Salomon, Stefan; (2008) Polysaccharide based nanocarriers for the delivery of antigens. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Most vaccines are administered parentally since they are absorbed poorly at mucosal sites. However, the nasal mucosa is a particularly attractive site for the delivery of vaccines due to its large absorption area and low proteolytic activity. The use of this route would result in both local and systemic immune responses. While most proteins are not well adsorbed from the nasal mucosa, particulate systems with protein antigens have shown great potential for nasal vaccination. The most commonly used polymers for the preparation of particulates are the polyesters PLGA and PLA. One of the major drawbacks of these polymers is their water insolubility necessitating the use of organic solvents which can cause problems with antigen stability. An alternative polymer to the polyesters is chitosan. Chitosan has been effectively used as a biomaterial for drug delivery to enhance mucosal absorption. However, a major disadvantage of chitosan is its insolubility at physiological pH whereas it is soluble and active as an absorption enhancer in acidic media. Recently, N-trimethyl chitosan (TMC), a derivative of chitosan has been used as a particulate and as a soluble adjuvant for vaccines. It is highly water soluble at neutral pH and protonated. This quaternised polysaccharide has a permanent positive charge and its toxicity is dependant on the degree of quaternisation. The aim of this research has been to investigate the potential of TMC nanoparticles for their utilisation as a mucosal vaccine adjuvant. Low molecular weight TMCs with different conversion degrees were synthesised and characterised by NMR and GPC. Nanoparticles were then prepared by a novel mild polyelectrolyte complexation with different polyanions such as poly(γ-glutamic acid), fucoidan, carboxymethylated laminarin and carboxymethylated pullulan. These polymers were selected due to their known adjuvanticities. The nanoparticles were characterised with respect to their size, charge and loading efficiency. Antigens employed in this study were bovine serum albumin, diphtheria toxoid and tetanus toxoid. The antigen integrity was investigated by SDS page. Both the polymers and the nanoparticles were tested for their toxicity by MTT assay (Calu-3 and HEK 293). In addition, the nanoparticles were prepared with FITC-BSA and cellular uptake studies were performed with a macrophage cell line (J774A.1). After the in-vitro characterisation female BALB/c mice were primed and boosted with antigen loaded nanoparticles via intramuscular, subcutaneous and intranasal route. Different formulations resulted in the particle sizes between 150 and 250 nm and with a positive surface charge. All particulate formulations were able to encapsulate almost 100% of the antigens used (1% (m/m) theoretical loading with respect to the polymers). This result was independent on the chosen antigen. The antigens retained full integrity due to the mild preparation method chosen. The nanoparticles appeared to be nontoxic up to a concentration of 5 mg/ml compared to TMC alone 0.1 mg/ml. In vivo studies revealed that the nanoparticles induced high serum IgG antibody titres. Furthermore, antibody isotyping showed high IgG1 antibody titres whereas the IgG2a serum antibody titres were much lower indicating a Th2 biased immune response. Some of the formulations were also able to elicit a significant disseminated mucosal immunity as significant levels of IgA response in the vaginal washes indicating a common mucosal response. Additionally, the co-encapsulation of CpG motifs enhanced the levels of IgG2a isotype titre. The amounts of IL-2, IL-4, IL-6 and INF-γ and TNF-α were measured following in-vitro spleen stimulation. They correlated well with the measured antibody titres for example showing higher amounts of IL-6. In conclusion, this study showed that it was possible to prepare non toxic TMC nanoparticles with a size range between 150 to 250 nm. All tested particulate formulations were able to elicit significant antibody titres as well as cytokine levels which revealed a Th2 biased immune response.

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