Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification.
BACKGROUND
Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications.


OBJECTIVE
The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells.


METHODS
We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay.


CONCLUSIONS
The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000™ showed decreased cell viability.


CONCLUSIONS
This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.
NanoBioCel Group, University of the Basque Country , Vitoria-Gasteiz , Spain and.
Mireia
M
Agirre
Jon
J
Zarate
Gustavo
G
Puras
Edilberto
E
Ojeda
José Luis
JL
Pedraz
D002460:Cell Line; D002470:Cell Survival; D002686:Chitin / Q000008:administration & dosage / Q000031:analogs & derivatives*; D048271:Chitosan; D004247:DNA / Q000008:administration & dosage*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000494:pharmacology; D009844:Oligosaccharides; D010316:Particle Size; D010957:Plasmids; D055213:Retinal Pigment Epithelium / Q000166:cytology; D014162:Transfection / Q000379:methods*
D008055:Lipids; C086724:Lipofectamine; D009844:Oligosaccharides; C485184:enhanced green fluorescent protein; C493484:oligochitosan; D002686:Chitin; D049452:Green Fluorescent Proteins; D004247:DNA; D048271:Chitosan
Gene therapy; nonviral vectors; oligomeric chitosan; pCMS-EGFP; pH influence