Joule heating and electroosmotic flow in cellular micro/nano electroporation.

Localized micro/nano-electroporation (MEP/NEP) shows tremendous potential in cell transfection with high cell viability, precise dose control, and good transfection efficacy. In MEP/NEP, micro or nanochannels are used to tailor the electric field distribution. Cells are positioned tightly by a micron or nanochannel, and the cargoes are delivered into the cell via the channel by electrophoresis (EP). Such confined geometries with micro and nanochannels are also widely used in sorting, isolation, and condensing of biomolecules and cells. Theoretical studies on the electrokinetic phenomena in these applications have been well established. However, for MEP/NEP applications, electrokinetic phenomena and their impact on the cell transfection efficiency and cell survival rate have not been studied comprehensively. In this work, we reveal the coupling between electric field, Joule heating, electroosmosis (EO), and EP in MEP/NEP at different channel sizes. A microfluidic biochip is used to investigate the electrokinetic phenomena in MEP/NEP on a single cell level. Bubble formation is observed at a threshold voltage due to Joule heating. The bubble is pushed to the cargo side due to EO and grows at the outlet of the nanochannel. As the voltage increases, the cargo transport efficiency decreases due to more intense EO, particularly for plasmid DNAs (3.5 kbp) with a low EP mobility. An 'electroporation zone' is defined for NEP/MEP systems with different channel sizes to avoid bubble formation and excessive EO velocity that may reduce the cargo delivery efficiency.

A rapid and efficient method using electroporation for releasing intracellular microcystin toxins from cultured and naturally occurring cyanobacterial cells in lake water.

In cyanotoxin measurements, effective release of intracellular cyanotoxins through cell lysis is pivotal. The conventional method for cell lysis is repeated freeze-thaw (F-T), which has several disadvantages, including poor reproducibility since it is operator and equipment dependency and time-consuming. In this study, a rapid and sensitive method was developed using irreversible electroporation, reducing quantification time by over 6 h compared to F-T. Focusing on microcystins (MCs), we developed the most optimal electroporation medium (50 mM Tris (pH 7.0) with 0.5 % SDS) and determined the optimal intensity of electroporation using Microcystis culture. Microcystis cell rupture was validated by scanning electron microscopy. COMSOL simulations mirrored experimental conditions. Compared to F-T, this new method generated an average 13.7 % (6.7 ppb) more MCs from lake water samples (p ≥ 0.05). This innovation, surpassing the time-consuming F-T process, emerges as a valuable tool for timely decision-making in water safety advisory and cyanotoxin management in various settings.

Development of a novel immunoFET technology-based POC assay for detection of Leishmania donovani and Leishmania major.

Leishmaniasis is considered as one of the 20 neglected tropical diseases. Current methods of leishmanial diagnosis depend on conventional laboratory-based techniques, which are time-consuming, costly and require special equipment and trained personnel. In this context, we aimed to provide an immuno field effect transistors (ImmunoFET) biosensor that matches the conventional standards for point-of-care (POC) monitoring and detection of Leishmania (L.) donovani/Leishmania major. Crude antigens prepared by repeated freeze thawing of L. donovani/L. major stationary phase promastigotes were used for ELISA and ImmunoFETs. Lesishmania-specific antigens were serially diluted in 1× PBS from a concentration of 106 -102 parasites/mL. A specific polyclonal antibody-based sandwich ELISA was established for the detection of Leishmania antigens. An immunoFET technology-based POC novel assay was constructed for the detection of Leishmania antigens. Interactions between antigen-antibody at the gate surface generate an electrical signal that can be measured by semiconductor field-effect principles. Sensitivity was considered and measured as the change in current divided by the initial current. The final L. donovani/L. major crude antigen protein concentrations were measured as 1.50 mg/mL. Sandwich ELISA against the Leishmania 40S ribosomal protein detected Leishmania antigens could detect as few as 100 L. donovani/L. major parasites. An immunoFET biosensor was constructed based on the optimization of aluminium gallium nitride/gallium nitride (AlGaN/GaN) surface oxidation methods. The device surface was composed by an AlGaN/GaN wafer with a 23 nm AlGaN barrier layer, a 2 µm GaN layer on the silicon carbide (SiC) substrate for Leishmania binding, and coated with a specific antibody against the Leishmania 40S ribosomal protein, which was successfully detected at concentrations from 106 to 102 parasites/mL in 1× PBS. At the concentration of 104 parasites, the immunoFETs device sensitivities were 13% and 0.052% in the sub-threshold regime and the saturation regime, respectively. Leishmania parasites were successfully detected by the ImmunoFET biosensor at a diluted concentration as low as 150 ng/mL. In this study, the developed ImmunoFET biosensor performed well. ImmunoFET biosensors can be used as an alternative diagnostic method to ELISA. Increasing the sensitivity and optimization of immuno-FET biosensors might allow earlier and faster detection of leishmaniasis.

