2 | 500μg/mL | 100μg/mL | O/W | 200μL |
疫苗3 | 500μg/mL | 100μg/mL | W/O | 200μL |
疫苗4 | 500μg/mL | 100μg/mL | W/O/W | 200μL |
制备FIP/OVA水剂型疫苗(记为疫苗1):以0.1M的PBS(pH 7.4)缓冲液作为溶剂,分别配制1000μg/mL的OVA溶液和200μg/mL的FIP(即FL-γ-PGA-R837-LA)溶液,然后将两溶液等体积混合,制备成FIP/OVA水剂型疫苗(记为疫苗1)。疫苗1中,FIP浓度为100μg/mL,OVA浓度为500μg/mL。按疫苗1的制备方法制备阳性对照疫苗1,不同之处仅在于以水替代FIP溶液。
制备FIP/OVA水包油剂型疫苗(记为疫苗2):以中国专利ZL201310021011.3中配方4作为油相;以0.1M的PBS(pH 7.4)缓冲液作为溶剂,配制含有OVA和FIP的水相;将水相与油相按照体积比1:1混合、高压均质化处理,得到FIP/OVA水包油剂型疫苗(记为疫苗2)。该疫苗中FIP的浓度为100μg/mL,OVA浓度为500μg/mL。按照FIP/OVA水包油剂型疫苗的制备方法制备阳性对照疫苗2,不同之处仅在于该疫苗中不含有FIP。
制备FIP/OVA油包水剂型疫苗(记为疫苗3):以0.1M的PBS(pH 7.4)缓冲液作为溶剂,配制含有OVA和FIP的水相;将水相与白油按照体积比为1:3混合、乳化,得到FIP/OVA油包水剂型疫苗(记为疫苗3)。疫苗3中OVA的浓度为500μg/mL,FIP的浓度为100μg/mL。按疫苗3的制备方法制备阳性对照疫苗3,不同之处仅在于水相中不含有FIP。
制备水包油包水(W/O/W)剂型疫苗:以0.1M的PBS(pH 7.4)缓冲液作为溶剂,配制含有FIP和OVA的水相;将水相与ISA201按照体积比为1:1混合、乳化,得到FIP/OVA水包油包水剂型疫苗(记为疫苗4),该疫苗中FIP的浓度为100μg/mL,OVA浓度为500μg/mL。按照疫苗4的制备方法制备阳性对照疫苗4,不同之处仅在于水相中不含有FIP。
二、小鼠免疫接种方案
将6-8周龄的雌性Balb/c小鼠(20-25g)随机分成9组,每组小鼠有6只,其中8组小鼠分别免疫阳性对照疫苗1-4、疫苗1-4(表1),剩余一组小鼠接种0.1M、pH 7.4的PBS缓冲液作为阴性对照。各疫苗的接种方法如下:总共接种两次,在第1天和14天接种疫苗,接种剂量均为200μL疫苗/只。
三、各项生化和免疫指标的测定
(1)首免后28天组织H&E染色毒副作用测定
将接种PBS缓冲液和疫苗1的两组小鼠,在首免后28天,进行接种位点、肝、肾、脾组织切片,观察是否有损伤和病变等问题。具体方法如下:每组取3只小鼠进行安乐死处理,并手术分离出注射部位皮肤及内脏(如肝、肾、脾),用4%多聚甲醛(购买于雷 根生物,产品货号DF0135)固定浸泡,石蜡包埋,切片处理,进行免疫组织化学染色。结果如图11所示,与接种PBS缓冲液的小鼠对比,接种疫苗1小鼠的注射部位皮肤、肝、脾和肾均未发现病变问题,证明实验组小鼠体征指标正常,无不良副反应。
(2)IgG抗体效价测定
首免后每隔两周眼眶采血,分离血清,按照实施例2中方法测定各小鼠血清中的特异性抗体(IgG)水平。
结果如图12所示,通过与不同剂型、仅含有OVA的阳性对照疫苗对比,可以发现含有FIP的疫苗具有更强的免疫应答能力。针对兽用疫苗的市场中剂型类型进行抗体效价检测,通过对小鼠进行免疫效价评估,在首免后2周、6周采血,利用ELSIA法进行判定其抗体效价,对比抗体效价,可以发现O/W>W/O/W>W/O>W,随着免疫时间的增加,含有FIP的不同剂型的疫苗效价水平均有明显的上升,约达到相应对照阳性疫苗的2倍,说明FIP是一种很好的水剂型佐剂,安全性更好,是一种较为理想多剂型佐剂。
