Dry-powder form of chitosan nanospheres containing influenza virus and adjuvants for nasal immunization.

The objective of this study was to develop and statistically optimize chitosan nanospheres. For this purpose chitosan powder was turned into nanospheres using tripolyphosphate as a crosslinker and through ionic gelation. D-optimal response surface design was applied to optimize the nanospheres. Their size and polydispersity index (PDI) were measured as the dependant variables. Then the inactivated influenza virus and/or CpG ODN or Quillaja saponin (QS) were incorporated into the chitosan nanospheres. The release profiles of the antigen and both adjuvants were obtained. The toxicity of the formulations was tested by XTT using Calu 6 cell lines. The size distribution and PDI of plain chitosan nanospheres was 581.1 ± 32.6 and 0.478 ± 0.04. After 4 h the release of antigen, QS and CpG from the chitosan matrix were 33, 36 and 62%, respectively. The inactivated virus remained intact during preparation, as revealed by the SDS-PAGE method. Differential scanning calorimetry and Fourier Transform Infrared Spectroscopy indicated no serious structural changes in the chitosan carrier in the presence of either the antigen or the immunoadjuvants. Although the antigen loaded into chitosan nanospheres showed slight cytotoxicity on lung-cancer cells, co-encapsulation of the adjuvant (especially CpG) lowered this effect. The results demonstrated that chitosan as a carrier and immunostimulator, along with CpG or QS adjuvants, creates a potential influenza vaccine delivery system which can be administered nasally.

Immunization against leishmaniasis by PLGA nanospheres loaded with an experimental autoclaved Leishmania major (ALM) and Quillaja saponins.

Immune responses against the Leishmania antigens are not sufficient to protect against a leishmania challenge. Therefore these antigens need to be potentiated by various adjuvants and delivery systems. In this study, Poly (d,l-lactide-co-glycolide (PLGA) nanospheres as antigen delivery system and Quillaja saponins (QS) as immunoadjuvant have been used to enhance the immune response against autoclaved Leishmania major (ALM). PLGA nanospheres were prepared by a double-emulsion (W/O/W) technique. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. Mean diameter for nanospheres loaded with ALM+QS was 294 ± 106 nm. BALB/c mice were immunized three times in 3-weeks intervals using ALM plus QS loaded nanospheres [(ALM+QS)PLGA], ALM encapsulated with PLGA nanospheres [(ALM)PLGA], (ALM)PLGA + QS, ALM + QS, ALM alone or PBS. The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection, showed by significantly (P < 0.05) smaller footpad, were observed in mice immunized with (ALM)PLGA. The (ALM+QS)PLGA group showed the least protection and highest swelling, while the (ALM)PLGA+QS, ALM+QS and ALM showed an intermediate protection with no significant difference. The mice immunized with ALM and ALM+QS showed the highest IgG2a/IgG1 ratio (P < 0.01), followed by (ALM)PLGA+QS. The highest IFN-γ and lowest IL-4 production was seen in (ALM)PLGA+QS, ALM+QS groups. The highest parasite burden was observed in (ALM)PLGA+QS and (ALM+QS)PLGA groups. It is concluded that PLGA nanospheres as a vaccine delivery system could increase the protective immune responses, but QS adjuvant has a reverse effect on protective immune responses and the least protective responses were seen in the presence of this adjuvant.

Nasal immunization studies by cationic, fusogenic and cationic-fusogenic liposomes encapsulated with tetanus toxoid.

Particulate antigens are more effective than soluble antigens in induction of systemic and mucosal immunity; possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization with tetanus toxoid (TT) entrapped in cationic, fusogenic and cationic-fusogenic liposomes. Liposomes containing TT were prepared by dehydration-rehydration method. The volume mean diameter of cationic, fusogenic and cationic-fusogenic liposomes were 3.4 +/- 0.6, 4.3 +/- 2.3 and 3.4 +/- 1.5 microm, respectively. Encapsulation efficiency of TT in cationic, fusogenic and cationic-fusogenic liposomes was respectively determined as 49.1 +/- 8.4%, 48.5 +/- 2.1% and 50.8 +/- 4.9%. After 3 months, the leaking of encapsulated TT from liposomes ranged between 2.02 - 5.46%. Immunoreactivities of encapsulated TT in all kinds of liposomes were completely preserved, as studied by Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Enzyme-Linked Immunosorbent Assay (ELISA). The highest serum immunoglobulin G (IgG) and antitoxin titers were observed in groups immunized with solution formulation (P< 0.001). However, the highest mucosal secretory IgA (sIgA) titers were achieved by fusogenic liposomes (five times more titers compared with TT solution, and 15 times more titers compared with i.m. vaccine), followed by cationic-fusogenic liposomes. No hemolysis was occurred on incubation of liposomes and human erythrocytes. Also after nasal administration of plain liposomes to human volunteers, no local irritation was seen. This study suggests that intranasal administration of fusogenic and cationic-fusogenic liposomes encapsulated with vaccines could be an effective way for inducing mucosal immune responses.

Formulation, characterization and release studies of alginate microspheres encapsulated with tetanus toxoid.

Alginate is a safe, non-immunogenic and inexpensive natural polymer with high mucoadhesive properties. Alginate microspheres can be used as a delivery system for antigens to mucosal surfaces. In the present study alginate microspheres were prepared by an emulsification technique. The effects of sonication time, concentration of alginate, emulsifier and calcium chloride, and also the volume of calcium solution, were evaluated on mean size, size range, surface roughness and porosity, sphericity and clumping of microspheres using an optical microscope and particle size analyzer. The most desirable conditions were 90 s sonication, 3% alginate solution, 2% surfactant and 60 ml of 0.33% CaCl2 in octanol. The resulting microspheres had a mean size of 1.34 +/- 0.3 microm and size range of 0.3 +/- 2.0 microm, with no surface roughness and porosity, low clumping and high sphericity. The encapsulation efficiency was about 47.7%. All batches showed nearly the same release profiles with a low burst release. The stability of the model antigen (tetanus toxoid (TT)) extracted from microspheres was confirmed by SDS-PAGE; and the antigenicity of TT was studied by ELISA and found to be 91 +/- 5% of the original TT. It can be concluded that, with regard to the size and morphological characteristics of the prepared microspheres and their ability in preserving the antigenicity of the encapsulated TT, they could be used as a delivery system for mucosal delivery of TT.