Topical transfection using plasmid DNA in a water-in-oil nanoemulsion.
Expression plasmids encoding chloramphenicol acetyltransferase (CAT) or human interferon-alpha2 cDNA were formulated in water-in-oil nanoemulsions and applied to murine skin. The histological location of transfected cells was assessed by in situ DNA PCR and showed that the deposition of plasmid DNA was primarily in follicular keratinocytes. Transgene expression in the skin was monitored for 24-72 h, following topical application of either single or multiple daily doses by quantitative RT-PCR and ELISA. It was found that transgene expression was optimal at 24 h following topical application of a single dose of water-in-oil nanoemulsion containing plasmid DNA. Dose-response studies using a total dose of 3, 10 or 30 microg of plasmid DNA suggested that topical transfection using nanoemulsions is subject to both threshold and saturation effects. None of the cationic liposome formulations tested as controls mediated transgenic protein expression at levels higher than background values of the ELISAs used to assay transgenic protein. Single and multiple dose experiments using human interferon-alpha2 as a transgene indicated that the efficiency of nanoemulsion mediated transfection was most effective in the context of normal versus atrophic hair follicles. In addition, the total amount of human interferon-alpha2 present in skin appeared to accumulate as a consequence of multiple dosing. Histologic evaluation of treated skin showed no overt signs of toxicity or irritation associated with the short-term application of the nanoemulsions. The results suggest that water-in-oil nanoemulsions can be used to facilitate transfection of follicular keratinocytes in vivo.
Wu
H
H
Department of Pharmaceutics, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.
Ramachandran
C
C
Bielinska
A U
AU
Kingzett
K
K
Sun
R
R
Weiner
N D
ND
Roessler
B J
BJ
D000287:Administration, Topical; D000818:Animals; D002626:Chemistry, Pharmaceutical; D015500:Chloramphenicol O-Acetyltransferase / Q000008:administration & dosage*; D004251:DNA Transposable Elements / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D004655:Emulsions; D004797:Enzyme-Linked Immunosorbent Assay; D008297:Male; D051379:Mice; D008812:Mice, Hairless; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000473:pathology*; D012869:Skin Absorption; D014162:Transfection; D019076:Transgenes / Q000235:genetics*
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D004655:Emulsions; D015500:Chloramphenicol O-Acetyltransferase