CN107200788B - Quaternary phosphonium chitosan and application thereof as vaccine immunologic adjuvant - Google Patents
Quaternary phosphonium chitosan and application thereof as vaccine immunologic adjuvant Download PDFInfo
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- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
The invention discloses quaternary phosphonium chitosan and application thereof as vaccine immunologic adjuvant, and a preparation method of the quaternary phosphonium chitosan comprises the following steps: adding chitosan and 1-hydroxybenzotriazole into a solvent, carrying out ice-bath reaction, and then adjusting the pH value to 4.8-5.5; then adding 3-carboxypropyl triphenyl phosphonium bromide, and stirring until the 3-carboxypropyl triphenyl phosphonium bromide is completely dissolved; dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in a solvent, adding the solution into the reaction system, precipitating the product after reaction, drying, dialyzing, and freeze-drying to obtain the quaternary phosphonium chitosan. The invention adopts quaternary phosphonium chitosan as an immunologic adjuvant for the first time, and the quaternary phosphonium chitosan can promote the uptake of DC2.4 cells to antigens, promote the proliferation of spleen cells and induce higher levels of IgG antibody titer, IFN-gamma, IL-4 and IL-10 with antigen specificity, thereby having important application value in the field of vaccine treatment.
Description
Technical Field
The invention belongs to the field of biomedical materials, and relates to quaternary phosphonium chitosan and application thereof as a vaccine immunologic adjuvant.
Background
Vaccination is one of the most important inventions recognized in recent modern human medical development history and is still the most effective method for human to prevent, fight and control infectious diseases such as influenza, hepatitis, tuberculosis and the like.
The original vaccine was made of the complete pathogen or its lysate and was well immunogenic but resulted in adverse symptoms and infection. Thus, several safer vaccines are being researched and developed, including DNA, subunit, and recombinant vaccines. However, the novel vaccines still have the defects of low immunogenicity, weak immune response induction, weak targeting property and the like. Therefore, the exploration and development of the technology for enhancing the efficacy of the vaccine are of great significance, and the design of the immune adjuvant which is beneficial to enhancing the immunogenicity, improving the safety and enhancing the specific immune response of the organism becomes a primary task.
Since 1925, immune adjuvant (Immunoadjuvant) has attracted the researchers' extensive attention and great interest. Immunoadjuvants are a class of non-specific immunostimulatory molecules that help deliver antigen, activate Antigen Presenting Cells (APCs), and trigger activation and differentiation of immune cells in the body.
An ideal immunoadjuvant must have the following characteristics: 1. the safety is good; 2. enhancing the immunogenicity of a weakly immunogenic vaccine; 3. enhancing immune contact and enhancing immune response of the body; 4. reducing the antigen inoculation dose and the inoculation times; 5. speeding up the speed and extending the duration of the immune response, etc.
Common immunological adjuvants include aluminum adjuvant and Freund's adjuvant. Aluminum adjuvant is the first human adjuvant approved by FDA for clinical treatment, but it is less able to induce cellular immunity and can cause local inflammation. The Freund's adjuvant is mainly used as an animal vaccine adjuvant and has large toxic and side effects. Therefore, the research and development of novel and safe immunologic adjuvants capable of effectively inducing cellular and humoral immunity are of great significance.
The chitosan and the derivatives thereof have the advantages of good biocompatibility, biodegradability, biological adhesion, immunostimulation activity, slow release and controlled release, targeting property and the like, and have become a hot research point of immune adjuvants worldwide in recent years. However, chitosan is poorly water soluble and only soluble in dilute acids, which severely limits its application in the biomedical field.
Disclosure of Invention
In order to overcome the problem of poor water solubility of the existing chitosan and derivatives thereof, the invention aims to provide quaternary phosphonium chitosan which is synthesized by an amide reaction, has good water solubility and can be dissolved in water and physiological saline.
In order to overcome the defects of weak cell immunity induction capability and large toxic and side effects of the existing immunologic adjuvant, the invention also aims to provide the application of the quaternary phosphonium chitosan as the vaccine immunologic adjuvant, and the quaternary phosphonium chitosan as the vaccine immunologic adjuvant can improve specific immune response of organisms.
