Study On Protamine Coating PLGA Nanoparticles As Vaccine Delivery Vector

In recent years, biocompatible and biodegradable polymers such as poly(D, L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol)-poly(D, L-lactic-co-glycolic acid) (PEG-PLGA) have been widely studied as one of the effective delivery systems for sustained-release of antigens including DNA, proteins and peptides. Nanoparticles are paticles within the size range of 10-1000 nm. PLGA nanoparticles encapsulate antigens, which act as depot at the site of injection and enhance the uptake of antigens by antigen presenting cells (APC). PEG-PLGA nanoparticles have been commonly used as the delivery system to transport antigens through mucosal surfaces, especially via transnasal or oral route. Previous studies showed that modification of polymer particle surface with polycations such as protamine sulfate (PS), a FDA-approved arginine-rich peptide with strong cationic charge, resulting in a strong antibody response to the encapsulated antigen possibly due to the enhanced adjuvant effect. However, the effect of PS coating PLGA or PEG-PLGA nanoparticles as vaccine vector has not been studied. In this study, the immune effects of exogenous antigen encapsulated in PS coating PLGA nanoparticles and PEG-PLGA nanoparticles were investigated.Ovalbumin (OVA) was used as the model antigen and encapsulated into PLGA nanoparticles or PEG-PLGA nanoparticles, with (OVA-PLGA/PS, OVA-PEG-PLGA/PS) or without PS coating (OVA-PLGA, OVA-PEG-PLGA). These nanoparticles were then used to incubated with murine bone marrow-derived dendritic cells (BMDC). The phenotype of BMDC (CD80, CD83, CD86, MHC I and MHC II) and cellular uptake of nanoparticles were measured by flow cytometry analysis (FACS). The cytokines secretion (IL-4, IL-10 and IL-12p70) were measured by standard ELISA methods. The cross-presentation was studied with B3Z cells. The research was mainly concerned with the following aspects:(1) PLGA nanoparticles and PEG-PLGA nanoparticles were prepared by the double emulsion method. The nanoparticles were characterized in terms of particle size, PDI, zeta potential, encapsulation efficiency, loading efficiency and morphology. The preparation procedures were optimized to obtain the nanoparticles with the desired characteristics with PLGA as polymer material and OVA as a model antigen. We obtained the optimal conditions for preparation of nanoparticles that the OVA concentration was 100 mg/mL, polymer material was 50 mg/mL, the volume ratio of internal water phase to organic phase (W1/O) was 1:5, the volume ratio of organic phase to external water phase (O/W2) was 1:20, the concentration of emulsifier PVA was at 0.5%, and the sonication power was at 600 W. The average size, zeta potential, PDI, encapsulation efficiency and loading efficiency of optimized OVA-PLGA nanoparticles were 303.3±6.1 nm,-7.36±1.02 mV,0.113±0.031,62.46±0.32% and 23.80±0.09%; while those of OVA-PEG-PLGA nanoparticles were 271.4±9.2 nm,-6.04±0.23 mV, 0.311±0.032,61.51±2.9% and 23.51±0.85%, respectively. We then obtained PS coating nanoparticles by mixing PS with prepared nanoparticles. The average size and zeta potential of OVA-PLGA/PS nanoparticles were 347.0±10.6 nm,8.01±1.05 mV while those of OVA-PEG-PLGA/PS nanoparticles were 341.2±55.8 nm and 8.48±3.42 mV, respectively. The morphology of all prepared nanoparticles were examined by transmission electron microscope (TEM) which appeared that the nanoparticles were spherical in shape with smooth surface and without any aggregation or adhesion. All these nanoparticles were prepared to interact with BMDC for surface marker study, ctokine secretion assay and cross-presentation assay. We also prepared different nanoparticles with different research purpose, such as FITC-OVA-PLGA, FITC-OVA-PEG-PLGA, FITC-OVA-PLGA/PS and FITC-OVA-PEG-PLGA/PS for studying cellular uptake of BMDC; OsO4-PLGA, OsO4-PEG-PLGA, OsO4-PLGA/PS and OsO4-PEG-PLGA/PS for studying intracellular distribution uptake by BMDC. The zeta potential of all these nanoparticles transferred from negative to positive after PS coating.