Role of specific endocytic pathways in electrotransfection of cells

Abstract

Electrotransfection is a technique utilized for gene delivery in both preclinical and clinical studies. However, its mechanisms are not fully understood. The goal of this study was to investigate specific pathways of endocytosis involved in electrotransfection. In the study, three different human cell lines (HEK293, HCT116, and HT29) were either treated with ice cold medium postelectrotransfection or endocytic inhibitors prior to electrotransfection. The inhibitors were pharmacological agents (chlorpromazine, genistein, and amiloride) or different small interfering RNA (siRNA) molecules that could knockdown expression of clathrin heavy chain (CLTC), caveolin-1, and Rab34, respectively. The reduction in gene expressions was confirmed with western blot analysis at 48-72h post-siRNA treatment. It was observed that treatments with either ice cold medium, chlorpromazine, or genistein resulted in significant reductions in electrotransfection efficiency (eTE) in all three cell lines, compared to the matched controls, but amiloride treatment had insignificant effects on eTE. For cells treated with siRNA, only CLTC knockdown resulted in eTE reduction for all three cell lines. Together, these data demonstrated that the clathrin-mediated endocytosis played an important role in electrotransfection.

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…48 In addition, we demonstrated that a clathrin-mediated endocytosis mechanism could actively transport plasmids across the membrane and facilitate electrotransfection. 77 The accumulating evidence supports the notion that electrotransfection of pDNA is mediated by endocytosis. In the present work, we further confirmed that electrotransfection could indeed induce actin remodeling, which is an essential requirement for macropinocytosis.…”

“…Cells were obtained from Duke University Cell Culture Facility (CCF) and cultured as previously described. 48,77 In brief, cells were grown as monolayers in high-glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and Antibiotic-Antimycotic (Invitrogen) at 37 C in a humidified incubator with 5% CO 2 and passaged every 36-48 hr.…”

“…77 In brief, cells were collected and re-suspended in 200-300 mL PBS and then stained with propidium iodide (PI) (5 mg/ mL). Flow cytometry analysis was performed with a BD FACSCanto II flow cytometer (Becton Dickinson).…”

GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells

“…These include pinocytosis, phagocytosis, clathrin-mediated endocytosis or caveolae-mediated uptake process15. To investigate the mechanism of GapmeR cellular uptake in primary human T-cells, we treated cells separately with specific endocytosis pathway inhibitors, including amiloride (pinocytosis inhibitor17), cytochalasin D (phagocytosis inhibitor18), filipin (caveolae-mediated uptake inhibitor19), or chlorpromazine (clathrin-mediated endocytosis inhibitor1920). Pre-treatment of T-cells with increasing concentrations of amiloride (0.5 to 5 mM), prior to incubation with FAM-GapmeR, showed dose-dependent inhibition of GapmeR internalization, with >95% inhibition at 5 mM concentration (Fig.…”

ALPPL2 Aptamer-Mediated Targeted Delivery of 5-Fluoro-2′-Deoxyuridine to Pancreatic Cancer

“…Conversely, a slight increase in the SQ2 internalization was observed. It is well known that the inhibition of one endocytic pathway can lead to upregulation of other alternative pathways as a compensatory mechanism [18,27]. If such alternative pathways are utilized for SQ2 internalization, then the increased cellular uptake can be explained.…”