In vivo cutaneous interferon-gamma gene delivery using novel dicationic (gemini) surfactant-plasmid complexes.
BACKGROUND
Localized scleroderma (morphea and linear scleroderma) is a connective tissue disease, accompanied by excessive proliferation and deposition of collagen within the skin, inflammation, vasculopathy and a deranged immune system. Interferon gamma (IFNgamma), an inhibitor of collagen synthesis and an immunomodulator, could be a potential therapeutic agent if it could be delivered into or expressed locally in affected skin in a non-invasive manner. In this study, the feasibility of topical delivery of the IFNgamma gene and expression of IFNgamma were investigated in mice.


METHODS
Novel dicationic (gemini) surfactant (spacer length n=2-16; alkyl chain m=12 or 16)-DNA complexes were formulated and characterized by circular dichroism and atomic force microscopy to select gemini analogues with the highest transfection efficiency (TE). Transfection and cellular expression of IFNgamma from the bicistronic pGTmCMV.IFN-GFP plasmid were evaluated in PAM 212 keratinocyte culture by ELISA and fluorescence microscopy. Topical delivery of plasmid using liposomal and nanoemulsion systems, based on gemini surfactant 16-3-16, was evaluated in mice by IFNgamma expression analysis.


RESULTS
In vitro TE was found to be dependent on the spacer length of the gemini surfactant, with the C3 spacer showing the highest activity (both 12-3-12 and 16-3-16). Both gemini cationic liposomes and gemini nanoemulsion (3x25 microg DNA/animal) produced significantly higher levels of IFNgamma in the skin (359.4 and 607.24 pg/cm2) compared to naked DNA (135.69 pg/cm2) or a liposomal Dc-chol formulation (82.15 pg/cm2). IFNgamma expression in the lymph nodes was higher in the animals treated with gemini liposomes (422.74 pg/animal) compared to the nanoemulsion formulation (131.27 pg/animal) or the Dc-chol formulation (82pg/animal).


CONCLUSIONS
The feasibility of topical delivery of pGTmCMV.IFN-GFP plasmid in mice using gemini cationic surfactant based delivery systems was demonstrated. IFNgamma expression after treatment with gemini-DNA formulations in the skin was 3-5-fold higher compared to the treatment with naked DNA (p<0.05), and 4-6-fold higher than the Dc-chol-DNA complex, indicating a significant advance in topical DNA delivery across intact skin in vivo.
Badea
Ildiko
I
College of Pharmacy and Nutrition, University of Saskatchewan, SK. S7N 5C9, Canada.
Verrall
Ronald
R
Baca-Estrada
Maria
M
Tikoo
Suresh
S
Rosenberg
Alan
A
Kumar
Praveen
P
Foldvari
Marianna
M
D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D003587:Cytomegalovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013501:Surface-Active Agents; D014162:Transfection
D013501:Surface-Active Agents; D007371:Interferon-gamma