(2) Bone marrow cells were isolated and bone marrow devrived dendritic cells (BMDC) were generated successfully. The morphology and phenotype indentification of BMDC revealed the successful differentiation by GM-CSF and IL-4 stimulating. The purity of the BMDC harvested was between 85 and 95% based on the expression of CD11c at day 7. The sussessful generation of BMDC was the most important condition for the next experiments. Different nanoparticles and related factors were used to stimulate BMDC, and phenotype of BMDC (CD80, CD83, CD86, MHC I and MHCⅡ) was meassured by fluorescence activated cell sorting (FACS). The rusults revealed a significantly increased surface marker expression of BMDC in OVA-PLGA/PS stimulated group. ELISA assay suggested OVA-PLGA/PS stimulation could increase secretion of IL-12p70 and decrease production of IL-4 by BMDC, while OVA-PEG-PLGA/PS stimulation could increase secretion of IL-4 by BMDC. These results suggested that PS coating on the surface of different polymer material might induce different effect on BMDC. In details, PLGA/PS might induce BMDC secreting Th1-polarization cytokine (IL-12p70) while PEG-PLGA/PS induce BMDC secreting Th2-polarization cytokin (IL-4).(3) The BMDC uptake of nanoparticles encapsuled FITC-OVA were meassured by FACS. As shown in this study, the internalization of all kinds of nanoparticles were in a time-dependent manner by BMDC at 37℃. The results of significantly reduced internalization at 4℃suggested that all nanoparticles uptake by BMDC could be the energy-dependent phagocytosis. PS coating significantly enhanced the uptake rate of FITC-OVA-PLGA/PS and FITC-OVA-PEG-PLGA/PS by BMDC by remarkably increasing the phagocytosis capacity of BMDC. This suggested that coating with PS could further improve the delivery efficiency of both nanoparticles.(4) OsO4 is a widely used electron dense agent. To study the intracellular distribution of nanoparticles, BMDC were incubated with PS coating (OsO4-PLGA/PS, OsO4-PEG-PLGA/PS) or uncoating nanoparticles (OsO4-PLGA, OsO4-PEG-PLGA). TEM study suggested that PS coating PLGA nanoparticles and PS coating PEG-PLGA nanoparticles escaped from lysosomes through the interaction with lysosomal membrane.(5) The cross-presentation of encapsulated exogenous antigen (OVA) was studied by CD8+ B3Z T cell in vitro. Nanoparticles coating (OVA-PLGA/PS, OVA-PEG-PLGA/PS) or uncoating (OVA-PLGA, OVA-PEG-PLGA) with PS were used to stimulated BMDC, then B3Z T cell was used to coincubate with BMDC to assay the cross-presentation. Results showed OVA-PLGA/PS and OVA-PEG-PLGA/PS induced higher cross-presentation of exogenous antigens than uncoating nanoparticles in B3Z cell, suggesting that PS facilitated cross-presentation of encapsulated OVA via MHC I. The cross-presentation assay showed significantly higher in OVA-PLGA/PS stimulated BMDC. There was a significantly positive correlative relation at concentration of 50-300μg/mL and coincubation time of 2-8 h in OVA-PLGA/PS stimulated BMDC, while the positive correlative relation of OVA-PEG-PLGA/PS stimulated BMDC was at the concentration of 50-400μg/mL and coincubation time of 2-10 h.In conclusion, PS coating nanoparticles (PLGA/PS and PEG-PLGA/PS) had the ability of increasing cellular uptake, promoting maturation, stimulating cytokines secretion and enhancing cross-presentation of BMDC. The results demonstrated the feasibility of PS coating nanoparticles as a potent vaccination carrier for both injection and trans-mucosal delivery.The present study revealed the mechanism of PS as adjuvant of cellular vaccine, and provided new information for the design of polycation coating polymer nanoparticles vaccine delivery system. It is necessary to investigate the pharmacokinetics and tissue distribution of antigen encapsulated in PS coating nanoparticles and in vivo assessment in the future.