ABSTRACT
Regards to the weak immunigenicity of tumor cells as well as recombinant antigenic markers [like BLP25], adjuvants are needed for enhancement of immune response and it's skewing toward cellular immunity. In this research poly [lactide-co-glycolide] [PLGA] microspheres were used as delivery system for a recombinant mucin named mucin 1 [MUCl, BLP25] as a recombinant antigenic cancer marker, and CpG-ODN was used for enhancement of immune response and its biasing toward cellular immunity. PLGA microspheres encapsulated with BLP25 and CpG-ODN were prepared using a w/o/w emulsion method. Encapsulation of BLP25 was determined by a HPLC method and spectrophotometry at 260 nm was used for quantification of encapsulated CpG-ODN. In vivo immunization studies were performed by SC injections of 20 micro g BLP25 and 4 micro g CpG-ODN in mice [4 animal per group]. Group I] Mice immunized with microspheres co-encapsulated with BLP25 and CpG; Group II] Mice immunized with microspheres encapsulated with BLP25 mixed with CpG solution; Group III] Mice immunized with microspheres encapsulated with BLP25. For evaluation of specify of immune response, T lymphocytes separated from different groups of mice were incubated with different antigens [T Cell Proliferation Assay]. IFN-gamma and IL-4 cytokines were assayed by a sandwich ELISA method. T lymphocytes separated from group I, showed high proliferation [stimulation index = 25] and high levels of IFN-y interferon [11200+/-172 pg/ml] which were significantly higher than other two groups [P<0.0001]. Co-encapsulation and co-delivery of MUCl and CpG-ODN produced high cellular [Thl] immunity responses [high levels of IFN-gamma and no IL-4], indicating the high adjuvanticity potential of CpG-ODN for immunization against cancer markers. Importance of co-encapsulation of antigen and adjuvant in the same delivery system for better adjuvant effect was also approved
ABSTRACT
Poor absorption of protein antigens across mucosal membranes necessitates the use of drug delivery systems. Pre-formed dextran microspheres [sephadex] possess a good potential for delivery of protein and peptide drugs to mucosal membranes. However they have rarely been used for mucosal immunization. Regards the advantages of mucosal over parenteral immunization and also the penetration enhancement potential of dextran microspheres, we examined their efficacy as mucosal antigen delivery system. Dextran microspheres loaded with tetanus toxoid [TT] were prepared by suspending microspheres in TT solution followed by freeze-drying. Size and morphological features of microspheres were studied by optical and electron microscopes. Encapsulation efficiency of TT was determined by micro-BCA protein assay. In vitro release study was performed in a model, simulating nasal cavity. Immunoreactivity of encapsulated antigen was assayed by ELISA. Gamma-scintigraphy [GS] was used for determination of mucoadhesion and clearance rate of microspheres, by spraying of technetiumlabelled microspheres to the nose of human volunteers. Local irritating potential of microspheres was studied by nasal administration to human nose. Microspheres with a mean diameter of about 150 micro m and a TT encapsulation efficiency of 20.3% [n=3] were obtained. Antigenicity of encapsulated TT was determined as 90.5+/-1.8% [n=3] that of the original TT. Release studies showed that almost 100% of TT was released in 1 h. GS studies showed that after 4 h, 36.9+/-3.4% [n=4] of microspheres were cleared from nasopharynx region of volunteers. Considering the suitable release characteristics, desirable preservation of the antigenic activity of TT, appropriate mucoadhesive properties and also safety of TT loaded Sephadex microspheres, these microspheres could be used as a proper mucosal antigen delivery system. However further studies are needed regarding the immune response induced by these systems in an animal model