Photolyase production and nanoencapsulation for topical applications

• Autor(a) principal: Torres-Obreque, Karin Mariana Torres
• Data de Publicação: 2024
• Tipo de documento: Tese
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: https://www.teses.usp.br/teses/disponiveis/9/9135/tde-22072024-094811/
• topic: Biorreator de batelada alimentada; Fed-batch bioreactor; Fotoliase; Liposome; Llipossoma; Nanopartículas poliméricas; Photolyase; Polimerossoma; Polymeric nanoparticle; Polymersome

Ultraviolet (UV) radiation from sunlight can induce DNA damage, leading to the formation of photoproducts that can eventually result in skin cancer. Topical application of DNA repair enzymes, such as photolyase, represents an innovative strategy for active photoprotection. In this sense, photolyase nanoencapsulation offers a promising route to facilitate the access of enzymes to the deeper layers of the skin. This work aimed to produce the recombinant photolyase, characterize it, and investigate nanostructures for the enzyme encapsulation, namely polymersomes (PL), liposomes (LP) and polymeric nanoparticles (PNP). Photolyase production by recombinant E. coli was investigated in metabolic shaker and batch and fed-batch bioreactors. After extraction and purification by affinity chromatography, the photolyase exhibited a molecular weight of 47 kDa with a yield of 480 mg of pure protein per liter of fed-batch culture. Very low photolyase concentrations, such as 15 µg/mL, already presented 90% of CPD repair in DNA samples in vitro. Subsequently, nanostructures were developed based on design of experiments (DoE) to optimize the hydrodynamic diameter (Dh) and encapsulation efficiency (EE). Nanometric values of Dh were obtained for all nanostructures. EE of 23% was achieved for both PL and LP, and >90% for PNP. PL exhibited the best enzyme release profile to the external medium over time (up to 35% after 24h), while PNP presented the lowest release reaching only ≈15% after 10 days. Based on in vitro cytotoxicity studies, PL, LP and PNP were classified as non-irritating material. Finally, both free and photolyaseloaded PL and LP were able to protect cells from UV radiation, resulting in cell viability recoveries of 37% for the free enzyme and 50-60% when nanoencapsulated, compared to 28% without the enzyme. Overall, our study demonstrates efficient production of T. thermophilus photolyase and the enzyme ability to repair CPD damage, particularly when encapsulated in PL, offering a promising and innovative alternative as an active ingredient for incorporation into dermatological products.