Electrotransfer is an effective non-viral strategy to deliver exogenous cargo such as nucleic acids and proteins into living cells in ex vivo and in vivo scenarios.Next-generation electrotransfer ...strategies aim at enhancing efficiency through localized electroporation and hybrid methods involving microfluidics, mechanoporation, and sonoporation. In addition, there is a focus on creating affordable, single-use electroporation devices that will potentially expand the global reach of DNA vaccination.Continuous advances in electrotransfer, and the integration of these next-generation electroporation techniques into in utero and in vivo applications, hold the promise of significantly improving gene-editing efficiencies in these scenarios.Promising results from clinical trials utilizing DNA electrotransfer highlight its favorable safety profile and indicate encouraging prospects for its broader application.
Electrotransfer of nucleic acids and proteins has become crucial in biotechnology for gene augmentation and genome editing. This review explores the applications of electrotransfer in both ex vivo and in vivo scenarios, emphasizing biomedical uses. We provide insights into completed clinical trials and successful instances of nucleic acid and protein electrotransfer into therapeutically relevant cells such as immune cells and stem and progenitor cells. In addition, we delve into emerging areas of electrotransfer where nanotechnology and deep learning techniques overcome the limitations of traditional electroporation.
Electrotransfer of nucleic acids and proteins has become crucial in biotechnology for gene augmentation and genome editing. This review explores the applications of electrotransfer in both ex vivo and in vivo scenarios, emphasizing biomedical uses. We provide insights into completed clinical trials and successful instances of nucleic acid and protein electrotransfer into therapeutically relevant cells such as immune cells and stem and progenitor cells. In addition, we delve into emerging areas of electrotransfer where nanotechnology and deep learning techniques overcome the limitations of traditional electroporation.
Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules ...to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.
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•Electroporative uptake of propidium iodide increases when actin networks are chemically disrupted.•Electroporative uptake of propidium iodide is a temperature dependent process.•Activation energy barrier of electroporation is reduced when the actin networks are disrupted.•The observed reduction in activation energy barrier is possibly due to altered cell membrane mechanical properties.
The present review focuses on the effects of pulsed electric fields on lipid vesicles ranging from giant unilamellar vesicles (GUVs) to small unilamellar vesicles (SUVs), from both fundamental and ...applicative perspectives. Lipid vesicles are the most popular model membrane systems for studying biophysical and biological processes in living cells. Furthermore, as vesicles are made from biocompatible and biodegradable materials, they provide a strategy to create safe and functionalized drug delivery systems in health-care applications. Exposure of lipid vesicles to pulsed electric fields is a common physical method to transiently increase the permeability of the lipid membrane. This method, termed electroporation, has shown many advantages for delivering exogenous molecules including drugs and genetic material into vesicles and living cells. In addition, electroporation can be applied to induce fusion between vesicles and/or cells. First, we discuss in detail how research on cell-size GUVs as model cell systems has provided novel insight into the basic mechanisms of cell electroporation and associated phenomena. Afterwards, we continue with a thorough overview how electroporation and electrofusion have been used as versatile methods to manipulate vesicles of all sizes in different biomedical applications. We conclude by summarizing the open questions in the field of electroporation and possible future directions for vesicles in the biomedical field.
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•Lipid vesicle is an important model for studying biological cells in electric field.•The intriguing responses of vesicles to electric pulses are discussed.•There is a need to increase the complexity of vesicles as model cell systems.•Electric field is a versatile method for manipulating lipid vesicles.•Overview of vesicle electroporation in biomedical application is provided.
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.
Due to increased energy demand, it is vital to enhance the recovery from existing oilfields. Polymer flooding is the most frequently used chemical enhanced oil recovery (cEOR) method in field ...applications that increases the oil sweep and displacement efficiencies. In recent years, there has been growing interest to assess the use of polymer flooding in an increasing number of field applications. This is due to the improved properties of polymers at high-salinity and high-temperature conditions and an increased understanding of the transport mechanisms of water-soluble polymers in porous media. In this review, we present an overview of the latest research into the application of polymers for cEOR, including mechanisms of oil recovery improvement and transport mechanisms in porous media. We focus on the recent advances that have been made to develop polymers that are suitable for high-salinity and high-temperature conditions and shed light on new insights into the flow of water-soluble polymers in porous media. We observed that the viscoelastic behavior of polymers in porous media (e.g., shear thickening and elastic turbulence) is the most recently debated polymer flow mechanism in cEOR applications. Moreover, advanced water-soluble polymers, including hydrophobically modified polymers and salt- and temperature-tolerant modified polyacrylamides, have shown promising results at high-salinity and high-temperature conditions.