The purpose of the invention is realized by the following technical scheme:
a preparation method of quaternary phosphonium chitosan comprises the following steps:
(1) adding Chitosan (CS) and 1-hydroxybenzotriazole (HOBt) into the water/dimethyl sulfoxide mixed solution, stirring for 2-3h under an ice bath condition, and then adjusting the pH value of the mixture to 4.8-5.5; adding 3-Carboxypropyl Triphenyl Phosphonium Bromide (CTPB), and stirring until the CTPB is completely dissolved;
(2) dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC & HCl) in a water/dimethyl sulfoxide mixed solution, adding the solution into the reaction system in the step (1), reacting for 1-2 days, precipitating the product by using an acetone/diethyl ether mixed solution, drying the precipitated product in vacuum for 1-2 days, dissolving the product in ultrapure water, dialyzing for 3-4 days in the ultrapure water, and freeze-drying to obtain quaternary phosphonium chitosan (NPCS);
in the step, the molar ratio of the sugar unit of the chitosan to the HOBt, CTPB and EDC & HCl is 1:2:1: 2;
in the water/dimethyl sulfoxide mixed solution, the volume ratio of water to dimethyl sulfoxide is preferably (1-2) to 1;
in the acetone/diethyl ether mixed solution, the volume ratio of acetone to diethyl ether is preferably 2: 1.
The quaternary phosphonium chitosan prepared by the method has improved water solubility and can be dissolved in water and physiological saline.
Because of good biocompatibility and immunostimulation activity and improved water solubility, the quaternary phosphonium chitosan can be used as a vaccine immunologic adjuvant, and can obviously improve the specific immune response of an organism;
in the vaccine, the mass ratio of the quaternary phosphonium chitosan to the antigen is 2: 1;
the antigen is preferably Ovalbumin (OVA), and the immunization mode of vaccine therapy is leg intramuscular injection.
Chitosan has unusual application potential in the field of immune adjuvants, but the poor water solubility limits the application of the chitosan. Therefore, the water solubility of chitosan grafted with small molecular quaternary phosphonium salt is greatly improved.
The quaternary phosphonium chitosan used as adjuvant in vaccine therapy has the following advantages: 1. the source is rich, the preparation is simple, and the biological safety is good; 2. the water solubility is good, so that the production and preparation of the immune vaccine preparation are facilitated; 3. the biological adhesion is good, the biological adhesion is rich in positive charge, the antigen with negative charge can be efficiently wrapped and adhered to a cell membrane with negative charge through electrostatic interaction, so that the antigen is more effectively recognized and taken up by an Antigen Presenting Cell (APC); 4. the nano-size is easy to be recognized and taken by APC (APC), is easy to be phagocytized by cells, and is beneficial to inducing the immune response of an organism; 5. can effectively protect the antigen from being damaged by acid, alkali, protease and the like in vivo and improve the utilization rate of the antigen.
The research of the invention finds that when the quaternary phosphonium chitosan/antigen vaccine preparation is immunized in an organism, the vaccine delivery system can slowly release the antigen, so that the organism continuously receives immune stimulation to induce long-term effective immune response.
Compared with the prior art, the invention has the following advantages and effects:
1. the quaternary phosphonium chitosan has good biocompatibility, simple synthesis, low cost, economy and environmental protection. Meanwhile, the modified quaternary phosphonium chitosan has good water solubility and is convenient for preparing vaccine preparations.
2. The invention adopts quaternary phosphonium chitosan as an immunologic adjuvant for the first time, and the quaternary phosphonium chitosan can promote the uptake of DC2.4 cells to antigens, promote the proliferation of spleen cells and induce higher levels of IgG antibody titer, IFN-gamma, IL-4 and IL-10 with antigen specificity, thereby having important application value in the field of vaccine treatment.
Drawings
FIG. 1 is a graph showing the results of contact angle experiments for quaternary phosphonium chitosan and chitosan.
FIG. 2 is a graph showing the results of OVA uptake by DC2.4 cells.
FIG. 3 is a graph of the results of immunohistochemical staining.
FIG. 4 is a graph showing the results of measurement of IgG antibody titer.
FIG. 5 is a graph showing the results of in vitro proliferation experiments of splenocytes.
FIG. 6 is a graph showing the results of IFN-. gamma.secretion experiments.
FIG. 7 is a graph showing the results of IL-12 secretion experiments.
FIG. 8 is a graph showing the results of IL-4 secretion experiments.
FIG. 9 is a graph showing the results of IL-10 secretion experiments.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
A quaternary phosphonium chitosan is prepared by the following steps:
into a 250mL three-necked flask, 400mg of Chitosan (CS) and 664mg of HOBt were added, and 30mL of H was added2A mixture of O/DMSO (v/v: 2/1) was stirred for 2.5h under ice-bath conditions. Subsequently, the pH is adjusted to 4.8-5.5. Then, 1.052g of CTPB (3-carboxypropyltriphenylphosphonium bromide) was added and stirred until completely dissolved. 940mg EDC & HCl was dissolved in 10mL H2A mixture of O/DMSO (v/v: 1/1) was added to the flask and the reaction was stirred for 24 h. The product was precipitated with a mixture of acetone/diethyl ether (v/v-2/1), and the precipitated product was vacuum-dried for 24h, dissolved in ultrapure water, dialyzed against ultrapure water for 3 days, and freeze-dried to give quaternary phosphonium chitosan (NPCS).
