Relatórios de Estágio e Monografia intitulada "Chitosan-based Nanoparticles for Gene Delivery"

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de FarmáciaA terapia genética caracteriza-se pela entrega de ácidos nucleicos às células de um indivíduo para compensar genes anormais ou com o objetivo de produzir uma proteína benéfica. Além disso, a terapia genética apresenta grandes vantagens para a área da vacinação, uma vez que é esperado que as vacinas de DNA gerem uma resposta imune tanto humoral, como celular, propiciando estratégias de vacinação profiláticas e também terapêuticas. Comparativamente aos sistemas de entrega viral, os vetores não-virais são uma alternativa mais segura, com poucos efeitos secundários, muito estáveis e suscetíveis a modificações físico-químicas. Entre estes sistemas transportadores, o quitosano, um polímero catiónico, tem suscitado interesse como vetor não-viral devido à sua biodegradabilidade, biocompatibilidade, baixa toxicidade e capacidade de interagir com as moléculas de DNA carregadas negativamente, formando facilmente poliplexos. No entanto, alguns autores afirmam que a interação entre as cargas opostas dos grupos amino do quitosano e dos grupos fosfato no ácido nucleico origina complexos muito estáveis, impedindo a libertação do DNA e causando baixa transfecção. Para superar esta limitação, formulámos a hipótese de que a adição de caseína, assim como o glucano, na estrutura das nanopartículas facilitaria uma transferência do DNA mais adequada.O trabalho aqui apresentado visa produzir Nanopartículas de Quitosano-α-Caseína (ChiCas NPs) e testar sua capacidade como um sistema de entrega de DNA através de estudos in vitro de transfecção. Os resultados obtidos demonstram que as ChiCas NPs, apresentando tamanhos pequenos e homogéneos com pequenas variações entre as diferentes formulações (variando entre 230 nm e 390 nm) e potenciais zeta marcadamente positivos (> 32 mV), possuem características e estabilidade adequadas para serem um bom candidato para um sistema de entrega génica. Deste modo, os resultados dos ensaios de complexação de DNA confirmaram a produção com sucesso de complexos NPs:DNA, seja com DNA adsorvido à superfície das NPs, ou com DNA incorporado nas NPs. Nos estudos preliminares com várias formulações constituídas por diferentes rácios de NPs:DNA observou-se uma baixa eficiência de transfecção, comparativamente ao controlo positivo.Para concluir, os resultados apresentados sugerem que as ChiCas NPs têm a capacidade de atuar como um sistema de entrega de genes, no entanto, é necessária uma investigação mais aprofundada para otimizar os rácios ChiCas NPs:DNA e assim obter uma formulação de DNA plasmídico bem-sucedida.Gene therapy is designated to introduce nucleic acids into the cells of a patient to compensate for abnormal genes or to produce a beneficial protein. Moreover, gene therapy presents great advantages for the vaccine field, since DNA vaccines are expected to generate both humoral and cellular immune responses, supporting prophylactic, as well as therapeutic vaccination strategies. Comparatively to viral gene delivery systems, non-viral vectors are a safer alternative, with minimal side effects, highly stable and susceptible to physical/chemical modifications. Among these gene carriers, the cationic polymer chitosan has gained attention as a non-viral gene delivery system due to its biodegradability, biocompatibility, low toxicity and ability to interact with the negatively charged DNA molecules, easily forming polyplexes. Nevertheless, some authors claim that the interaction between the opposing charges of the amino groups of chitosan and the phosphate groups in the nucleic acid originates very stable complexes, precluding the unloading of the DNA and causing low transfection. To overcome this limitation, we hypothesized that the addition of casein, as well as glucan, into the nanoparticle’s structure would facilitate a proper gene transfer. The work herein presented aims to produce Chitosan-α-Casein Nanoparticles (ChiCas NPs) and to test their ability as a gene delivery system using preliminary in vitro transfection studies. Results demonstrate that ChiCas NPs, presenting small and homogenous sizes with minor variations between different formulations (ranging between 230 nm and 390 nm) and highly positive zeta potentials (> 32 mV), have suitable characteristics and stability to be a good candidate for a gene delivery system. In fact, results from DNA complexation assays confirmed the successful production of NPs-DNA complexes, either with DNA adsorbed to the NPs surface, or with DNA incorporated within the NPs. Preliminary studies with several NPs-DNA formulations and ratios suggested a low transfection efficiency, comparatively to the positive control. To conclude, the results presented suggest that ChiCas NPs have high ability to form complexes with DNA, however, further investigation is needed to optimize ChiCas NPs: DNA ratios for successful plasmid DNA-based vaccination

Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 μg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans