| Adjuvants are one of the essential components of commercial vaccines,which can improve the specific immune response to antigens.Previous studies have shown that resveratrol can improve the immune response after vaccination and has an adjuvant effect.The nano-delivery system can effectively deliver resveratrol to improve its physicochemical properties.If the antigen and adjuvant can be co-localized in the same antigen presenting cells(APCs),the APCs can be more effectively activated and the immune response can be enhanced.In this study,Quaternized chitosan-coated liposomes were used as carriers to achieve antigen and adjuvant co-delivery and enhance immune response through internal loading of resveratrol and external adsorption of antigen.The main research contents are as follows:1.Preparation of quaternized chitosan-coated resveratrol liposomesThe preparation of quaternized chitosan-coated resveratrol liposomes mainly includes the investigation of resveratrol methodology,the optimization of resveratrol liposome formulation,and the screening of the optimal concentration of quaternized chitosan.First,the detection method of resveratrol was investigated;secondly,the optimal prescription and preparation process of resveratrol liposomes were screened through single-factor and orthogonal experiments;finally,the optimal concentration of quaternized chitosan was screened through Zeta potential distribution.The results showed that:(1)High-performance liquid chromatography(HPLC)is suitable for the detection of resveratrol content.Resveratrol has a good linear relationship in the concentration range of 2.5-80μg/m L.The standard curve equation is C=81.861A+24.675,and the curve correlation coefficient R~2=0.9997.(2)The optimal prescription and preparation process of resveratrol liposomes are as follows:10 mg resveratrol,200 mg phospholipids,and 10 mg cholesterol were weighed and dissolved in 20 m L chloroform-methanol(1:1,v:v).The solvent was removed by rotary evaporation under reduced pressure at 35℃,followed by adding 10 m L PBS(p H 7.4)and rotating hydration for 30 min.The cell ultrasonic crusher was used to ultrasonicate for 10 min under ice bath conditions with an interval of 5 s and ultrasound for 5 s at a power of 600 W.Finally,it was repeatedly extruded five times through a 0.1μm liposome extruder to obtain semi-transparent resveratrol liposomes with a light blue milk-like color.(3)Quaternized chitosan at a concentration of 8mg/m L can completely encapsulate resveratrol liposomes with uniform Zeta potential distribution.2.Characterization of quaternized chitosan-coated resveratrol liposomesThe physicochemical properties of Quaternized chitosan-coated resveratrol liposomes were characterized,including their infrared spectra,morphology,particle size,polydispersity index(PDI),zeta potential,and antigen adsorption rate.Results showed that:(1)The infrared spectra revealed the presence of hydrogen bonding interactions between quaternary ammonium chitosan and liposomes,antigens,and Quaternized chitosan-coated resveratrol liposomes Transmission electron microscopy showed quaternary ammonium chitosan particles coating the surface of Resveratrol Liposomes and adsorbed ovalbumin antigens.(2)The Z-average particle size of Quaternized chitosan-coated resveratrol liposomes was 96.3±3.52 nm with a low PDI value(<0.3),indicating a homogeneous size distribution.The Zeta potential was9.6±0.356 m V,and the liposomes could encapsulate 76.22±1.02%of resveratrol and adsorb 88.2±16.3%of antigens.These results suggest that quaternized chitosan-coated resveratrol liposomes are nanoparticles with a homogeneous size distribution,capable of encapsulating resveratrol and adsorbing antigens.3.Effects of quaternized chitosan-coated resveratrol liposomes on the function of antigen-presenting cellsUsing mouse dendritic cells as a model,the delivery efficiency and immune adjuvant activity of quaternized chitosan-coated resveratrol liposomes were evaluated in vitro.The results showed that:(1)At a concentration of 1.25μg/m L(based on resveratrol concentration),the quaternized chitosan-coated resveratrol liposomes had no cytotoxicity to mouse bone marrow dendritic cells(BMDCs).