Study On The Intracellular PH-Responsive Astragalus Polysaccharide-Encapsulated PLGA Nanoparticles As Adjuvant For Antigens

Astragalus,the dry roots of Astragalus L.(Leguminosae).Modern medicine science believes that astragalus has the effect of enhancing immunity,lowering blood pressure,protecting liver and diuresis,resisting stress and the action of broad-spectrum antibiotic.Studies have found that astragalus polysaccharides,the main active ingredient of astragalus membranaceus,plays a vital role in improving immunity.However,astragalus polysaccharide have the properties of short plasma half-life,rapid metabolism and weak targeting,which has greatly reduced its clinical applications.Therefore,the development of new dosage forms appears to be particularly important.Poly(lactic-co-glycolic acid)(PLGA)is a synthetic carrier material with outstanding biocompatibility and biodegradability,biosecurity,non-toxic,and non-specific immunogenicity,which is used as a nanomaterial to prepare drug-loaded nanoparticles in order to achieve sustained release behavior and targeted effects on drugs,protect the biological activity of drugs,improve the bioavailability and reduce the side effects of drugs.In this study,the astragalus polysaccharides-loaded PLGA nanoparticles(APSP)were prepared by double emulsion solvent evaporation method.The preparation conditions of APSP were optimized by response surface methodology.The APSP were then modified as pH-responsive APSP based on the optimized preparation conditions.The qualities of pH-responsive astragalus Polysaccharides-loaded PLGA Nanoparticles(pH-responsive APSP)were evaluated and the immunoenhancing activities of them were studied by in vitro and in vivo experiments.The study was mainly divided into the following four parts:Experiment 1 Preparation and optimization of intracellular pH-responsive astragalus polysaccharides-loaded PLGA nanoparticles In order to screen out the optimal conditions for the preparation of pH-responsive astragalus polysaccharide-loaded PLGA nanoparticles(pH-responsive APSP),the pH-responsive APSP were prepared by double emulsion solvent evaporation method.The free APS and the polysaccharides-loaded nanoparticles were separated by Sephadex G-50 microcolumn gel.Phenol-sulfuric acid method was used to test the contents of APS loaded in nanoparticles,then the encapsulation efficiency and drug loading of the nanoparticles were calculated.On this basis,6 factors were selected:the ratio of oil phase to internal water phase(W1:O),the ratio of external water phase to primitive emulsion(W2:WPE),the concentration of F68(%),the mass ratio of drug to material,the ultrasound power on primitive emulsion,and the concentration of ammonium bicarbonate(mg·mL-1).Three of them were picked up for their greater influences on the encapsulation efficiency of pH-responsive APSP:the ratio of oil phase to internal water phase(W1:O),the ratio of external water phase to primitive emulsion(W2:WPE),and the concentration of F68(%).Furthermore,the response parameters were used to optimize the preparation conditions scientifically and rationally,then the optimal conditions were obtained,the ratio of oil phase to internal water phase(W1:O),the ratio of external water phase to primitive emulsion(W2:WPE),and the concentration of F68(%)are 7:1,5:1,and 1.1%,respectively.The experimental results showed that the actually average encapsulation efficiency of the pH-responsive nanoparticles was 65.23±0.51%,and the model fitted with the response surface BBD method design as well.Experiment 2 Characterizations and quality assessments of intracellular pH-responsive astragalus polysaccharides-loaded PLGA nanoparticles In order to evaluated the potential ability of the nanoparticles to be the drug-carrier,characterizations of blank PLGA nanoparticles(BP),normal astragalus polysaccharides-loaded PLGA nanoparticles(APSP),and pH-responsive astragalus polysaccharides-loaded PLGA nanoparticles(pH-responsive APSP)were measured.The nanoparticles were prepared according to the optimal preparation conditions and the hydrated particle sizes of APSP and pH-responsive APSP were 100.6± 1.1 nm and 127.0± 1.2 nm,respectively.The surfaces of the nanoparticles were negatively charged and the PDI of these two nanoparticles were 0.441±0.016 and 0.127±0.015,both of which are less than 0.5,indicating that the nanoparticles were evenly distributed and had good dispersibility.The ultrafine morphologies of each kind of nanoparticles were observed under transmission electron microscope.The nanoparticles were uniformly spherical,and the surfaces were smooth and compact.The results of in vitro polysaccharides and OVA release showed that APSP showed slow release