RESUMO
Inefficient autologous tissue recovery in diverse skin injuries increases the susceptibility of patients to infections caused by multiresistant microorganisms, resulting in a high mortality rate. Nonviral transfection is an attractive alternative for these patients, where genetically modified cells incorporated into skin substitutes could release additional antimicrobial agents into the native skin. In this work, we have modulated the conditions of using a nonviral system for transfection of primary human keratinocytes and fibroblasts, consisting of a polymer/plasmid DNA (pDNA) complex called polyplex and its effects on the expression of LL-37 antimicrobial peptide. Linear and branched polyethylenimine (PEI) polymers in different weight concentrations were varied for evaluating the formation and colloidal characteristics of the polyplexes. The PEI/pDNA polyplexes with 19 nitrogen/phosphate ratio are nanometric particles (400 and 250 nm with linear and branched PEI, respectively) exhibiting positive surface (+30 ± 2 mV). Both kinds of polyplexes allowed the expression of a reporter gene and increased the human cathelicidin antimicrobial peptide gene expression in transfected keratinocytes and fibroblasts; however, greater cytotoxicity was observed when polyplexes formed with branched PEI were used. Moreover, cell culture supernatants from transfected cells with linear PEI/pDNA polyplexes showed enhanced antimicrobial activity (decrease of bacterial growth in 95.8%) against a Staphylococcus aureus strain in vitro. The study of the PEI/pDNA polyplexes formation allowed us to develop an improved transfection strategy of skin cells, promoting the production of LL-37 antimicrobial peptide. In the future, this strategy could be used for the construction of skin substitutes to prevent, reduce, or eliminate bacterial infections. Impact statement The results of this study contribute to the understanding of the polyplexes system in the genetic modification of skin cells and its effects on the expression of the LL-37 antimicrobial peptide. In the future, three-dimensional skin substitutes built with these cells could be an efficient way to decrease bacterial growth and prevent the infections in skin wounds.
Assuntos
Proteínas Citotóxicas Formadoras de Poros/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Iminas/química , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Polietilenos/química , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Staphylococcus aureus/efeitos dos fármacos , CatelicidinasRESUMO
Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (human cathelicidin antimicrobial peptide) and two reporter genes (red or green fluorescent protein) that included or not an internal ribosome entry site (IRES). The efficiency was measured by flow cytometry in terms of percentage of cells expressing the reporter gene; viability of transfected cells was also evaluated. It was found that nucleofection was more efficient than polyplexes for transfecting fibroblasts, while no significant differences were found between both systems of transfection when applied to keratinocytes. Regarding the viability of fibroblasts after transfection, values were high in both systems. In contrast, keratinocytes were more sensitive to nucleofection. It was also noted that both types of cells decreased reporter gene expression when IRES sequence was located upstream of the reporter gene, suggesting a negative effect on the expression of this gene. These results confirm that the transfection efficiency depends on the type of cells and the system used.