Shengnian Wang

Micro-/nanofluidics based cell electroporation

Non-viral gene delivery has been extensively explored as the replacement for viral systems. Among various non-viral approaches, electroporation has gained increasing attention because of its easy operation and no restrictions on probe or cell type. Several effective systems are now available on the market with reasonably good gene delivery performance. To facilitate broader biological and medical applications, micro-/nanofluidics based technologies were introduced in cell electroporation during the past two decades and their advances are summarized in this perspective. Compared to the commercially available bulk electroporation systems, they offer several advantages, namely, (1) sufficiently high pulse strength generated by a very low potential difference, (2) conveniently concentrating, trapping, and regulating the position and concentration of cells and probes, (3) real-time monitoring the intracellular trafficking at single cell level, and (4) flexibility on cells to be transfected (from single cell to large scale cell population). Some of the micro-devices focus on cell lysis or fusion as well as the analysis of cellular properties or intracellular contents, while others are designed for gene transfection. The uptake of small molecules (e.g., dyes), DNA plasmids, interfering RNAs, and nanoparticles has been broadly examined on different types of mammalian cells, yeast, and bacteria. A great deal of progress has been made with a variety of new micro-/nanofluidic designs to address challenges such as electrochemical reactions including water electrolysis, gas bubble formation, waste of expensive reagents, poor cell viability, low transfection efficacy, higher throughput, and control of transfection dosage and uniformity. Future research needs required to advance micro-/nanofluidics based cell electroporation for broad life science and medical applications are discussed.

Semicontinuous flow electroporation chip for high-throughput transfection on mammalian cells.

We have recently developed a semicontinuous flow electroporation (SFE) device for in vitro DNA delivery. Cells mixed with plasmid DNA continuously flowed through a serpentine channel, the side walls of which also serving as electrodes. With the use of pWizGFP plasmid and K562 cells as a model system, SFE showed better transgene expression (10-15%) compared to a commercial electroporation system. Quantitative results via MTS assay also revealed a 50% or higher cell viability. Similar observations were also found with pWizGFP transfection to mouse embryonic stem cells. Such improvements were attributed to less gas formation and Joule heating in SFE.

Micronozzle Array Enhanced Sandwich Electroporation of Embryonic Stem Cells

Electroporation is one of the most popular nonviral gene transfer methods for embryonic stem cell transfection. Bulk electroporation techniques, however, require a high electrical field and provide a nonuniform electrical field distribution among randomly distributed cells, leading to limited transfection efficiency and cell viability, especially for a low number of cells. We present here a membrane sandwich electroporation system using a well-defined micronozzle array. This device is capable of transfecting hundred to millions of cells with good performance. The ability to treat a small number of cells (i.e., a hundred) offers great potential to work with hard-to-harvest patient cells for pharmaceutical kinetic studies. Numerical simulation of the initial transmembrane potential distribution and propidium iodide (PI) dye diffusion experiments demonstrated the advantage of highly focused and localized electric field strength provided by the micronozzle array over conventional bulk electroporation.

Targeted nanoparticles enhanced flow electroporation of antisense oligonucleotides in leukemia cells

Liposome nanoparticles (LNs) with a targeting ligand were used in a semi-continuous flow electroporation (SFE) device to enhance in vitro delivery of exogenous oligonucleotides (ODN). Nanoparticles comprising transferrin-targeted lipoplex encapsulating ODN G3139 were mixed with K562 cells (a chronic myeloid leukemia cell line) and incubated for half an hour to accomplish nanoparticle binding. The mixture was then flowed through a SFE channel where electric pulses were given. Better ODN delivery efficiency was achieved with an increase of ∼24% to the case in combination of non-targeted LNs and SFE, and ∼60% to the case using targeted LNs alone, respectively. The MTS assay results confirmed cell viability greater than 75%.

Conformation dependence of DNA electrophoretic mobility in a converging channel

The electrophoresis of λ‐DNA is observed in a microscale converging channel where the center‐of‐masses trajectories of DNA molecules are tracked to measure instantaneous electrophoretic (EP) mobilities of DNA molecules of various stretch lengths and conformations. Contrary to the usual assumption that DNA mobility is a constant, independent of field and DNA length in free solution, we find DNA EP mobility varies along the axis in the contracting geometry. We correlate this mobility variation with the local stretch and conformational changes of the DNA, which are induced by the electric field gradient produced by the contraction. A “shish‐kebab” model of a rigid polymer segment is developed, which consists of aligned spheres acting as charge and drag centers. The EP mobility of the shish‐kebab is obtained by determining the electrohydrodynamic interactions of aligned spheres driven by the electric field. Multiple shish‐kebabs are then connected end‐to‐end to form a freely jointed chain model for a flexible DNA chain. DNA EP mobility is finally obtained as an ensemble average over the shish‐kebab orientations that are biased to match the overall stretch of the DNA chain. Using physically reasonable parameters, the model agrees well with experimental results for the dependence of EP mobility on stretch and conformation. We find that the magnitude of the EP mobility increases with DNA stretch, and that this increase is more pronounced for folded conformations.