behaviors under different pH conditions,and the cumulative release rates were much lower than the ones of pH-responsive APSP,while pH-responsive APSP showed explosive release bahaviors in acidic environment,and the cumulative release rates were higher than APSP.Then the ultrastructures of pH-responsive APSP in vitro were observed under an electron microscope.As the results showed,in the acidic environment(pH 5.0),there were a lot of damages on the surface of the nanoparticles.However,in the neutral environment(pH 7.4),the surfaces of the nanoparticles were still relatively smooth and rounding,which indicated the successful loading of ammonium bicarbonate.Then pH-responsive APSP were saved under normal storage conditions for 28 days,the stability of the nanoparticles was tested and the results showed that pH-responsive APSP had good stability.Experiment 3 Effects of intracellular pH-responsive astragalus polysaccharides-loaded PLGA nanoparticles on mouse spleen lymphocytes and peritoneal macrophages In order to investigate the in vitro activation of APSP and pH-responsive APSP on mouse spleen lymphocytes and peritoneal macrophages,the nanoparticles were used to co-incubate with cells.The effects of nanoparticles on the maximum safe concentrations and proliferation activities of spleen lymphocytes were tested by MTT assay and the effects of nanoparticles on lymphocyte CD4+and CD8+changes were detected by flow cytometry.Then the maximum safe concentrations of nanoparticles on peritoneal macrophages were tested by MTT method.The phagocytosis ability of macrophage and the expression of macrophage surface molecules MHC-II,CD80 and CD86 were measured by flow cytometry.And the antigen uptake abilities of macrophage were observed under laser confocal microscopy.The results showed that pH-responsive APSP could reduce the toxicity of drugs to cells,stimulate lymphocyte proliferation,stimulate the differentiation and maturation of CD4+T cells,and enhance the immune activity of spleen lymphocytes.It could also significantly stimulate the maturation of mouse peritoneal macrophages,up-regulate the expression of MHC-II,CD80 and CD86 on the surface of cells,significantly enhance the phagocytic ability and antigen uptake capacity of macrophages,and have significant cell activation compared with polysaccharide alone.Experiment 4 Study on the adjuvant activity of intracellular pH-responsive astragalus polysaccharides-loaded PLGA nanoparticles In order to evaluate the adjuvant activities of the APS-loaded nanoparticles,the immune responses of mice immunized with OVA-loaded intracellular pH-responsive APSP and APSP were studied.168 8-week-old ICR female mice were randomly divided into 7 groups,24 in each group.The first immunization was performed on day 0 by subcutaneous immunization,and the second immunization was performed on the 14th day.Vaccines are grouped into:①PBS group;②OVA group;③OVA/FIA group;④OVA/APS group;⑤OVA/BP group;⑥OVA/pH-responsive APSP group;⑦OVA/APSP group.At 96 h after the first immunization,4 mice were randomly selected from each group,their drainage lymph nodes were then collected.Flow cytometry was used to detect CD80,MHC-II,and CD86 molecules on surface of dendritic cells in lymph nodes.After the second immunization,4 mice were randomly selected from each group on day 7(D21),14 days(D28),21 days(D35),and 28 days(D42),and blood was taken from the orbital venous plexus to detect the content of OVA-specific IgG,IgG2a,and IgGl antibody in serum and the concentrations of cytokines IL-4,IL-6,IFN-y and TNF-α.On the 7th day(D21)and the 35th day(D49)after the second immunization,four mice were randomly selected from each group,spleens were then aseptically collected and the mouse spleen lymphocytes were isolated and cultured in vitro for 44-48 hours.The proliferation of lymphocytes was tested by MTT assay,and the activations of T lymphocytes were detected by flow cytometry.From the test results,the APSP and the pH-responsive APSP as vaccine adjuvants could significantly promote the maturation of dendritic cells in draining lymph nodes,stimulate the body to produce more OVA-specific antigen,express higher levels of IgG antibodies and cytokines,and enhance more proliferation and differentiation of T lymphocytes compared with polysaccharides alone and the control group.The results further showed that in the early stage of immunization,the pH-responsive APSP promoted stronger immune response than the normal APSP,which was consistent with the results of the quality assessment,and pH-responsive APSP showed magnificent targeting ability than APSP.On the contrary,normal APSP had the property of sustained release,which can prolong the action time of the drug.