Development and characterization of chitosan coated poly-(ɛ-caprolactone) nanoparticulate system for effective immunization against influenza.

In this study surface coated poly-(ɛ-caprolactone) (PCL) nanoparticles with chitosan (CS) were developed as a carrier system for nasal immunization using recombinant Influenza A virus (A/California/07/2009) H1N1 hemagglutinin (HA) protein, for the induction of humoral, cellular and mucosal immunity. CS coated PCL (CS-PCL) nanoparticles were characterized in vitro for their percent yield, size, shape, entrapment efficiency, loading capacity and zeta potential. The in vitro release and antigen integrity were also evaluated. Particles were prepared by an emulsion-diffusion-solvent evaporation method. The coated cationic nanoparticles of average size 125.64±6.51 nm with a narrow size distribution (pdi: 0.185±0.032) and a positive surface charge (+22.88 mV) were obtained. HA antigen was efficiently entrapped in CS-PCL nanoparticles (entrapment efficiency 74.84±4.51%, loading capacity 14±2% (w/w)). The molecular weight and antigenicity of the entrapped HA was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. In vitro release study of antigen showed that about 66.47% of entrapped antigen was released within 63 days. The immune-stimulating activity was studied by measuring hemagglutination inhibition (HAI) titer, IgG, IgG1 and IgG2a titer, secretory IgA level in nasal and lung lavage (mucosal secretions) following nasal administration of modified CS-PCL nanoparticles in Balb/c mice and compared with soluble HA antigen administered intramuscular (IM) and with PCL (uncoated) nanoparticles administered intranasal (IN). The numbers of IFN-γ or IL-4 secreting cells in spleen homogenates were also measured 21 day after third immunization. Single IN or IM immunization with antigen-loaded CS-PCL nanoparticles resulted in strong HAI and total IgG responses. These responses were higher than those achieved after booster IM administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The IN administered antigen-CS-PCL nanoparticles induced higher immune responses compared to the other IN antigen formulations, and these responses were enhanced by IN booster vaccinations. Moreover, IM administered soluble HA antigen did not elicit s-IgA in mucosal secretions as it was induced and measured in the case of nasal administration of CS-PCL nanoparticles. In contrast to IM administered antigen CS-PCL nanoparticles induced a balanced Th1 and Th2 response. CS-PCL nanoparticles (cationic nanoparticles) thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. These findings demonstrate high potential of CS-PCL nanoparticles for their use as a carrier adjuvant for nasal administered influenza antigens.