Exosomes are attractive nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and of their ability to penetrate physiological barriers that are impermissible to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs (mRNAs), 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 to bulk electroporation and to other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and more than a 10 3 -fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic PTEN-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced tumourgrowth inhibition, and increased animal survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

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)1–6. 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-processing7,8. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection9,10. 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 methods11,12. 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.

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.

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.

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, we report a high-throughput nano-electroporation technique based on a nanochannel array platform, which enables massively parallel delivery of genetic cargo (microRNA, plasmids) into mouse primary cardiomyocytes in a controllable, highly efficient and benign manner. A simple ‘dewetting’ approach was implemented to precisely position a large number of cells on the nano-electroporation platform. With dosage control, our device precisely titrated the level of miR-29, a potential therapeutic agent for cardiac fibrosis, and determined 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 was monitored. Altogether, our nanochannel array platform provides efficient trapping and transfection of primary mouse cardiomyocyte, which could improve the quality control for future microRNA therapy in heart diseases.

In order to create a high-throughput electroporation based cell transfection system, it is required that each cell has localized delivery and minimal membrane damage to ensure optimal transfection and longevity post-biomolecule delivery. To meet these requirements, a three-dimensional (3D) nanochannel device was fabricated on a Si platform due its ease of etching, wide industrial availability, and mechanical stability. The device is designed to shoot desired biomolecules into a seated array of target cells to achieve the high-throughput of bulk electroporation, but with greatly reduced cell mortality. To accomplish this, a wafer-scale Bosch etching process was optimized to etch a 3D array of channels consisting of larger microchannels feeding into smaller nanochannels that cells are ultimately seated on for transfection. The microchannel array consists of 50 μm wells spaced 50 μm apart, which are etched from the “back side.” The wafer is then flipped over to etch the smaller 650 nm channels on the “front side.” In the creation of the 3D silicon device, other feature sizes were explored, and their Bosch etching was characterized for comparison. The results show that when etching samples with the same feature sizes, but different densities, there was no relation between feature density and etch rate for our recipe. However, when etching features, or more specifically, circular channels of different sizes (650 nm–150 μm), the results show a positive correlation with etch rate (1.10–4.06 μm/min). Standard deviations indicate very uniform etching with an average value of 0.1 μm/min across all etches. After optimization, the 3D Si device was tested to ensure successful cell seating and transfection via electroporation, using fluorescence as the tool of evaluation. Fluorescent imaging (postelectroporation) indicates a transfection efficiency of approximately 70% with a cell viability of roughly 90%.

Nano-channel electroporation (NEP) is a new technology for cell transfection, which provides superior gene delivery and cell viability to conventional bulk electroporation (BEP). In NEP, cells laid on a porous...

Little has been studied on how the electrochemical noise impacts the limit of detection of field effect transistor (FET) biosensors. Herein, we investigate low frequency noise associated with phosphate-buffered saline (PBS) solutions at varying ionic strengths (Ni) under both weak and strong gate biases corresponding to saturation and sub-threshold regimes, respectively, in AlGaN/GaN heterojunction FET biosensors. We show that the electrochemical noise is strongly dependent on the ionic strength and gate biasing conditions. In the saturation regime (low bias), varying the ionic strength (a range of 10−6× PBS to PBS 1 × stock solutions used for testing) has little to no effect on the characteristic frequency exponent β(β=1), indicating a predominately diffusion-based process. Conversely, under higher biases (sub-threshold regime), the β parameter varies from 1 to 2 with ionic strength exhibiting both diffusion and drift characteristics, with a “cut point” at approximately 10−5× PBS (Ni≈9×1014/mL). Under a high bias, once the PBS concentration reaches 10−3×, the behavior is then drift dominant. This indicates that the higher bias likely triggers electrochemical reactions and by extension, faradaic effects at most physiologically relevant ionic strengths. The signal-to-noise ratio (SNR) of the device has an inverse linear relationship with the low frequency current noise. The device exhibits a higher SNR in the sub-threshold regime than in the saturation regime. Specifically, within the saturation regime, an inversely proportional relationship between the SNR and the ionic concentration is observed. The electrochemical noise induced from ionic activities is roughly proportional to Ni−1/2.