AL/CS/DNA And OVA/PLGA/GC Nanoparticles As Vaccine Delivery System For Nasal Mucosal Immunity

External pathogens (such as bacteria, viruses and parasites, etc.) invading mucosa can cause respiratory, gastrointestinal or systemic diseases. Mucosal immunity is the most effective way for building up the local long-term immune memory at the invading site of pathogens, and it is the first line of defense which prevents pathogens invasion in the body. In all mucosal immune strategies, nasal mucosal immune is the most attractive and potential route. It can induce both proximal and distal mucosa immunization. Nanoparticle delivery system is a kind of nano-scale particles for drug delivery. Several features of nanopaticles make it an excellent mucosal vaccine adjuvant. Firstly, nanopaticles could control the release of the antigen. It greatly increases the residence time of the antigen in the mucosal sites. Secondly, nanopaticles can integrate their antigen presentation function and other effects, such as immune regulation and immune stimulation. Thirdly, nanopaticles can overcome physiological barriers in mucosa, which effectively increases the amount of the antigen to reach the immune system.According to the property of DNA vaccine and protein vaccine, we designed two different naoparticles for enhancing the immunogenicity of antigen. DNA vaccine pGJA-P/VAX against S.mutan which is the main reason leading to detal caries was used, and OVA was used as protein vaccine model. As a result of the existence of various physiological barriers, the single DNA vaccine pGJA-P/VAX displayed low efficacy via intranasal (i.n.) administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan (CS) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles could deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (sIgA) than that of CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity.On the other hand, protein vaccine loaded polylactic-glycolic acid (PLGA) nanocapsules is a potential vaccine vector for mucosal immunization. It can significantly enhance the immune response of loaded protein vaccine. Meanwhile, it exhibits good biodegradability and biocompatibility. However, the inert and negative charged surface of PLGA nanocapsules limited its interaction with the negative charged cell membrane, so as to inhibit the intracellular uptake of PLGA nanocapsules. We tried to use glycol chitosan (GC) to modify the surface of PLGA nanocapsules, in order to enhance the immunogenicity. The role of GC coating was showed as follows:Firstly, GC coating was able to increase the uptake of PLGA nanocapsules; Secondly, GC coating was able to increase the interaction with lysosomes, so as to facilitate the escape of PLGA nanocapsules from lysosomes; Thirdly, GC coating was able to increase the nasal residence time of PLGA nanocapsules. These outstanding features made OPG nanocapsules induce effective mucosal immune responses, systemic immune responses and cellular immune response. The research was mainly focused on the following aspects:(1) AL/CS/DNA was prepared with a self-assembling method, it was confirmed that the optimal N/P ratio and lipid/DNA (w/w) was7and3, accordingly. TEM results revealed that the AL/CS/DNA was homogeneous spherical structure.(2) Gel retardation and circular dichroism assay proved that AL/CS/DNA could effectively compact DNA without changing the B-DNA conformation. Meanwhile, DNA release from AL/CS/DNA was pH-mediated manner.(3) Compared to CS/DNA, the AL/CS/DNA exhibit longer nasal residence time and stronger power in penetrating the mucosal layer. The incorporation of AL did not affect the power of mucin asorption and opening tight conjuction between cells. However, through enhancing the uptake of AL/CS/DNA in epithelial cells, it can prolong the nasal residence time on nasal mucosa.(4) Compared to the CS/DNA, the AL/CS/DNA could induce significantly higher level of saliva IgA via i.n. manner, which last at least for more than12weeks. The AL/CS/DNA could induce proximal mucosa immune response and distal mucosa immune response at the same time. Thus, the AL/CS/DNA has the potential as a delivery system for inducing systemic mucosal immune response.(5) OP (OVA/PLGA) and OPG (OVA/PLGA/GC) nanocapsules were prepared with double emulsion solvent evaporation techniques. The modification of GC on PLGA nanocapsule increased the encapsulation efficiency greatly. Both OPG and OP nanocapsule was homogeneous spherical, and OPG exhibit core-shell structure.(6) Cellular uptake and colocalization experiments proved that GC coating could enhance the uptake of PLGA nanocapsule in DCs (Dendritic cell), without affecting the intracellular localization of PLGA nanocapsule. Both OPG and OP nanocapsules did not enter the nuclear in2h, and they both had the power of escaping from lysosomes.(7) In vivo fluorescent imaging proved that GC coating could prolong the nasal residence time of PLGA nanocapsules. Two reasons might be responsible for the results. Firstly, more OPG nanocapsules might enter the epithelial cells than the OP nanocapsules; Secondly, GC coating increased the adhesion ability of OPG nanocapsules. Frozen sections of nasal mucosa results revealed that both OPG and OP nanocapsules entered the mucosa layer, while OVA only located outside of mucosa layer.(8) In this paper, the mucosal immunity, systemic immunity and cellular immunity induced by OPG and OP nanocapsules were evaluated. The results showed that OPG nanocapsules were more power in inducing serum IgG response, proximal mucosa response, distal mucosa response and Thl cellular immune response. These results revealed that GC coated PLGA nanocapsules had multiple immune enhancement effect via i.n. manner.These results suggest that the AL/CS/DNA nanoparticles and OPG nanocapsules could increase the immune responses of loaded DNA and protein vaccine at nasal mucosa sites, and have the potential as a novel mucosa vaccine carrier.