Abstract:
Novel carriers for nasal application of Japanese encephalitis (JE) vaccine utilized by poly(D,L lactic-co-glycolic acid) (PLGA) and surface conjugating with chitosan (CS) and Al(OH)₃ were prepared by means of multiple emulsion solvent evaporation technique to obtain various sizes and surface charge of particles. The influences of preparation conditions on physicochemical characteristics and on integrity of JE virus were evaluated. The results demonstrated that size and uniformity increased by increasing primary w/o ratio, secondary w/o/w ratio, volume of PVA or by decreasing the percentage of PVA as well as the sonication output. All microparticles were spherical with smooth surface comprising the positively surfaced charge after conjugating with CS and Al(OH)₃. Integrity and structural conformation of epitope E protein was fine retained during the preparation process. The efficacy of these particles as nasal JE vaccine carrier whose particles diameter and surface charge were suitable for nasal route was first introduced in this study. The use of various size of PLGA, PLGA conjugated CS and Al(OH)₃ as nasal JE vaccine carrier was investigated after application into porcine nasal mucosa. CLSM suggested that particulate uptake of particles up to approximately 1µm was rapidly taken up by and permeated through nasal mucosa even though the adhesion on tissue surface was relatively lower compared to those from 5 and 15 µm. The tissue adhesion was considerably improved after the conjugation of 1 µm particles with CS and Al(OH)₃ However, higher amount of particulate uptake and permeation occurred only in Al(OH)₃ formulation. CS formulation showed conversely different result as the uptake mechanism of CS was mainly by paracellular path, consequence in the lower percentage of particulate uptake but the higher percentage of particulate permeation within the same period of time since paracellular process was a more rapid process than transcellular pathway. All formulations selected to further study in experimental animal were relatively non-toxic to nasal tissue. Induction of nasal immune response following nasal administration of various dose of JE vaccine and JE vaccine entrapped in different size and surface charge of PLGA particles was evaluated. The immunogenicity of 10,40 and 80µg dose of JE in Balb/C mice was relatively equal but considerably lower compared to subcutaneous route with the same frequency of administration. The 10µg dose entrapped in 1,5 and 15µm PLGA particles was selected to inoculate in Balb/C mice and found that particles could enhance more level of immunological titer compared to vaccine alone and 1µm could elicit the highest level but still not comparable to subcutaneous route and repeated vaccination dose remained necessitate. After conjugation by CS and Al(OH)₃, the titer seemed to persist for a longer period of time with much higher level compared to unconjugated particles and the booster dose need not be required.