综上所述,申请人首次利用酰胺键将咪喹莫特与γ-聚谷氨酸共价键偶联,并嫁接了脂溶性基团和荧光发色团修饰的FL-γ-PGA-R837-LA聚合物。FL-γ-PGA-R837-LA具有良好的生物相容性和双亲溶解性。小鼠免疫研究表明,OVA作为模型抗原,使用γ-PGA-R837-LA或FL-γ-PGA-R837-LA作为免疫佐剂,能够高效、持久地促进抗原特异性体液和细胞免疫应答;可以通过荧光标记追踪疫苗的进入、刺激、转运及代谢过程;可以制成不同的剂型,用于皮下注射、肌肉注射、鼻腔或口服给药,为免疫剂型设计和选择提供了可行性方案。因此,γ-PGA-R837-LA和FL-γ-PGA-R837-LA作为免疫佐剂在免疫治疗领域有着重要的应用价值。
Claims (10)
- 双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,采用包括如下步骤的方法制备:(1)在无水氛围下,将γ-聚谷氨酸分散在非质子溶剂中,然后加入催化剂N,N-二甲基甲酰胺,在搅拌状态下,滴入氯化剂,反应10-40小时;(2)将咪喹莫特、脂溶性醇和缚酸剂加入步骤(1)反应后所得溶液中,反应42-54小时;(3)纯化后,得到双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯材料。
- 根据权利要求1所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,其特征在于γ-聚谷氨酸的分子量为1-200万,优选为30-70万;所述氯化剂为氯化亚砜、草酰氯或五氯化磷,优选氯化亚砜和草酰氯;脂溶性醇为C8-C24醇、脂环醇或者甾醇,优选正月桂醇或胆固醇;缚酸剂为三乙胺、4-N,N-二甲氨基吡啶、吡啶、无水碳酸铯、无水碳酸钾、无水碳酸钠、氢氧化钠和氢氧化钾中的一种。
- 根据权利要求1或2所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,其特征在于非质子溶剂中为二氯甲烷、氯仿、乙腈、二甲亚砜、N,N-二甲基甲酰胺、四氢呋喃、1,4-二氧六环和甲苯中的一种;所述非质子溶剂优选为二氯甲烷或乙腈。
- 根据权利要求3所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,其特征在于γ-聚谷氨酸、咪喹莫特、脂溶性醇和缚酸剂的摩尔比为10:0.5-1.5:1-3:10-15;步骤(1)中γ-聚谷氨酸与氯化剂的物质的量之比为1:(1~2.5),优选为1:1,反应时间为20-25小时。
- 根据权利要求4所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,其特征在于步骤(1)、(2)的反应温度均为10-40℃;优选20-25℃。
- 根据权利要求5所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,其特征在于步骤(1)中每克γ-聚谷氨酸分散在5~70mL的非质子溶剂中。
- 根据权利要求6所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,其特征在于所述制备方法还包括采用荧光素进行标记的步骤;所述荧光素为氨基荧光素或氨基罗丹明,优选5-氨基荧光素。
- 根据权利要求7所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯,其特征在于纯化步骤如下:除去溶剂,残留固体用无水丙酮、甲醇、乙醇或乙腈浸泡,过滤、洗涤和真空干燥。
- 权利要求1所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯作为疫苗佐剂方面的用途。
- 根据权利要求9所述用途,其特征在于所述疫苗为手足口病、禽流感、新城疫、伪狂犬、猪细小、猪瘟和猪蓝耳疫苗;所述双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯与抗原的质量比为(0.01~1):(0.5~1)。
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