A novel approach to a fine particle coating using porous spherical silica as core particles.

Abstract The applicability of porous spherical silica (PSS) was evaluated as core particles for pharmaceutical products by comparing it with commercial core particles such as mannitol (NP-108), sucrose and microcrystalline cellulose spheres. We investigated the physical properties of core particles, such as particle size distribution, flow properties, crushing strength, plastic limit, drying rate, hygroscopic property and aggregation degree. It was found that PSS was a core particle of small particle size, low friability, high water adsorption capacity, rapid drying rate and lower occurrence of particle aggregation, although wettability is a factor to be carefully considered. The aggregation and taste-masking ability using PSS and NP-108 as core particles were evaluated at a fluidized-bed coating process. The functional coating under the excess spray rate shows different aggregation trends and dissolution profiles between PSS and NP-108; thereby, exhibiting the formation of uniform coating under the excess spray rate in the case of PSS. This expands the range of the acceptable spray feed rates to coat fine particles, and indicates the possibility of decreasing the coating time. The results obtained in this study suggested that the core particle, which has a property like that of PSS, was useful in overcoming such disadvantages as large particle size, which feels gritty in oral cavity; particle aggregation; and the long coating time of the particle coating process. These results will enable the practical fine particle coating method by increasing the range of optimum coating conditions and decreasing the coating time in fluidized bed technology.

Enhanced CTL response by controlled intracellular trafficking of antigen in dendritic cells following DNA vaccination.

To elicit a cytotoxic T lymphocytes (CTL) response efficiently after DNA vaccination, we constructed several plasmid DNA (pDNA) vectors encoding the major histocompatibility complex (MHC) class I-restricted epitope peptide (SIINFEKL) of ovalbumin (OVA) or OVA protein with modified intracellular trafficking. An in vitro antigen presentation assay was carried out using DC2.4 cells, a dendritic cell line, to examine the potentials of the constructs following direct transfection. Among the vectors, pPep-ER, pDNA encoding antigen peptide combined with an endoplasmic reticulum (ER)-retention signal, exhibited a significant ability of antigen presentation compared with the counterpart without the signal. Based on the in vitro results, we carried out in vivo immunization experiments using pPep-ER via the intradermal or intramuscular route in combination with electroporation in mice. pPep-ER showed an efficient antigen-specific CTL induction and the effect was superior to that exhibited by the positive control, OVA in complete Freund's adjuvant (CFA). The levels of interferon gamma (IFN-gamma) released from spleen cells were significantly increased by pPep-ER compared with pPep-free. Immunization with pPep-ER also exhibited a high inhibitory effect on the growth of E.G7 tumor. These results indicate that DNA vaccination with the pDNA vector expressing a MHC class I epitope peptide with controlled intracellular trafficking is a promising method of inducing an antigen-specific CTL response via direct presentation.

Enhanced antigen-specific antibody production following polyplex-based DNA vaccination via the intradermal route in mice.

DNA vaccination is an attractive approach with various advantages over conventional vaccination. The present study was undertaken to examine whether polyplex-based DNA vaccination could be used to modulate immune responses by plasmid DNA (pDNA). Methylated bovine serum albumin (mBSA) was used as a model of a cationic macromolecular carrier of pDNA encoding obalbumin (OVA) and the effects of polyplex formation of pDNA with mBSA on the antigen-specific immune responses were examined. Anti-OVA IgG antibody production was significantly increased following intradermal immunization with the polyplex compared with naked pDNA, although the induction of cytotoxic T lymphocyte activity was lowered by polyplex formation. We also demonstrated that the disposition and gene expression of pDNA following intradermal injection could be manipulated by polyplex formation. Intriguingly, we also found that the migration of dendritic cells to the injected site could be induced by polyplex formation probably due to a high level of tumor necrosis factor alpha production from the keratinocytes treated with mBSA/pDNA complexeses. Thus, the present study has demonstrated that the immune responses could be biased towards a Th2-type response by polyplex-based DNA vaccination through manipulation of not only pDNA disposition but also dendritic cell migration.

Manipulation of local disposition and gene expression characteristics of plasmid DNA following intramuscular administration by complexation with cationic macromolecule.

To modulate the immune responses of DNA vaccine, it is very important to control the disposition and gene expression of plasmid DNA (pDNA) after local administration. We chose methylated bovine serum albumin (mBSA), a cationic macromolecule, as a carrier of pDNA. We examined the effects of complexation of pDNA with mBSA on the disposition and gene expression in mice after intramuscular administration. The elimination from injection site was retarded and the accumulation to lymph nodes was increased at the positively charged mBSA/pDNA complexes. As the charge ratios of mBSA/pDNA complexes were higher, the levels of gene expression were reduced. Antigen specific immune responses were evaluated using pDNA encoding ovalbumin (OVA), pCMV-OVA, as a model antigen-expressing pDNA. However, significant levels of production of anti-ovalbumin IgG antibody were obtained in mice immunized with a positively charged complex, mBSA/pCMV-OVA (8:1) (weight ratio). In vitro experiments using DC2.4 cells, a murine dendritic cell line, demonstrated that the levels of gene expression and cytokine release were increased by complexation. These results suggest that the immune responses might be manipulated by complexation presumably due to the altered disposition and gene expression of pDNA.