Gene Electrotransfer into Mammalian Cells Using Commercial Cell Culture Inserts with Porous Substrate.

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modeling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on the numerical results, we design a simple device combining an insert with substrate containing pores with 0.4 µm or 1.0 µm diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with conventional bulk electroporation, but at better cell viability and with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability.

Revisiting the role of pulsed electric fields in overcoming the barriers to in vivo gene electrotransfer.

Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.

DNA-membrane complex formation during electroporation is DNA size-dependent.

Size of DNA molecules governs their interaction with the cell membrane during electroporation and their subsequent transport inside the cell. In order to investigate the effect of DNA size on DNA-membrane interaction during electroporation, cells are electro-pulsed with DNA molecules; 15 bp, 25 bp, 50 bp, 100 bp and 1000 bp (bp = base pairs). Within the experimental parameter space, DNA-membrane complexes or DNA aggregates are observed at the cell membrane for DNA molecules containing 25 or more base pairs. No aggregates are observed for DNA molecules containing 15 bp. For all DNA sizes, direct access to the cytoplasm is observed, however the amount translocated decays with the size. The observed dependency of DNA aggregate formation on the size of the DNA molecules is consistent with the Onsager's theory of condensation of anisotropic rod-like molecules.

DNA translocation to giant unilamellar vesicles during electroporation is independent of DNA size.

Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25-20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.

Bridging the scales in high-throughput dielectrophoretic (bio-)particle separation in porous media.

Dielectrophoresis (DEP) is a versatile technique for the solution of difficult (bio-)particle separation tasks based on size and material. Particle motion by DEP requires a highly inhomogeneous electric field. Thus, the throughput of classical DEP devices is limited by restrictions on the channel size to achieve large enough gradients. Here, we investigate dielectrophoretic filtration, in which channel size and separation performance are decoupled because particles are trapped at induced field maxima in a porous separation matrix. By simulating microfluidic model porous media, we derive design rules for DEP filters and verify them using model particles (polystyrene) and biological cells (S. cerevisiae, yeast). Further, we bridge the throughput gap by separating yeast in an alumina sponge and show that the design rules are equally applicable in real porous media at high throughput. While maintaining almost 100% efficiency, we process up to 9 mL min-1, several orders of magnitude more than most state-of-the-art DEP applications. Our microfluidic approach provides new insight into trapping dynamics in porous media, which even can be applied in real sponges. These results pave the way toward high-throughput retention, which is capable of solving existing problems such as cell separation in liquid biopsy or precious metal recovery.

Polymer conformation during flow in porous media.

Molecular conformations of individual polymers during flow through porous media are directly observed by single-DNA imaging in microfluidics. As the Weissenberg number increases during flow (Wi > 1), we observe two types of elastic instabilities: (a) stationary dead-zone and (b) time-dependant dead-zone washing. When stretched polymer chains enter a dead-zone, they first re-coil and, once inside the dead-zone, they rotate and re-stretch again. The probability distribution of DNA chains under the stretched condition inside the dead-zone is found to be heterogeneous with a broad distribution.

Molecular Processes Leading to "Necking" in Extensional Flow of Polymer Solutions: Using Microfluidics and Single DNA Imaging.

We study the necking and pinch-off dynamics of liquid droplets that contain a semidilute polymer solution of polyacrylamide close to overlap concentration by combining microfluidics and single DNA observation. Polymeric droplets are stretched by passing them through the stagnation point of a T-shaped microfluidic junction. In contrast with the sudden breakup of Newtonian droplets, a stable neck is formed between the separating ends of the droplet which delays the breakup process. Initially, polymeric filaments experience exponential thinning by forming a stable neck with extensional flow within the filament. Later, thin polymeric filaments develop a structure resembling a series of beads-on-a-string along their length and finally rupture during the final stages of the thinning process. To unravel the molecular picture behind these phenomena, we integrate a T-shaped microfluidic device with advanced fluorescence microscopy to visualize stained DNA molecules at the stagnation point within the necking region. We find that the individual polymer molecules suddenly stretch from their coiled conformation at the onset of necking. The extensional flow inside the neck is strong enough to deform and stretch polymer chains; however, the distribution of polymer conformations is broad, and it remains stationary in time during necking. Furthermore, we study the dynamics of single molecules during formation of beads-on-a-string structure. We observe that polymer chains gradually recoil inside beads while polymer chains between beads remain stretched to keep the connection between beads. The present work effectively extends single molecule experiments to free surface flows, which provides a unique opportunity for molecular-scale observation within the polymeric filament during necking and rupture.