New insights into the mechanism of electrotransfer of small nucleic acids.

RNA interference (RNAi) is a powerful and rapidly developing technology that enables precise silencing of genes of interest. However, the clinical development of RNAi is hampered by the limited cellular uptake and stability of the transferred molecules. Electroporation (EP) is an efficient and versatile technique for the transfer of both RNA and DNA. Although the mechanism of electrotransfer of small nucleic acids has been studied previously, too little is known about the potential effects of significantly larger pDNA on this process. Here we present a fundamental study of the mechanism of electrotransfer of oligonucleotides and siRNA that occur independently and simultaneously with pDNA by employing confocal fluorescence microscopy. In contrast to the conditional understanding of the mechanism, we have shown that the electrotransfer of oligonucleotides and siRNA is driven by both electrophoretic forces and diffusion after EP, followed by subsequent entry into the nucleus within 5 min after treatment. The study also revealed that the efficiency of siRNA electrotransfer decreases in response to an increase in pDNA concentration. Overall, the study provides new insights into the mechanism of electrotransfer of small nucleic acids which may have broader implications for the future application of RNAi-based strategies.

Accumulation of defense systems in phage-resistant strains of Pseudomonas aeruginosa.

Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains.

Electrotransfer for nucleic acid and protein delivery.

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.

Nucleophile responsive charge-reversing polycations for pDNA transfection.

Polycationic carriers promise low cost and scalable gene therapy treatments, however inefficient intracellular unpacking of the genetic cargo has limited transfection efficiency. Charge-reversing polycations, which transition from cationic to neutral or negative charge, can offer targeted intracellular DNA release. We describe a new class of charge-reversing polycation which undergoes a cationic-to-neutral conversion by a reaction with cellular nucleophiles. The deionization reaction is relatively slow with primary amines, and much faster with thiols. In mammalian cells, the intracellular environment has elevated concentrations of amino acids (∼10×) and the thiol glutathione (∼1000×). We propose this allows for decationization of the polymeric carrier slowly in the extracellular space and then rapidly in the intracellular milleu for DNA release. We demonstrate that in a lipopolyplex formulation this leads to both improved transfection and reduced cytotoxicity when compared to a non-responsive polycationic control.

Models of electroporation and the associated transmembrane molecular transport should be revisited.

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.

Microtrap array on a chip for localized electroporation and electro-gene transfection.

We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).

A Programmable Multifunctional 3D Cancer Cell Invasion Micro Platform.

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-ß) 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.

Actin networks regulate the cell membrane permeability during 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.

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.

Response of an actin network in vesicles under electric pulses.

We study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

High Frame Rate Ultrasound Particle Image Velocimetry for Estimating High Velocity Flow Patterns in the Left Ventricle.

Echocardiographic determination of multicomponent blood flow dynamics in the left ventricle remains a challenge. In this paper, we compare contrast enhanced, high frame rate (HFR) (1000 frames/s) echo-particle image velocimetry (ePIV) against optical particle image velocimetry (oPIV, gold standard), in a realistic left ventricular (LV) phantom. We find that ePIV compares well to oPIV, even for the high velocity inflow jet (normalized RMSE = 9% ± 1%). In addition, we perform the method of proper orthogonal decomposition, to better qualify and quantify the differences between the two modalities. We show that ePIV and oPIV resolve very similar flow structures, especially for the lowest order mode with a cosine similarity index of 86%. The coarser resolution of ePIV does result in increased variance and blurring of smaller flow structures when compared to oPIV. However, both modalities are in good agreement with each other for the modes that constitute the bulk of the kinetic energy. We conclude that HFR ePIV can accurately estimate the high velocity diastolic inflow jet and the high energy flow structures in an LV setting.

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.