Alginate-chitosan particulate delivery systems for mucosal immunization against tuberculosis

Although vaccination is still the most cost-effective strategy for tuberculosis control, there is an urgent need for an improved vaccine. Current BCG vaccine lacks efficacy in preventing adult pulmonary tuberculosis, the most prevalent form of the disease. Targeting nasal mucosa, Mycobacterium tuberculosis infection site, will allow a simpler, less prone to risk of infection and more effective immunization against disease. Due to its biodegradable, immunogenic and mucoadhesive properties, chitosan particulate delivery systems can act both as carrier and as adjuvant, improving the elicited immune response. In this study, BCG was encapsulated in alginate and chitosan microparticles, via a mild ionotropic gelation procedure with sodium tripolyphosphate as a counterion. The particulate system developed shows effective modulation of BCG surface physicochemical properties, suitable for mucosal immunization. Intracellular uptake was confirmed by effective transfection of human macrophage cell lines.

Chitosan-alginate microparticulate delivery system for an alternative route of administration of BCG vaccine

Immunisation against M. tuberculosis with current available BCG vaccine lacks efficacy in preventing adult pulmonary tuberculosis. Targeting nasal mucosa is an attractive option for a more effective immunization. The delivery of BCG via the intranasal route involves overcoming barriers such as crossing the physical barrier imposed by the mucus layer and ciliar remotion, cellular uptake and intracellular trafficking by antigen presenting cells. Due to its biodegradable, immunogenic and mucoadhesive properties, chitosan particulate delivery systems can act both as vaccine carrier and adjuvant, improving the elicited immune response. In this study, different combinations of Chitosan/Alginate/TPP microparticles with BCG were produced as vaccine systems. The developed microparticle system successfully modulates BCG surface physicochemical properties and promotes effective intracellular uptake by human macrophage cell lines Preliminary immune responses were evaluated after s.c. and intranasal immunisation of BALB/c mice. BCG vaccination successfully stimulated the segregation of IgG2a and IgG1, where intranasal immunisation with chitosan/alginate particulate system efficiently elicited a more equilibrated cellular/humoral immune response.

Effect of experimental parameters on alginate/chitosan microparticles for BCG encapsulation

The aim of the present study was to develop novel Mycobacterium bovis bacille Calmette-Guérin (BCG)-loaded polymeric microparticles with optimized particle surface characteristics and biocompatibility, so that whole live attenuated bacteria could be further used for pre-exposure vaccination against Mycobacterium tuberculosis by the intranasal route. BCG was encapsulated in chitosan and alginate microparticles through three different polyionic complexation methods by high speed stirring. For comparison purposes, similar formulations were prepared with high shear homogenization and sonication. Additional optimization studies were conducted with polymers of different quality specifications in a wide range of pH values, and with three different cryoprotectors. Particle morphology, size distribution, encapsulation efficiency, surface charge, physicochemical properties and biocompatibility were assessed. Particles exhibited a micrometer size and a spherical morphology. Chitosan addition to BCG shifted the bacilli surface charge from negative zeta potential values to strongly positive ones. Chitosan of low molecular weight produced particle suspensions of lower size distribution and higher stability, allowing efficient BCG encapsulation and biocompatibility. Particle formulation consistency was improved when the availability of functional groups from alginate and chitosan was close to stoichiometric proportion. Thus, the herein described microparticulate system constitutes a promising strategy to deliver BCG vaccine by the intranasal route.

Cellular uptake of BCG-loaded chitosan microparticles and in vivo evaluation of immune response following intranasal immunisation

Attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only currently available vaccine against tuberculosis. It is highly effective in pre-exposure immunisation against TB in children when administered by subcutaneous route to newborns. However, it does not provide permanent protection in adults. In this work, polymeric chitosan-alginate microparticles have been evaluated as potential nasal delivery systems and mucosal adjuvants for live attenuated BCG. Chitosan (CS) has been employed as adjuvant and mucosal permeation-enhancer, and, together with alginate (ALG), as additive to enhance BCG-loaded microparticles (MPs) cellular uptake in a human monocyte cell line, by particle surface modification. The most suitable particles were used for vaccine formulation and evaluation of immune response following intranasal immunisation of BALB/c mice.

Solid-state sensory properties of Calix-Poly(Phenylene Ethynylene)s toward nitroaromatic explosives

This study is primarily focused in establishing the solid-state sensory abilities of several luminescent polymeric calix[4]arene-based materials toward selected nitroaromatic compounds (NACs), creating the foundations for their future application as high performance materials for detection of high explosives. The phenylene ethynylene-type polymers possessing bis-calix[4]arene scaffolds in their core were designed to take advantage of the known recognition abilities of calixarene compounds toward neutral guests, particularly in solid-state, therefore providing enhanced sensitivity and selectivity in the sensing of a given analyte. It was found that all the calix[4]arene-poly(para-phenylene ethynylene)s here reported displayed high sensitivities toward the detection of nitrobenzene, 2,4-dinitrotoluene and 2,4,6-trinitrotoluene (TNT). Particularly effective and significant was the response of the films (25-60 nm of thickness) upon exposure to TNT vapor (10 ppb): over 50% of fluorescence quenching was achieved in only 10 s. In contrast, a model polymer lacking the calixarene units showed only reduced quenching activity for the same set of analytes, clearly highlighting the relevance of the macrocyclics in promoting the signaling of the transduction event. The films exhibited high photostability (less than 0.5% loss of fluorescence intensity up to 15 min of continuous irradiation) and the fluorescence quenching sensitivity could be fully recovered after exposure of the quenched films to saturated vapors of hydrazine (the initial fluorescence intensities were usually recovered within 2-5 min of exposure to hydrazine).

Produção enzimática de biodiesel a partir de resíduos agro-industriais usando a lipase imobilizada de origem microbiana

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica