Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells.
Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 microg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin.
Fimmel
S
S
Department of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Berlin, Germany.
Saborowski
A
A
Orfanos
C E
CE
Zouboulis
C C
CC
D000199:Actins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000502:physiology; D004847:Epithelial Cells / Q000502:physiology; D005145:Face; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000502:physiology; D008856:Microscopy, Fluorescence; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism; D012627:Sebaceous Glands / Q000166:cytology / Q000502:physiology; D012879:Skin Physiological Phenomena; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D000199:Actins; D005229:Fatty Acids, Monounsaturated; D016376:Oligonucleotides, Antisense; D000644:Quaternary Ammonium Compounds; D011944:Receptors, Androgen; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane