Electric field mediated gene delivery or electrotransfection is a widely used method in various studies ranging from basic cell biology research to clinical gene therapy. Yet, mechanisms of electrotransfection are still controversial. To this end, we investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane and how treatment of cells with three endocytic inhibitors (chlorpromazine, genistein, dynasore) or silencing of dynamin expression with specific, small interfering RNA (siRNA) would affect the eTE. Our data demonstrated that the presence of divalent cations (Ca2+ and Mg2+) in electrotransfection buffer enhanced pDNA adsorption to cell membrane and consequently, this enhanced adsorption led to an increase in eTE, up to a certain threshold concentration for each cation. Trypsin treatment of cells at 10 min post electrotransfection stripped off membrane-bound pDNA and resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake of pDNA (between 10 and 40 min) far exceeded the lifetime of electric field-induced transient pores (∼10 msec) in the cell membrane. Furthermore, treatment of cells with the siRNA and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. These findings suggest that electrotransfection depends on two mechanisms: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.
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.
Electrotransfection has been widely used as a versatile, non-viral method for gene delivery. However, electrotransfection efficiency (eTE) is still low and unstable, compared to viral methods. To understand potential mechanisms of the unstable eTE, we investigated effects of electrode materials on eTE and viability of mammalian cells. Data from the study showed that commonly used metal electrodes generated a significant amount of particles during application of pulsed electric field, which could cause precipitation of plasmid DNA from solutions, thereby reducing eTE. For aluminum electrodes, the particles were composed of aluminum hydroxide and/or aluminum oxide, and their median sizes were 300 to 400 nm after the buffer being pulsed 4 to 8 times at 400 V cm−1, 5 ms duration and 1 Hz frequency. The precipitation could be prevented by using carbon (graphite) electrodes in electrotransfection experiments. The use of carbon electrodes also increased cell viability. Taken together, the study suggested that electrodes made of inner materials were desirable for electrotransfection of cells in vitro.
A bioactive platform for the quantitative observation of cell migration is presented by 1) presenting migration factors in a well-defined manner on 2-D substrates, and 2) enabling continuous cell tracking. Well-defined substrate presentation is achieved by correctly orienting immobilized proteins (chemokines and cell adhesion molecules), such that the active site is accessible to cell surface receptors. A thiol-terminated self-assembled monolayer on a silica slide was used as a base substrate for subsequent chemistry. The thiol-terminated surface was converted to an immobilized metal ion surface using a maleimido-nitrilotriacetic acid (NTA) cross-linker that bound Histidine-tagged recombinant proteins on the surface with uniform distribution and specific orientation. This platform was used to study the influence of surface-immobilized chemokine SDF-1α and cell adhesion molecule ICAM-1 on murine splenic B lymphocyte migration. While soluble SDF-1α induced trans-migration in a Boyden Chamber type chemotaxis assay, immobilized SDF-1α alone did not elicit significant surface-migration on our test-platform surface. Surface-immobilized cell adhesion protein, ICAM-1, in conjunction with activation enabled migration of this cell type on our surface. Controlled exposure to UV light was used to produce stable linear gradients of His-tagged recombinant SDF-1α co-immobilized with ICAM-1 following our surface chemistry approach. XPS and antibody staining showed defined gradients of outwardly oriented SDF-1α active sites. This test platform can be especially valuable for investigators interested in studying the influence of surface-immobilized factors on cell behavior and may also be used as a cell migration enabling platform for testing the effects of various diffusible agents.
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