Cell membrane damage and cargo delivery in nano-electroporation.

Nanochannel electroporation (NEP) is a new technology for cell transfection, which provides superior gene delivery and cell viability to conventional bulk electroporation (BEP). In NEP, the cells laid on a porous substrate are subjected to an asymmetric electric field which induces asymmetric membrane poration. The cell membrane near the channel outlet ('transfection membrane') is porated intensely, allowing direct delivery of genetic materials, while the rest of the cell membrane ('non-transfection membrane') remains much less perturbed for low cellular damage. In this work, the transfection window of NEP for the delivery of different sized molecules is systematically investigated. The results show that small molecules (∼0.6 kDa) can be delivered into cells at a relatively lower voltage without significantly impacting the non-transfection membrane. To deliver larger molecules (∼6 kDa), a higher working voltage is required at the cost of cell viability due to more severe damage of the non-transfection membrane. Through numerical analysis of both transient transmembrane potential (t-TMP) and dynamic transmembrane potential (d-TMP), here we show that the membrane damage on both transfection and non-transfection sides of the cell membrane can be predicted. The agreement between experimental results and numerical analysis provides a comprehensive understanding of cell membrane damage and cargo delivery in NEP.

Author Correction: Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

A Correction to this paper has been published: https://doi.org/10.1038/s41551-021-00725-w.

Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue.

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency). In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

Controllable Large-Scale Transfection of Primary Mammalian Cardiomyocytes on a Nanochannel Array Platform.

While electroporation has been widely used as a physical method for gene transfection in vitro and in vivo, its application in gene therapy of cardiovascular cells remains challenging. Due to the high concentration of ion-transport proteins in the sarcolemma, conventional electroporation of primary cardiomyocytes tends to cause ion-channel activation and abnormal ion flux, resulting in low transfection efficiency and high mortality. In this work, a high-throughput nanoelectroporation technique based on a nanochannel array platform is reported, which enables massively parallel delivery of genetic cargo (microRNA, plasmids) into mouse primary cardiomyocytes in a controllable, highly efficient, and benign manner. A simple "dipping-trap" approach was implemented to precisely position a large number of cells on the nanoelectroporation platform. With dosage control, our device precisely titrates the level of miR-29, a potential therapeutic agent for cardiac fibrosis, and determines the minimum concentration of miR-29 causing side effects in mouse primary cardiomyocytes. Moreover, the dose-dependent effect of miR-29 on mitochondrial potential and homeostasis is monitored. Altogether, our nanochannel array platform provides efficient trapping and transfection of primary mouse cardiomyocyte, which can improve the quality control for future microRNA therapy in heart diseases.

3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy.

Current transfection technologies lead to significant inter-clonal variations. Previously we introduced a unique electrotransfection technology, Nanochannel-Electroporation (NEP), which can precisely and benignly transfect small cell populations (~100-200 cells) with single-cell resolution. Here we report on the development of a novel 3D NEP system for large scale transfection. A properly-engineered array of nanochannels, capable of handling/transfecting ~60 000 cells cm(-2), was fabricated using cleanroom technologies. Positive dielectrophoresis was used to selectively position cells on the nanochannels, thus allowing highly efficient transfection. Single-cell dosage control was demonstrated using both small and large molecules, and different cell types. The potential clinical relevance of this system was tested with difficult-to-transfect natural killer cell suspensions, and plasmids encoding for the chimeric antigen receptor (CAR), a model of high relevance for adoptive immunotherapy. Our results show significantly higher CAR transfection efficiencies for the DEP-NEP system (>70% vs. <30%), as well as enhanced cell viabilities.