Browsing by Author "Rodolfo, Carlos Afonso"
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- Optimization of chitosan-based nanosystems to deliver plasmid DNA vaccinesPublication . Rodolfo, Carlos Afonso; Sousa, Ângela Maria Almeida de; Costa, Diana Rita Barata; Ferreira, Helena Isabel Fialho Florindo RoqueCancer is the second leading cause of death worldwide and it is estimated that soon it could become the first. Cervical cancer is the fourth most common cancer among women worldwide and in underdeveloped countries, it is the most common cancer among women. This cancer is most often caused by the Human Papilloma Virus (HPV). The incidence of this cancer has decreased in countries with good health systems due to a vaccination and prevention programme against this virus. Currently, vaccines are based on viruslike particles constructed from the recombinant expression of the L1 protein, which belongs to the HPV capsid. However, existing vaccines are only preventive and have no th erapeutic effect if the vaccine is administered to a person who has previously contracted the infection. Currently , DNA vaccines constitute an innovative tool with great potential to help in the battle against viral infections or from other pathogens , and against various types of cancer. To deliver the DNA to eukaryotic cells, a delivery system must be created to protect, transport and deliver the DNA to the target cells. DNA vaccines aim to encode genes, characteristic of pathogens or cancer cells, in orde r to activate and generate specific immune responses. In this context, the development of a DNA vaccine encoding the HPV E7 oncoprotein, responsible for interfering with the tumour suppressor retinoblastoma protein, which is responsible for regulating the cell cycle of eukaryotic cells, may be an effective tool against HPV potential and therapeuticinduced cervical cancer, due to its preventive effects. To maximise the efficiency of these vaccines, the development of an appropriate delivery system is critic al. Thus, the present work was based on the optimization of the formulation of delivery systems based on the chitosan polymer, and four types of chitosan with different molecular weights were explored: high molecular weight chitosan (HMW, 200500 kDa), low molecular weight (LMW, 50 to formulate the systems was the 190 kDa), 20 kDa and 5 kDa. The technique used ionotropic gelation, based on ionic crosslinking. The nanoparticle formulation resulted from the interaction positively charg ed chitosan polymers, and the established negatively charged between the crosslinker sodium tripolyphosphate (TPP) and the plasmid DNA (pADN), encoding mutated E7. The inclusion of TPP aims to compact the formed nanoparticles and increase their stability , thus favouring a more de fined round shape. To achieve the best delivery systems, a design of experiments (DoE) tool was used. This tool allowed combining several parameters/inputs simultaneously (chitosan and TPP concentrations) to obtain the optimal formulation conditions of the systems based on the chosen responses (size, polydispersity index (PDI) and zeta potential). After performing the proposed tests and characterizing the properties of the obtained formulations, the respective responses were introduced in the DoE program an d a statistical analysis was performed. The linear and quadratic models obtained were statistically significant (pvalue <0,05) and the "lack of al points for each chi fit" was not significant, with an adequate determination coefficient. The predicted optim tosan polymer were all successfully validated. Subsequently, further studies were conducted to evaluate the properties of the delivery systems formulated with the conditions defined at the optim al points. Scanning electron microscopy (SEM) analyses were performed to evaluate the morphology, Fourier transform infrared spectroscopy was used to evaluate the chemical properties and specific functional groups, while different stability tests were perfor med to evaluate the strength and DNA release, and finally cytotoxicity tests were performed to ensure the biocompatibility of the chitosan nanoparticles. The four delivery systems developed , presented satisfactory characteristics , such as spherical or oval stability and strength, DNA retention and good biocompatibility. nanoparticles, good In this sense, the DoE tool proved to be a powerful tool to explore and tailor the characteristics of chitosan/TPP/pADN nanosystems, allowing the development of a system suitable for the encapsulation, transport and delivery of pADN, thus providing the advancement of the DNA vaccine delivery research field.