Efficient Gamma-Retroviral Transduction of Primary Human Skin Cells Using the EF-c Peptide as a Transduction Enhancer.

Efficient gene transfer into cultured fibroblasts and keratinocytes during retroviral transduction is a critical step toward the treatment of genodermatoses such as epidermolysis bullosa. However, achieving high transduction rates is still a difficult task, particularly for the insertion of large coding sequences for which high viral titers cannot always be obtained. Multiple polycationic molecules, such as polybrene, which has been used in several clinical trials, have the ability to boost ex vivo retroviral gene transfer. However, the use of polybrene has been associated with a reduction of the proliferation and growth potential of human keratinocytes in culture. We developed a method for the efficient retroviral transduction of primary fibroblasts and keratinocytes using EF-c, a polycationic nanofibril-forming peptide. In comparison with polybrene, we found that the retroviral transduction efficiency with EF-c was increased 2.5- to 3.2-fold for fibroblasts, but not for keratinocytes. Moreover, the use of EF-c did not affect fibroblast proliferation and keratinocyte stem cell content, whereas polybrene induced a decrease in both. This method could have a positive impact on the development of ex vivo gene correction of genodermatoses, allowing for more efficient gene transfer into primary skin cells with little to no effect on proliferation and stem cell content. © 2022 Wiley Periodicals LLC. Basic Protocol: Fibroblast and keratinocyte transduction Support Protocol: Assessment of transduction efficiency through flow cytometry analysis.

Influence of cosmetics vehicles on 4-methylbenzylidene-camphor's skin penetration, in vitro

The aim of this work was to compare the skin penetration of 4-methylbenzylidene-camphor (4-MBC) in two vehicles, an oil-in-water (O/W) emulsion and an alcoholic gel. The penetration of this UVB filter through these vehicles was determined in vitro (Franz cells) using pig ear skin. The 4-MBC permeated through the skin both with the emulsion o/w as the alcoholic gel. However, with the alcoholic gel, 5 h after application of the product, the 4-MBC was found in the receptor fluid, while with the emulsion o/w, 24 h after it was detected. In both vehicles, the 4-MBC was present in the viable epidermis, dermis but most part of it, was found in the stratum corneum, being more remarkable for alcoholic gel. The right choice of the vehicle could decrease the potential toxicological risk and increase the efficacy of sunscreens products.

O objetivo deste estudo foi avaliar a influência de diferentes veículos cosméticos na permeação cutânea da 4-metilbenzilideno cânfora através de experimento in vitro utilizando células de difusão de Franz. Os veículos avaliados foram um gel alcoólico e uma emulsão não iônica O/A. Durante o experimento, foi doseada a presença da 4-MBC no fluido receptor em µg/cm², desde o tempo 0 h até 24 h após a aplicação dos veículos, bem como sua concentração na superfície cutânea, estrato córneo e [epiderme sem estrato córneo + derme]. No estudo realizado com o gel alcoólico, a partir de 5 h após o início do experimento, já foi detectada a presença da 4-MBC no fluido receptor, ao passo que, utilizando a emulsão O/A, a 4-MBC foi detectada apenas após 24 h. Em ambos os veículos a 4-MBC estava presente na [epiderme sem estrato córneo + derme] não havendo diferença estatística entre eles. A maior concentração de 4-MBC foi detectada na superfície cutânea, estando em maior quantidade quando utilizada a emulsão O/A como veículo. Já no estrato córneo, a concentração de 4-MBC foi maior na utilização do gel alcoólico. Analisando os resultados obtidos, observa-se que a penetração e a retenção cutânea da 4-metilbenzilideno cânfora são dependentes da natureza do veículo utilizado.