Pandit, Sreenivas; (2008) Particulate Carriers for Vaccines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Soluble antigens and antigens synthesized by recombinant DNA techniques are weakly immunogenic and need protection against passage through the mucosal epithelium. Encapsulation of antigens into polymeric carriers is one of the techniques developed for delivering these agents. However, this technology needs further improvements to enhance the immune responses and stabilization of the antigen within the carriers. The incorporation of adjuvants to enhance immune responses is well known, and therefore incorporation of adjuvants with the polymeric carriers could also be beneficial. The focus of this study, therefore, was to co-encapsulate known and unknown adjuvants with polymeric particles. Polymeric particulate carriers were prepared using the solvent evaporation or spray drying technique. The biodegradable polymers selected were polylactic acid and polycaprolactone. Diphtheria toxoid (DT) and hepatitis B surface antigen (HBsAg) were used as model antigens. The adjuvants used for co-encapsulation with antigens were penetration enhancers and mineral compounds. These adjuvants were either mixed with the polymer in the organic phase or in the internal phase during the preparation of the particles. The penetration enhancers used in the study were tocopherol acetate (TA), tocopherol nicotinate (TN), D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), cholic acid, lithocholic acid and melittin. Mineral compounds such as alum, zinc sulphate and zinc oxide were also co-encapsulated to evaluate their effect on the physicochemical properties and adjuvancy of the carriers. As surface charge plays an important role in the particle uptake, chitosan was used as an external phase stabilizer in some formulations to impart a positive charge on the particles. The penetration enhancers used here have not been used to co-encapsulate antigens in polymeric particles before, and there is not much data available for co-encapsulation of mineral compounds. The formulations were characterized with respect to size, surface charge, encapsulation efficiency, uptake in the cell lines and toxicity. These formulations were also evaluated in mice for antibody responses and cytokine analysis. Incorporation of tocopherol derivatives into the particulate formulations significantly increased the encapsulation efficiency of the antigen. The in vitro toxicity studies showed that these formulations were non-toxic. In vivo, the HBsAg-loaded particles containing TPGS significantly improved the serum antibody levels when compared with free HBsAg. TA and TN also improved the serum antibody levels when administered intramuscularly and intranasally. However, the intramuscular responses were higher than the intranasally administered formulations. DT-loaded particles containing TPGS significantly improved the antibody levels. Co-encapsulation of zinc sulphate and alum in nanoparticles improved the encapsulation efficiency of HBsAg. Sizes were in the range of 300 to 500nm for these formulations. The serum immune responses of particles containing zinc sulphate were equivalent to particles containing alum. However, the intranasal route elicited much lower antibody responses when compared with the intramuscular groups. Particles coated with chitosan significantly improved the antibody levels. The co-encapsulation of bile salts (cholic acid or lithocholic acid) and melittin along with HBsAg did not have any effect on the encapsulation efficiency. Particles containing bile salts had a higher cellular uptake compared with particles without bile salts. Particles containing bile salts or melittin significantly improved the antibody levels when compared with free antigen and antigen plus cholera toxin B.

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