Desenvolvimento de um microgel de alginato carreado com vetores adeno-associados para liberação sustentada

• Main Author: Cinel, Victor Dal Posolo [UNIFESP]
• Publication Date: 2020
• Format: Master thesis
• Language: por
• Source: Repositório Institucional da UNIFESP
• Download full: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=8464742; https://hdl.handle.net/11600/64275
• topic: Gene Therapy; Microfluidics; Adeno-Associated Vector; Alginate; Gene Carrier; Terapia Gênica; Microfluídica; Vetor Adeno-Associado; Alginato; Carreador Gênico

Gene therapy consists in manipulating and inserting exogenous genetic material in a patient to treat diseases. Although gene transfer technologies have greatly improved, there are still several limitations such as uncontrolled vector release that leads to immediate and excessive release of vectors at the site of administration. The consequence of this uncontrolled release is the leakage of vectors to other tissues, reducing gene transfer efficiency in the desired tissues and increasing the transfer of vectors to unwanted tissues. One solution to this problem is the use of vectors encapsulated in a biodegradable gel for slow and continuous release. Droplet microfluidics enabled the production of small and homogeneous microgels as carriers. Using alginate in a microfluidic device, it is possible to produce biocompatible alginate microgels with a sustained release kinetics of molecules. Despite being a promising carrier for gene therapy, there are no studies in the literature on the encapsulation of non-integrative viral vectors using microfluidics. In this work, 1.2% alginate microgel delivery system with type 2 adeno-associated vectors (AAV) was evaluated for its use for gene therapy. To this end, microgels encapsulate with model nanoparticles and AAVs were produced by microfluidics using the competitive ligand exchange crosslinking (CLEX) method. The microgels were produced and their encapsulation and controlled release capacity were evaluated by fluorescence spectroscopy, realtime quantitative PCR and fluorescence microscopy techniques, the latter to analyze the in vitro transduction efficiency of encapsulated AAVs. For the characterization, techniques of phase contrast microscopy, scanning electron microscopy and atomic force microscopy were used. Results showed that the pure 1.2% alginate microgels, microgels loaded with nanoparticles and microgels loaded with AAVs presented an average size of 125 µm, 106 µm and 116 µm, respectively. While pure and AAV microgels were monodisperse and with a regular topography, microgels with nanoparticles were monodisperse and showed an irregular and porous topography. The average encapsulation efficiency was 70.9% for nanoparticles and 13.2% for AAVs. Release kinetics studies have shown that the microgels produced can release encapsulated nanoparticles and AAVs in a continuous manner. In vitro transduction studies with HeLa cells using microgels with encapsulated AAVs showed that despite the low encapsulation efficiency, the AAVs released from the microgels were able to transduce cells, validating this delivery system for use in gene therapy studies.