Liposome-oligonucleotides interaction for in vitro uptake by COS I and HaCaT cells.
Liposomes are considered very promising delivery systems for antisense therapeutic approach, offering drug protection and facilitating oligonucleotide cell internalization. The present study was aimed to investigate the influence of phospholipid composition of the liposomal systems both on the encapsulation and on the oligonucleotide carrier capacity in vitro. Liposomes composed of neutral (phosphatidylcholine, cholesterol and dioleoylphosphatidylethanolamine) and/or cationic lipids (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride salt, DOTAP) with different molar ratios were complexed with 5' fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN). The interaction was evaluated using atomic force microscopy (AFM), gel electrophoresis and HPLC analysis. Cytofluorimetric analysis and fluorescence microscopy were applied to evaluate the uptake and intracellular distribution of fluorescently labelled PS-ODN after transfection in two cell lines, COS I (fibroblast cell) and HaCaT (immortalized keratinocyte cell). The AFM studies reveal that the liposome/PS-ODN interaction leads the formation of a new irregular structure that completely hides the PS-ODN. Gel electrophoresis experiments and HPLC analysis have clearly demonstrated that also neutral liposomes are able to keep a little amount of PS-ODN but without strain to the complexation; the interaction was weak and rapidly destabilized when the complex was added to the cells. Transfection experiments performed with different incubation times show that DOTAP liposomes increase the rate of cellular uptake of PS-ODN and seem to influence its intracellular distribution in COS I cells where the oligonucleotide looks localized in nucleoli. Similar behaviour, at a lesser extent, is exhibited in HaCaT cells.
Ruozi
Barbara
B
University of Modena and Reggio Emilia, Department of Pharmaceutical Sciences, Via Campi 183, 41100, Modena, Italy.
Battini
Renata
R
Tosi
Giovanni
G
Forni
Flavio
F
Vandelli
Maria Angela
MA
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D004337:Drug Carriers; D004339:Drug Compounding; D016503:Drug Delivery Systems; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects; D008081:Liposomes; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D010316:Particle Size; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D004337:Drug Carriers; D008081:Liposomes; D009841:Oligonucleotides