(2)The co-delivery efficiency of the antigen and resveratrol by quaternized chitosan-coated resveratrol liposomes was as high as 81.08%±0.090%,which was more than 50 times higher than that of resveratrol liposomes and 188 times higher than that of free resveratrol.Laser confocal microscopy showed that the quaternized chitosan-coated resveratrol liposomes exhibited a large amount of bright green fluorescent antigen and red fluorescent resveratrol particles in the cells,and the red and green fluorescence was co-localized.(3)The antigen could escape from lysosomes through the quaternized chitosan-coated resveratrol liposomes carrier.(4)The quaternized chitosan-coated resveratrol liposomes could increase the levels of co-stimulatory factors CD80 and CD86,and induce the production of cytokines IL-2 and IL-12,which were superior to those of the LPS positive control group.These results indicate that the quaternized chitosan-coated resveratrol liposomes can enhance the delivery efficiency of resveratrol and antigen,activate dendritic cells,and enhance the immune-enhancing effect of resveratrol.4.Evaluation of immune adjuvant effect of quaternized chitosan-coated resveratrol liposomesThe hemolytic,delivery efficiency and immune adjuvant activity of quaternized chitosan-coated resveratrol liposomes were evaluated in vivo using mice as model animals.First,quaternized chitosan-coated resveratrol liposomes were incubated with mouse red blood cells to detect the hemolysis.Then,the ability of the liposomes to recruit antigen-presenting cells and target lymph nodes was tested by subcutaneously injecting fluorescent antigen-adsorbed liposomes in the tail.Additionally,using commercial aluminum hydroxide adjuvant as a positive control and free resveratrol,resveratrol liposomes,and blank quaternized chitosan liposomes as controls,the adjuvant activity of quaternized chitosan-coated resveratrol liposomes was evaluated.The results showed that:(1)quaternized chitosan-coated resveratrol liposomes did not cause hemolysis of red blood cells.(2)Quaternized chitosan-coated resveratrol liposomes effectively recruited antigen-presenting cells and targeted antigen-presenting cells that remained in the lymph nodes,promoting the uptake rate of antigens by antigen-presenting cells.(3)After immunization using quaternized chitosan-coated resveratrol liposomes adsorbed with the antigen,the levels of cytokines,specific Ig G levels within 21-42 days,spleen lymphocyte proliferation rate,CD3+CD4+T lymphocyte phenotype,and CD3+CD8+T lymphocyte phenotype were all increased,and the effect was better than that of free resveratrol,resveratrol liposomes,blank quaternized chitosan-coated liposomes,and the positive control aluminum hydroxide adjuvant(P<0.05),with the best effect observed at the medium dose of quaternized chitosan-coated resveratrol liposomes.The above results indicate that quaternized chitosan-coated liposomes loaded with resveratrol have good delivery efficiency and immunoadjuvant activity in vivo.In summary,this study characterized and evaluated the adjuvant effects of quaternized chitosan-coated resveratrol liposomes both in vitro and in vivo.Results showed that quaternized chitosan-coated resveratrol liposomes met the standards of nanomedicine in terms of encapsulation efficiency,drug loading,morphology,particle size,polydispersity index,and zeta potential.The quaternized chitosan-coated liposomes achieved the goal of co-delivering and co-localizing resveratrol and antigens as well as escaping from lysosomes by loading resveratrol inside and adsorbing antigens on the surface.The quaternized chitosan-coated resveratrol liposomes acted as an immunostimulatory adjuvant by enhancing the expression of co-stimulatory factors in dendritic cells,increasing mouse antigen-specific Ig G antibodies and cytokine levels,and elevating CD3+CD4+and CD3+CD8+T lymphocyte levels in the spleen.This study improved the limited delivery efficiency and biological activity of free resveratrol and systematically evaluated the adjuvant-related indicators during the antigen-presenting process,providing a theoretical basis for the application of nano-resveratrol in the field of veterinary adjuvants. |
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