Polymeric gene transfection on insulin-secreting cells: sulfonylurea receptor-mediation and transfection medium effect.
OBJECTIVE
In vitro transfection of secreting cells is regarded as one strategy for improved cell engineering/ transplantation. Insulin-secreting insulinoma cell lines or pancreatic beta-cells could be genetically engineered using designed polymeric vectors which are safer than viral vectors. This study investigates the effects of the constituents in transfection media on polymeric transfection.


METHODS
Polyplexes conjugated with sulfonylurea (SU) were evaluated under different transfection conditions for gene transfection and their effects on cytotoxicity and insulin secretion. Several components in transfection media specifically associated with the insulin secretion pathway were amino acids, vitamins, Ca2+ and K+. The interactions of the polyplexes with insulin were monitored by surface charge and particle size to monitor how insulin as a protein influences transfection.


RESULTS
For an insulin-secreting cell line (RINm5F), polyplexes in Ca2+--containing KRH medium (Ca2+(+)KRH) enhanced transfection and did not cause damage to biological functions. When adding amino acids, vitamins, or K+ or depleting Ca2+ from Ca2+(+)KRH, poly(L-lysine)/DNA complexes showed a greater reduction in transfection than SU receptor (SUR)-targeting polyplexes (SU-polyplex). Positively charged polyplexes interacted with insulin, developing a negative surface charge, and these interactions may cause a decrease in transfection.


CONCLUSIONS
The findings suggest that in vitro and ex vivo polymeric transfection of insulin-secreting cells can be modulated and enhanced by adjusting the transfection conditions.
Kang
Han Chang
HC
Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, 421 Wakara Way, Suite 318, Salt Lake City, Utah 84108, USA.
Bae
You Han
YH
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media; D004247:DNA / Q000235:genetics; D007328:Insulin / Q000378:metabolism*; D007340:Insulinoma / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D010316:Particle Size; D011107:Polylysine / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D051381:Rats; D011955:Receptors, Drug / Q000378:metabolism*; D015203:Reproducibility of Results; D013453:Sulfonylurea Compounds / Q000378:metabolism; D064233:Sulfonylurea Receptors; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection / Q000379:methods*; D014810:Vitamin E / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D003470:Culture Media; D007328:Insulin; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D013453:Sulfonylurea Compounds; D064233:Sulfonylurea Receptors; D013778:Tetrazolium Salts; D013844:Thiazoles; C505376:poly(L-lysine)-g-sulfonylurea; D014810:Vitamin E; D011107:Polylysine; D004247:DNA; C022616:thiazolyl blue