Monitoring liposome‐mediated delivery and fate of an antisense drug in cell extracts and in single cells by capillary electrophoresis with laser‐induced fluorescence

Abstract

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.

Drug–liposome distribution phenomena studied by capillary electrophoresis‐frontal analysis

“…CE has proved to be an efficient tool for studying molecular interactions [9][10][11][12][13][14][15][16][17] encompassing determination of stability constants, complexation stoichiometry and distribution, and partitioning coefficients. Also, CE has been used to assess liposomal drug delivery systems in a few cases [18][19][20][21][22]. Liposomes have, however, primarily been introduced into capillaries in studies directed at estimating drug lipophilicity in relation to membrane permeability properties [2][3][4][5][23][24][25][26][27][28].…”

Capillary electrophoresis at the omics level: Towards systems biology

“…The various mutation analyses that are referred to in this article showed that the use of -CE-LIF detection could make the process easier and faster than traditional methods [86][87][88][89][90][91]. CE was also used for antisense drug analysis, viral DNA detection, mRNA analysis, and gene expression [92][93][94][95][96][97]. Considering the importance of the CE work in this field, only recent studies are listed here.…”

Capillary electrophoresis and nanomaterials – Part I: Capillary electrophoresis of nanomaterials

“…Due to the biological properties of liposomes, these structures are currently under extensive study as nanocarriers for targeted drug delivery . It should come as no surprise that CE is used in this research to verify the encapsulation and selective release of the cargo carried in these nanovehicles . An important aspect of many preparations in biological processes is the presence of a pH gradient across the membrane.…”