Background: Outer membrane vesicles (OMVs) released from Salmonella Typhimurium can become the effective vaccine candidate. For efficient delivery through oral route, the OMVs are conjugated with some delivery systems. The current study was aimed at optimization of conditions required for conjugation of OMVs with nano- or micro-particles for maximum entrapment of OMV in terms of protein concentration. Methods: The OMVs of Salmonella Typhimurium were conjugated under three optimum conditions of pH, temperature and ratio (nanoparticles or microparticles: OMVs) predicted by response surface method. The efficiency of conjugation was determined by entrapment of OMVs in nano/microparticles. Result: The pH and temperature were not influential conditions in case of conjugation of OMVs with chitosan nanoparticles (Ch-NP) and poly-lactide co-glycolide microparticles (PLG-MP) while they had influence in case of poly(anhydride) nanoparticles. In case of Ch-NP and PLG-MP, the optimum ratio for maximum entrapment was found to be 1:10 and 1:9 respectively. The optimum pH and temperature was found to be 7 and 24 degree C respectively for conjugation of poly(anhydride) nanoparticles. The optimized conditions did not alter the protein profile and immunogenic potential of conjugated vaccines.

The present study was carried out to evaluate in-vitro toxicity associated with chitosan nanoparticles, Gantrez nanoparticles and poly-lactide co-glycolide (PLG) microparticle in Vero cell line. The cytotoxicity of all three micro/nano-particles was assessed using different concentration. For each concentration, the confluent monolayer was treated for a period of 36-48 hours and studied for morphological alteration, live and death count etc. after the treatment. It was observed that the different concentrations of chitosan nanoparticles and Gantrez nanoparticles did not have significant effect on the cell viability as evident from the non-significant difference between the OD540 of formazan product formed from MTT in treated and untreated cells. The concentration of chitosan nanoparticles and Gantrez nanoparticles up to 1000 micrograms/ml did not have any influence in cellular metabolic activities and viability. However, a reduction in the cellular viability and metabolic activities were observed when PLG microparticles were used at 1000 micrograms/ml. At concentrations below 1000 micrograms/ml, all the nanoparticles/microparticles were found to be safe in terms of cytotoxicity.