Epidermal cell culture model with tight stratum corneum as a tool for dermal gene delivery studies.
The purpose of this study was to evaluate the feasibility of organotypic cultures of rat epidermal cells as a tool to study non-invasive dermal gene delivery. Also, a novel transfection method employing liposomal pre-treatment of stratum corneum (SC) was evaluated. Rat epidermal cells were cultured on Transwell tissue culture inserts and formation of stratum corneum barrier was evaluated in permeability studies with two model compounds. Transfections were performed with naked pCMV-SEAP2 plasmid and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleyl-phosphatidylethanolamine (DOPE)/DNA lipoplexes. Naked DNA was administered on the stratum corneum of the cell culture model with or without prior treatment of the stratum corneum with DOTAP/DOPE liposomes. Transfection was evaluated non-invasively by monitoring concentrations of secreted alkaline phosphatase (SEAP) in the culture medium of the basolateral compartment at 24-h intervals. Transfection with lipoplexes produced significant gene expression in rat epidermal keratinocyte (REK) epidermal culture model. Likewise, delivery of naked DNA on stratum corneum after DOTAP/DOPE liposome pre-treatment produced gene expression. Naked DNA alone did not result in detectable gene expression. In dermal gene delivery studies REK epidermal culture model is a suitable tool that includes tight stratum corneum and allows transgene expression in viable epidermis and non-invasive sampling of secreted gene product in the basolateral compartment. Liposomal pre-treatment of the stratum corneum augments transfection of viable epidermis.
Paasonen
Lauri
L
University of Kuopio, Department of Pharmaceutics, FIN-70211 Kuopio, Finland. Lauri.Paasonen@uku.fi
Korhonen
Maarit
M
Yliperttula
Marjo
M
Urtti
Arto
A
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry / Q000494:pharmacology; D005240:Feasibility Studies; D018014:Gene Transfer Techniques; D015603:Keratinocytes; D008081:Liposomes / Q000737:chemistry; D010539:Permeability; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D012869:Skin Absorption; D013997:Time Factors; D014162:Transfection
D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C094877:dioleoyl phosphatidylethanolamine; D004247:DNA; D000469:Alkaline Phosphatase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane