Development of cationic polymeric nanoparticles for plasmid DNA vaccine delivery

• Autor(a) principal: Nunes, Renato Pereira
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/11284
• topic: Gelificação Ionotrópica; Infecção do Hpv; Nanopartículas; Quitosano; Vacinas de Dna Plasmídico; Domínio/Área Científica::Engenharia e Tecnologia::Bioengenharia

Cancer is the second most prevalent cause of death in the world and does not yet have a universal cure. Human papillomavirus (HPV) is among the main carcinogenic pathogens and its infection is related to several cancers, such as cervical and oropharyngeal cancers. Cervical cancer is the 4th largest cause of cancer in women worldwide. Nowadays it is considered one of the most common public health issue, principally in middle-aged women group, especially in less developed countries. The two major infectious papillomavirus genotypes are HPV16 and HPV18, and they are considered to be the most oncogenic and responsible for more than 70% of cervical cancers. Among the HPV virus oncoproteins the E7 oncoprotein inhibits the activity of the retinoblastoma protein (pRb), leading to a deregulation of the cell cycle and consequent uncontrolled growth of cells. Vaccination is considered to be the greatest contribution to global public health interventions of the latest centuries. The evolution and consequent contribution of vaccines are responsible for an impressive increase in general life expectancy. However, the current vaccines against the HPV do not have a therapeutic effect when the patient is already infected. This means that the vaccine can only prevent the infection by the HPV, but cannot treat or revert cancers induced by pre-existing and persistent HPV infections. In this way, DNA vaccines can be a promising solution for the effective treatment of HPV-infected individuals, since they can induce preventive and therapeutic immune responses. In order to be effective, the naked DNA needs to be internalized into the eukaryotic cell nucleus without degradation and the target gene expression is also dependent on the non-degradation of the DNA molecule. However, the internalization efficiency is low, due to the repulsion of the DNA by the membrane. Intramuscular delivery is one of the main routes for DNA vaccine administration. Nevertheless, it requires large amounts of the DNA administered and external stimulation to encourage the internalization of the DNA into the eukaryotic cells. In this work, we consider biocompatible drug delivery systems that can protect, carry and help the cellular internalization of DNA vaccines. This can lead to alternative and less invasive administration routes, such as intranasal administration. In this context, Chitosan (CS) polyplexes using sodium tripolyphosphate (TPP) as a crosslinker were designed and prepared using the ionotropic gelation method. Several parameters that may affect the systems formulation were investigated, including different TPP and CS volumes and concentrations, DNA concentrations, flow rate speed addition of TPP/DNA/pDNA solution. These nanocarriers were characterized in terms of size, surface charge, encapsulation efficiency, morphology, stability and cellular viability exploring two different cell lines. Ultimately, the verification of cell internalization and consequent target gene expression encoded in the DNA vaccine was also verified by reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription-quantitative real-time. The results showed that the variation of parameters allowed to overcome an ordinary challenge regarding the CS systems achieving monodisperse nanoparticles (NPs) with good size, bellow 200 nm and encapsulation efficiency higher than 60%. The nanoparticles analyzed by scanning electron microscopy (SEM) present spherical or oval shapes in nano sizes. Stability studies demonstrated that the polyplexes are able to protect encapsulated DNA from serine proteases, trypsin, DMEM-F12 medium supplemented with 10% of fetal bovine serum, and Dulbecco´s Modified Eagle´s Medium with High Glucose (DMEM-HG) supplemented with 10% not inactivated fetal bovine serum. Resazurin assay showed that the systems are biocompatible supporting that no cytotoxicity is induced. In addition, the PCR and RT-qPCR results showed that produced nanoparticles have the desired and intended effect, showing that they are suitable for cell uptake, internalization, and gene expression. In conclusion, the presented results revealed that the CS-TPP-pDNA polyplexes are suitable as a good nanocarrier for plasmid DNA vaccine delivery.