Gold Nanoparticles Electroporation Enhanced Polyplex Delivery to Mammalian Cells

Nonviral methods have been explored as the replacement of viral systems for their low toxicity and immunogenicity. However, they have yet to reach levels competitive to their viral counterparts. In this paper, we combined physical and chemical methods to improve the performance of polyplex delivery of DNA and small interfering RNA. Specifically, gold nanoparticles (AuNPs) were used to carry polyplex (a chemical approach) while electroporation (a physical approach) was applied for fast and direct cytosolic delivery. In this hybrid approach, cationic polymer molecules condense and/or protect genetic probes as usual while AuNPs help fix polycations to reduce their cytotoxicity and promote the transfection efficiency of electroporation. AuNPs of various sizes were first coated with polyethylenimine, which were further conjugated with DNA plasmids or small interfering RNA molecules to form AuNPs‐polyplex. The hybrid nanoparticles were then mixed with cells and introduced into cell cytosol by electroporation. The delivery efficiency was evaluated with both model anchor cells (i.e., NIH/3T3) and suspension cells (i.e., K562), together with their impact on cell viability. We found that AuNP‐polyplex showed 1.5∼2 folds improvement on the transfection efficiency with no significant increase of toxicity when compared to free plasmid delivery by electroporation alone. Such a combination of physical and chemical delivery concept may stimulate further exploration in the delivery of various therapeutic materials for both in vitro and in vivo applications.

Etching of Pyrex glass substrates by inductively coupled plasma reactive ion etching for micro/nanofluidic devices

The inductively coupled plasma (ICP) reactive ion etching of Pyrex glass was carried out using SF6∕Ar plasmas. The etch rate and surface and sidewall smoothnesses were investigated systematically through their dependence on bias voltage, ICP power, pressure, flow rate, and cathode temperature. Near vertical sidewalls and smooth etched surfaces were obtained by optimized etching parameters. The maximum etch rate, 0.65μm∕min, was achieved at a pressure of 5mTorr, a bias of 720V, and an ICP power of 2500W. Microfluidic devices with various sizes on Pyrex glass have been designed and fabricated. Two types of electrokinetic flow patterns, which are extensional and rotational flows under different biases, have been successfully demonstrated with five cross microfluidic devices.

Five-cross microfluidic network design free of coupling between electrophoretic motion and electro-osmotic flow

Electro-osmotic and electrophoretic interactions are a major concern in the electrokinetic-based microfluidics, in particular, when the electrophoretic mobility of charged particles is greater than the surface electro-osmotic mobility. In this letter, a microfluidic device without surface charge patterning on the channel walls is introduced. Unlike the previous microfluidic devices, the charged particle movements in this five-cross design are largely independent of their charge density and polarity. Different flow patterns (i.e., extensional, mixed shear, and rotational flows) can be generated in this single design and can be quickly switched by just changing the external voltage inputs. The observed flow patterns of charged polystyrene microspheres and λ-DNA molecules in buffer solutions agree well with simulation results.

Electrokinetic interactions in microscale cross-slot flow

The effects of electroosmosis (EO) and electrophoresis (EP) interactions on the movement of highly charged polystyrene microspheres and lesser-charged liposome nanoparticles were studied, using a microscale cross-slot flow device with different charge density and polarity on the channel surfaces. When the particle EP mobility is greater than the channel surface EO mobility, the flow pattern would be either extensional or extension dominated mixed-shear. The rotation flow only exists when the EO mobility dominates. The surface charge density of top and bottom lids of microchannels plays an important role in microfluidics. Experimental observations agreed fairly well with calculated flow patterns.

Electrokinetics induced asymmetric transport in polymeric nanonozzles

The asymmetric geometry of polymeric nanonozzles provides two different transport directions: a converging direction (from the large opening to the small opening) and a diverging direction (from the small opening to the large opening). Asymmetric transport was observed in such nanochannels for both rigid polystyrene nanoparticles and flexible DNA molecules under a DC electric bias. Small, hard nanoparticles migrate easily in the diverging direction and tend to pack inside the nanochannel in the converging direction. In contrast, large, flexible DNA molecules transport better in the converging direction than in the diverging direction. A high electric field and a high velocity gradient along the tapered region produce different geometric constrictions and vortex-like particle motions for rigid nanoparticles, and also generate various coil-stretching dynamics for DNA molecules. Such nanonozzle arrays are useful in high flux and high sieving efficiency devices for biomolecule delivery or separation, and for loading trace amounts of drugs or genes for controlled drug and gene delivery.