Contact Angle test of Chitosan (CS) with Quaternary phosphonium Chitosan (NPCS) prepared in this example: chitosan (CS) and quaternary phosphonium chitosan (NPCS) prepared in this example were mixed to prepare a solution, dropped onto a glass slide, spread evenly, vacuum dried to prepare a thin film, and measured with a contact angle measuring instrument (DSA100, KRUSS).
The results are shown in FIG. 1, with a CS contact angle of 74.5 ° and a NPCS contact angle of 26.6 °. The smaller the contact angle, the more hydrophilic, indicating that NPCS is more hydrophilic than CS.
Example 2
The quaternary phosphonium chitosan is used as the vaccine immunologic adjuvant:
preparation of vaccine preparation
Ovalbumin OVA was used as a model antigen, and vaccines of different formulations were formulated as shown in Table 1, and then intramuscular injection was performed to mice.
TABLE 1 vaccine formulation for intramuscular injection
Second, mouse immunization protocol
Intramuscular injection: female Balb/c mice at 6-8 weeks were randomly divided into 6 groups of 5 mice each; on days 0, 10 and 20, 100 μ L of vaccine solution was injected into the legs (50 μ g OVA per injection, half dose per leg). After immunization for 10 days for the third time, taking blood, separating serum, and storing at-20 ℃ for later use; spleen of a mouse is taken, ground and resuspended to prepare spleen cell suspensions with different concentrations for standby.
Third, determination of each immune index
Uptake of OVA by DC2.4 cells
Add DC2.4 cell suspension (1X 10) to 24-well plates5cells/well) were incubated at 37 ℃ for 24h with 5% CO 2. Cy5.5-labeled OVA is respectively mixed with DMEM basal medium and NPCS solution uniformly, then 500 mu L of the mixture is added into each well, the DMEM basal medium is used as a blank control, and each group of three is parallel to each other and cultured for 6h at 37 ℃ under 5% CO 2. Cells were digested and harvested, then washed with PBS and resuspended. The uptake of antigen by DC2.4 cells in vitro was examined by flow cytometry.
Results as shown in figure 2, the vaccine formulation of 1mg/mL NPCS/OVA significantly enhanced antigen uptake by DC2.4 cells compared to the negative control. This indicates that antigen uptake by DC2.4 cells is significantly increased in the presence of NPCS, which facilitates DC2.4 cell capture and presentation of more antigen, thereby inducing a more robust immune response.
2. Immunohistochemical assay
Divided into 4 groups (n-4) and immunized intramuscularly with 100 μ L of different vaccine formulations (50 μ g OVA per mouse, half dose per leg). Mice were euthanized 2 or 7 days after immunization and spleens were surgically isolated, fixed in 10% formaldehyde, paraffin embedded, and cut into 4 μm thick sections on polylysine coated slides. Immunohistochemical staining was performed according to the manufacturer's instructions.
As a result, as shown in FIG. 3, the vaccine preparation containing NPCS and Freund's adjuvant could detect a large amount of antigen in the spleen of the immunized mouse at both day 2 and day 7 after the immunization, while only a small amount of antigen was detected at day 7 in the spleen of the mouse immunized with the negative control. In combination with the fluorescence in vivo imaging results, the immunohistochemical results further indicate that with the help of NPCS, the antigen is slowly released from the injection site and gradually transferred into the spleen, thereby increasing the availability of the antigen in the spleen and further inducing a more effective immune response.
Determination of IgG antibody Titers
The 96-well plate was coated with 10. mu.g/mL OVA antigen coating solution and was coated overnight at 4 ℃. The plate was then washed 1 time with PBS-T and 200. mu.L of blocking solution was added to each well and incubated for 1h on a shaker at 37 ℃. The plates were then washed 3 times with PBS-T, 100. mu.L of mouse serum dilution was added, and incubated for 1h on a 37 ℃ shaker. The plates were then washed 3 times with PBS-T and 50 μ LHRP-labeled anti-mouse IgG secondary antibody was added to all wells and incubated for 1h on a shaker at 37 ℃. The plate was then washed 4 times with PBS-T, 100. mu.L of TMB developing solution was added to each well, and incubated for 15min at room temperature in the dark. Then 100. mu.L of stop solution was added to each well. The absorbance was immediately measured at 450nm with a microplate reader.
