Study On The Preparation Of Amomum Longiligulare Polysaccharides 1-PLGA Nanoparticle And Its Immunoregulatory Ability

As one of the four southern medicines in China,the fruits of Ammonium regularization T.L.Wu has aroused great interest in scientists due to their variety of pharmacological activities including anti-inflammation and immune regulation.The major biological macromolecules are Amomum longiligulare polysaccharide 1.Polysaccharides from traditional Chinese herbs have excellent immune regularization and hypoglycemia.However,polysaccharides from traditional Chinese herbs are macromolecular substances that have lots of limitations,such as fast metabolism,short biological half-life,low concentration of action,and low availability,which seriously limit the usage of Ammonium regularization polysaccharides(ALP).The development of ALP has been seriously restricted.It’s a good way to improve the bioavailability that polysaccharides were encapsulated into PLGA nanoparticles.PLGA(lactic-co-glycolic acid)is the most commonly used material for the fabrication of a nanocarrier system for extending the half-life to improve the availability of ALP1.In this study,the preparation of ALPP was optimized by the response surface method(RSM)with a Box-Behnken design.The surface morphology of nanoparticles was detected.Meanwhile,the effects of ALPP on RAW264.7and lymphocyte differentiation,secretion of cytokines,as well as on the expression of DCs surface-active substances,and pro-T cell proliferation ability were measured by cytotoxicity assay and qRT-PCR.Compared with ALP1,the immune-enhancing effect of ALPP was stronger.The results of the experimental study are as follows.1 Preparation and optimization of ALP1-PLGA nanoparticles The response surface method with a Box-Behnken design to optimal preparation conditions of ALPP.Based on the single-factor results,optimizing the preparation conditions of nanoparticles by Response Surface Method.Meanwhile,field emission electron microscopy was used to observe the characterization of ALPP.Additionally,a laser particle size analyzer was used to measure the particle size and zeta potential.The results showed that the encapsulation efficiency of ALPP reached a high level(79.88%)when W1:O was 1:7,PE:W2 was 1:7,and the concentration of F68 was 0.7%.ALPP showed a controlled and sustained release.Meanwhile,the scanning electron microscope results showed that ALPP was a kind of nanoparticle with a diameter of 89.77±27.94 nm.2 Effect of nanoparticles on Raw264.7 cells Flow cytometry was applied to detect the phagocytic activity of Raw264.7,and ELISA was used to detect the effects of ALP1/ALPP on the activity of i NOS enzymes and the secretion of NO,IL-6,IL-10,TNF-α,and TGF-β in macrophages.The results showed ALPP promoted the activation of Raw264.7 cells and polarized Raw264.7 cells to both M1 type and M2 type macrophages.3 Effect of nanoparticles on T lymphocytes The proliferation rates of T lymphocytes were treated with ALP1/ALPP were determined by using the MTT method.Meanwhile,the relative mRNA expressions of cytokines from T lymphocytes were assayed by using the qRT-PCR method.The results showed the promoting effect on the proliferation of T cells.Meanwhile,ALPP group and ALP1 group compared with the PHA group,the relative expression levels of IL-2 and IFN-γ mRNA in lymphocytes stimulated by ALP1 were 3.85 times and 3.50 times,respectively,ALPP group elevated the mRNA expression of IL-2(6.05 times)and IFN-γ(7.18 times).4 Effect of nanoparticles on chicken bone marrow dendritic cells(ch BM-DCs)The proliferation rates of chicken bone marrow dendritic cells(ch BM-DCs)that were treated with ALP1/ALPP were determined by using the MTT method.Meanwhile,qRT-PCR method was used to detect relative mRNA expressions of surface molecules of ch BM-DCs.Besides,the uptake capacity of ch BM-DCs and proliferation of T cells were was examined.The results showed that the promoting effect on the proliferation of im ch BM-DCs was better than that of ALP1.Also,ALPP was more effectively improved ch BM-DCs uptake capacity,compared with ALP1.Meanwhile,ALPP group,compared with ALP1 group,was promoted the expression of surface molecules,which CD11.c,CD40,CD86,CD80,CD83 and MHC class II mRNA were increased of 55.33±0.12%,62±0.03%,26.67±0.02%,51.67±0.12%,41.68±0.03%,42.64 ± 0.01%.Taken together,ALPP had a stronger immunomodulatory activity than ALP1.This paper showed that compared with ALP1,ALPP not only significantly enhanced macrophage activity and induced macrophage polarization into M1 and M2 types,but also significantly increased the expression level of lymphocyte cytokines and induced differentiation of T lymphocytes to Th1 type lymphocytes,meanwhile,ALPP also significantly increased dendritic cell activity,maturation,uptake capacity,expression of surface markers,then the PLGA shell reduced the toxic effects of ALP1 and the immune-enhancing effect of ALPP stronger than that of ALP1.Therefore,ALPP is a highly promising immunomodulator.