•Critical assessment of models describing electroporation and molecular transport.•None of the models can be universily applied to the full range of experimental data.•None of the models can be ...considered truly validated by experimental data.•Guidelines for further development of mechanistic models of electroporation.•Guidelines for reporting experimental measurements to support modeling.
Electroporation has become a powerful tool for nonviral delivery of various biomolecules such as nucleic acids, proteins, and chemotherapeutic drugs to virtually any living cell by exposing the cell membrane to an intense pulsed electric field. Different multiphysics and multiscale models have been developed to describe the phenomenon of electroporation and predict molecular transport through the electroporated membrane. In this paper, we critically examine the existing mechanistic, single-cell models which allow spatially and temporally resolved numerical simulations of electroporation-induced transmembrane transport of small molecules by confronting them with different experimental measurements. Furthermore, we assess whether any of the proposed models is universal enough to describe the associated transmembrane transport in general for all the different pulse parameters and small molecules used in electroporation applications. We show that none of the tested models can be universally applied to the full range of experimental measurements. Even more importantly, we show that none of the models has been compared to sufficient amount of experimental data to confirm the model validity. Finally, we provide guidelines and recommendations on how to design and report experiments that can be used to validate an electroporation model and how to improve the development of mechanistic models.
Tissue surface tension influences cell sorting and tissue fusion. Earlier mechanical studies suggest that multicellular spheroids actively reinforce their surface tension with applied force. Here we ...study this open question through high-throughput microfluidic micropipette aspiration measurements on cell spheroids to identify the role of force duration and spheroid deformability. In particular, we aspirate spheroid protrusions of mice fibroblast NIH3T3 and human embryonic HEK293T homogeneous cell spheroids into micron-sized capillaries for different pressures and monitor their viscoelastic creep behavior. We find that larger spheroid deformations lead to faster cellular retraction once the pressure is released, regardless of the applied force. Additionally, less deformable NIH3T3 cell spheroids with an increased expression level of alpha-smooth muscle actin, a cytoskeletal protein upregulating cellular contractility, also demonstrate slower cellular retraction after pressure release for smaller spheroid deformations. Moreover, HEK293T cell spheroids only display cellular retraction at larger pressures with larger spheroid deformations, despite an additional increase in viscosity at these larger pressures. These new insights demonstrate that spheroid viscoelasticity is deformation-dependent and challenge whether surface tension truly reinforces at larger aspiration pressures.
In the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three‐dimensional (3D) tumor models that recapitulate spatial architecture, spatiotemporal ...control of cell communication and signaling pathways, and integration of extracellular cues remains an open challenge. Here, a programmable multifunctional 3D cancer cell invasion microbuckets‐hydrogel (Mb‐H) platform is developed by integrating various function‐variable microbuckets and extracellular matrix (ECM)‐like hydrogels. Based on this Mb‐H micro platform, the aggregation of multi‐cancer cells is well controlled to form cancer cell spheroids, and the guiding relationship of single‐cell migration and collective cell migration during the epithelial‐mesenchymal transition (EMT) of cancer cell invasion are demonstrated. By programming and precisely assembling multiple functions in one system, the Mb‐H platform with spatial‐temporal controlled release of cytokine transforming growth factor beta (TGF‐β) and various functionalized Mb‐H platforms with intelligent adjustment of cell‐matrix interactions are engineered to coordinate the 3D invasive migration of cancer cell spheroids. This programmable and adaptable 3D cancer cell invasion micro platform takes a new step toward mimicking the dynamically changing (localized) tumor microenvironment and exhibits wide potential applications in cancer research, bio‐fabrication, cell signaling, and drug screening.
A micro platform is engineered for 3D cancer cell invasion. By integrating various functionalized microbuckets and ECM‐like hydrogels in one platform, the 3D invasive migration of cancer cell spheroids is researched by spatial‐temporal controlled release of cytokine and adjustment of the cell–matrix interactions. It displays a strategy with the synergy of multiple factors in cancer cell invasion that promotes the mimicking of the real tumor microenvironment.
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