Development of alternative micro and nanoparticulate polymeric systems for mucosal delivery of Streptococcus equi antigens

• Author(s): Florindo
• Date: 2008
• Persistent ID: http://hdl.handle.net/10451/262
• Origin: Repositório da Universidade de Lisboa
• Subject(s): Tecnologia farmacêutica; Teses de doutoramento - 2008
• Document Type: Doctoral thesis
• Language: English
• Advisor(s): Almeida, António José Leitão das Neves, 1963-; Alpar, Hazire Oya
• Contributor(s): Repositório da Universidade de Lisboa

Description

Tese de doutoramento em Farmácia (Tecnologia Farmacêutica), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008

S. equi is considered the causative agent of strangles, a very contagious disease of the upper respiratory tract of the Equidae. Although S. equi is sensitive to some antibiotics, most of the current treatments are ineffective. During the recovery period, horses develop a protective immune response mostly against the antiphagocytic cell wall-associated M-like protein SeM, which encourages the development of efficient vaccines. The main purpose of this thesis was to develop and characterise stable polymeric particles and study their potential as mucosal S. equi antigens adjuvant. Non-aggregated and easily dispersible micro and nanospheres based on poly-e-caprolactone (PCL) and poly(lactic acid) (PLA) polymers were formulated by the double emulsion (w/o/w) solvent evaporation method and fully characterised. BALB/c monocyte macrophages (cell line J774.1A) were used in toxicity tests and cellular uptake studies. The influence of mucoadhesive polymers (chitosan (CS), glycolchitosan (GCS) and alginate (ALG)) and absorption enhancers (spermine (SP) and oleic acid (OA)) in particle physicochemical characteristics and consequently in the humoral, mucosal and cellular immune responses was as well evaluated. The non-toxic cholera toxin B subunit (CTB) is a potent mucosal adjuvant, and the oligodeoxynuleotide with repeating C and G motives (CpG) can also be used to up-regulate mucosal and cellular immune responses, increasing preferentially the T helper type 1 (Th1) cell activity. Therefore, those adjuvants were co-administrated with soluble antigen and non-modified PLA and PCL particles, in order to assess their eventual synergic effect. Inspite of administration route (i.n. and i.m.), S. equi-loaded polymeric systems were able to significantly increase systemic and cellular immune responses, when compared with free antigens, isolated or co-admixed with CTB or CpG. PLA-loaded particles generally induced higher mucosal antibody levels when compared with the correspondent PCL. Similar results were attained when the immune responses induced by S. equi antigens-adsorbed particles were compared with those elicited by the correspondent entrapped ones. As per PCL nanospheres, those formulated with ALG and GCS, seem to induce a more balance Th1/Th2 immune response. This study also confirmed the potential adjuvant of S. equi antigen-loaded PLA nanospheres, and again the mucopolysaccharide GCS induced the most prominent immune response. SeM recombinant proteins were entrapped in PLA nanospheres and their adjuvant potential after i.m. administration in a mouse model was compared with that achieved with S. equi extract. In fact, it was possible to observe that PLA-GCS and PLA-OA nanospheres are alternative cost-effective preparations, able to induce a balanced IgG2a/IgG1 immune response. These studies bring new insights into the strangles prevention field as the particulate carriers developed during this PhD thesis, mainly those containing GCS, are promising adjuvants for a safe vaccine against strangles, with no toxicity issues associated to their utilisation, in contrast to other adjuvants that have been associated to S. equi antigens. Therefore, the main goal of this thesis was accomplished but challenge studies must be done in order to support their future utilisation.

Fundação para a Ciência e Tecnologia (FCT) e FEDER (SFRH/BD/14370/2003, POCI/BIO/59147/2004 e PPCDT/BIO/59147/2004)