Han Chang Kang1, You Han Bae. 1. Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, Salt Lake City, Utah 84108, USA.
Abstract
BACKGROUND: In vitro genetic modification has been regarded as one option to improve the viability and functionality of pancreatic islets when used for transplantation in patients with diabetes, either as naked islets or in a type of bioartificial pancreas. In this approach, vector safety and poor transfection efficiency are major concerns. METHODS: In this study, the influence of in vitro transfection conditions on polyplexes constructed of polyethyleneimine (PEI) and plasmid DNA (pDNA) on the transfection efficiency was investigated by varying the transfection medium, the pDNA dose, and the amines of polycation/phosphates of pDNA (N/P) ratio. RESULTS: Ca2+-containing Krebs-Ringer-HEPES medium was more effective than RPMI 1640 medium by increasing transfection efficiency (2.5-fold). An increase in pDNA dose slightly reduced the transfection efficiency but had minimal influence on islet loss. However, the N/P ratio had a large effect on islet viability and transfection efficiency. For example, the PEI/pDNA ratio at N/P = 10 caused greater islet loss (56% vs. 28%) and 30-fold less transfection efficiency than at N/P = 5. Even under a set of best conditions selected from this study, mostly a fraction of cells located in the peripheral regions of an islet were transfected, and the viability and insulin secretion from the treated islets were not altered. However, it was found that the extent of apoptosis was noticeably higher (approximately 16%) than in untreated islets (approximately 2%). CONCLUSIONS: These results suggest that the gene delivery efficacy to isolated islets can be improved by manipulating the transfection conditions. Polymeric vectors will broaden the options for islet transfection, which is currently limited to viral vectors.
n class="abstract_title">BACKGROUND: Inpan> vitro genpan>etic modificationpan> has beenpan> regarded as onpan>e optionpan> to improve the viability and funpan>ctionpan>ality of n class="Disease">pancreatic islets when used for transplantation in patients with diabetes, either as naked islets or in a type of bioartificial pancreas. In this approach, vector safety and poor transfection efficiency are major concerns. METHODS: In this study, the influence of in vitro transfection conditions on polyplexes constructed of polyethyleneimine (PEI) and plasmid DNA (pDNA) on the transfection efficiency was investigated by varying the transfection medium, the pDNA dose, and the amines of polycation/phosphates of pDNA (N/P) ratio. RESULTS:Ca2+-containing Krebs-Ringer-HEPES medium was more effective than RPMI 1640 medium by increasing transfection efficiency (2.5-fold). An increase in pDNA dose slightly reduced the transfection efficiency but had minimal influence on islet loss. However, the N/P ratio had a large effect on islet viability and transfection efficiency. For example, the PEI/pDNA ratio at N/P = 10 caused greater islet loss (56% vs. 28%) and 30-fold less transfection efficiency than at N/P = 5. Even under a set of best conditions selected from this study, mostly a fraction of cells located in the peripheral regions of an islet were transfected, and the viability and insulin secretion from the treated islets were not altered. However, it was found that the extent of apoptosis was noticeably higher (approximately 16%) than in untreated islets (approximately 2%). CONCLUSIONS: These results suggest that the gene delivery efficacy to isolated islets can be improved by manipulating the transfection conditions. Polymeric vectors will broaden the options for islet transfection, which is currently limited to viral vectors.
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