The results are shown in fig. 4, where there was a very significant difference between NPCS at 1mg/mL and the positive control (p < 0.001). This indicates that NPCS can effectively enhance the humoral immune response of the body and increase the level of IgG secretion.
4. Splenocyte proliferation assay
In 96-well plates, 100. mu.L of spleen cell suspension (2X 10) was added per well6cells/mL), 100. mu.L of ovalbumin solution (concentration 100. mu.g/mL, RPMI1640 basal medium) or RPMI1640 basal medium (blank control) was added, 3 replicates in each group. 37 ℃ and 5% CO2After 72h incubation, 20. mu.L of CCK-8 solution was added to each well, the 96-well plate was incubated in an incubator for 4h, and the absorbance at 450nm was measured with a microplate reader.
As shown in FIG. 5, the proliferation index of splenocytes under the action of the negative control group was about 1.2, while the proliferation index of splenocytes stimulated by NPCS/OVA reached about 1.5, which was comparable to that of the positive control group, and both were significantly different. This suggests that NPCS can effectively promote proliferation of splenocytes, thereby enhancing immune response.
ELISA determination of cytokine levels secreted by splenocytes
In a 24-well plate, 750. mu.L spleen cell suspension (2X 10) was added per well6cells/mL), respectively adding 750 mu L of egg white eggsWhite solution (100. mu.g/mL in RPMI1640 medium) was plated in 1 well per mouse spleen cell. 37 ℃ and 5% CO2Culturing for 60h, collecting cell suspension to 1.5mL EP tube, 2000r/min, centrifuging for 5min, collecting supernatant, and storing at-80 deg.C for use. Splenocytes IFN-. gamma.IL-12, IL-10 and IL-4 cytokine levels were determined by ELISA.
The results are shown in fig. 6-9, compared with the negative control, the NPCS/OVA preparation induces no statistically significant difference in the level of IL-12 secreted by splenocytes, while the IFN-gamma secretion level is significantly enhanced and has significant difference. In addition, NPCS/OVA agents induced secretion levels of IL-4 and IL-10 significantly higher than negative controls. In conclusion, NPCS induces significantly increased levels of IFN- γ, IL-4 and IL-10 secretion, especially slightly stronger Th1 cytokine (mainly IFN- γ) secretion than Th2 cytokine (mainly IL-10).
In conclusion, the quaternary phosphonium chitosan (NPCS) prepared by the invention has good water solubility, is prepared into different vaccine preparations with OVA, and is injected into mice intramuscularly. The results show that a 1mg/mL NPCS-based formulation can significantly enhance antigen uptake, splenocyte proliferation, IgG levels, and cytokine secretion by DC2.4 cells (IFN-. gamma., IL-10, and IL-4). This is mainly due to the protective and sustained release ability of NPCS against antigens, which facilitates the uptake and presentation of more antigens by DCs, resulting in a stronger immune response. In summary, NPCS has potential application in vaccine delivery systems for intramuscular immunization.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (3)
1. The application of the quaternary phosphonium chitosan in the preparation of vaccine immunoadjuvant is characterized in that:
the preparation method of the quaternary phosphonium chitosan comprises the following steps:
(1) adding chitosan and 1-hydroxybenzotriazole into the water/dimethyl sulfoxide mixed solution, stirring for 2-3h under an ice bath condition, and then adjusting the pH value of the mixture to 4.8-5.5; then adding 3-carboxypropyl triphenyl phosphonium bromide, and stirring until the 3-carboxypropyl triphenyl phosphonium bromide is completely dissolved;
(2) dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in a water/dimethyl sulfoxide mixed solution, adding the mixture into the reaction system in the step (1), reacting for 1-2 days, precipitating the product by using an acetone/diethyl ether mixed solution, drying the precipitated product in vacuum for 1-2 days, dissolving the product in ultrapure water, dialyzing for 3-4 days in the ultrapure water, and freeze-drying to obtain the quaternary phosphonium chitosan;
in the step, the molar ratio of the sugar unit of the chitosan, the 1-hydroxybenzotriazole, the 3-carboxypropyl triphenyl phosphonium bromide and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:2:1: 2;
in the vaccine, the mass ratio of the quaternary phosphonium chitosan to the antigen is 2: 1; the antigen is ovalbumin.
2. Use according to claim 1, characterized in that: in the water/dimethyl sulfoxide mixed solution, the volume ratio of water to dimethyl sulfoxide is (1-2) to 1.
3. Use according to claim 1, characterized in that: in the acetone/ether mixed solution, the volume ratio of acetone to ether is 2: 1.
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