Synthesis, Physicochemical and Biological Characterization of Stimuli-Responsive Nanomedicines towards the Intracellular Delivery of Therapeutics

• Ano de defesa: 2021
• Autor(a) principal: Albuquerque, Lindomar Jose Calumby
• Orientador(a): Giacomelli, Fernando Carlos
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal do ABC
• Programa de Pós-Graduação: Programa de Pós-Graduação em Biotecnociência
• Link de acesso: http://biblioteca.ufabc.edu.br/index.php?codigo_sophia=122256&midiaext=79582; http://biblioteca.ufabc.edu.br/index.php?codigo_sophia=122256&midiaext=79582/index.php?codigo_sophia=122256&midiaext=79581

The intracellular delivery of therapeutic molecules such as nucleic acids or drugs requires a nanocarrier to protect them from degradation and to increase the circulation time. These therapeutic-loaded nanocarriers (nanomedicines) can target different diseases by improving the pharmacokinetic behavior and pharmacodynamic profile, thus optimizing release and biodistribution. This thesis is divided into five chapters. The first one is dedicated to a general introduction to nanomedicines and the others address different platforms for gene and drug delivery. In the second chapter, we investigated the use of lactose-functionalized branched polyethyleneimine (BPEI-Lac) as a nonviral gene delivery vector towards reduced cytotoxicity of the well-known transfecting agent branched polyethyleneimine (BPEI). The substitution of primary amine groups by lactose residues in the BPEI chains leads to larger polyplexes presumably due to the higher polymer amount required for complete DNA condensation. Nevertheless, the sugar functionalization reduced substantially the cytotoxicity of the assemblies. The BPEI-Lac polyplexes are uptaken to higher extent by cells, whereas the levels of gene expression are maintained as compared to BPEI which is known for its high transfection efficiency. We accordingly demonstrate the preparation of less cytotoxicity polyplexes while maintaining the levels of gene expression. The third chapter was dedicated to the used of lipopeptides as gene vectors, which are other alternatives to PEI. We have explored the solid phase peptide synthesis to fabricate four lipopeptides containing succinyl-tetraethylene pentamine (Stp) building blocks capable to bind to DNA at physiological pH. The structural features of the produced lipopolyplexes were assessed by light scattering and the biological evaluations demonstrated efficient HeLa, Neuro-2a and ARPE-19 cell transfection combined to lower levels of cytotoxicity compared to PEI. The lipopeptide sequences demonstrated to be promising platforms for gene delivery. The fourth chapter was dedicated to the potential use of pH-responsive block copolymers for the intracellular delivery of doxorubicin (DOX). Several PHPMAm-b-PDPAn diblock copolymers were synthesized by RAFT polymerization and characterized by 1H NMR and SEC. Self-assembled vesicular structures (also known as polymersomes) with a desirable size for cellular uptake were produced by microfluidics. They were further characterized by SLS, ELS, TEM and cryo-TEM. DOX was further loaded in the polymersomes and subsequently the cellular uptake and cytotoxicity were evaluated in EL4 lymphoma cancer cells. A fluorescent dye (DBCO-Cyanine7) was covalently attached to the polymersomes by click chemistry and the in vivo biodistribution of the polymersomes performed in nude mice showed that the polymeric vesicles were preferably accumulated in the liver and tumor. The biodistribution studies demonstrated long blood circulation life-time of the polymersomes (~ 48h). In vivo antitumor efficacy was analyzed in mice bearing EL4 lymphoma tumor. The results evidenced tumor shrink and increased survival rate compared to the administration of free DOX. Side-effects characteristic of therapeutic treatments based on DOX, such as hair loss and cardiotoxicity, were also notably reduced. We report our final considerations in the fifth chapter of this Thesis.