Monitoring liposome-mediated delivery and fate of an antisense drug in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence.
Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used for evaluation of the effectiveness of delivery and fate of a model 25-mer DNA-based phosphorothiate antisense drug in cells. The antisense molecule was delivered to the cells through a simple incubation and by using a cationic liposome (Cytofectin GS 3815). The cationic lipid interacted with the negatively charged antisense to form a more positively charged complex. It was observed that uptake of the liposome-antisense complex by the cell was dependent on concentration of lipid, duration of transfection, and the cell type. The antisense drug interacted with intracellular components such as proteins and additional steps were needed to quantify the free antisense. Proteinase-K was able to release antisense from proteins. However, the addition of sodium dodecyl sulfate (SDS) to the sample or running buffer was more effective than Proteinase-K to release both naked and liposome-bound antisense from the cellular materials. Analysis of single HeLa cells for uptake of the unbound and liposome-complexed antisense revealed averages of 8.9x10(-19) moles and 4.9x10(-18) moles, respectively. The amount of uptake, however, varied greatly among individual cells and depended on the delivery method. With liposome-mediated delivery, the relative standard deviation (RSD) for the amount of antisense in individual cells was 130%, while the variation was much smaller (RSD = 45%) when the cells were incubated with the unbound antisense. These uptake variations agreed with those obtained from flow cytometry analysis.
Malek
Amir H
AH
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
Khaledi
Morteza G
MG
D002457:Cell Extracts / Q000737:chemistry*; D016373:DNA, Antisense / Q000008:administration & dosage / Q000032:analysis* / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D019286:Endopeptidase K / Q000378:metabolism; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D008081:Liposomes / Q000008:administration & dosage* / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002457:Cell Extracts; D016373:DNA, Antisense; D008081:Liposomes; D012967:Sodium Dodecyl Sulfate; D019286:Endopeptidase K