Deyao Zhao

Circulating microRNA-122 as a potential biomarker for liver injury.

Liver-specific microRNA-122 (miR-122) is involved in the replication of hepatitis C virus (HCV) and its potential as a target for antiviral intervention was recently assessed. However, the use of circulating miR-122 in the evaluation of liver function has never been reported. In the present study, changes of serum miRNA levels were first evaluated in acute human hepatotoxicity due to paraquat exposure. Serum samples were collected and analyzed using real-time reverse transcription PCR. The results showed a positive correlation between serum miR-122 and alanine aminotransferase, a clinical biomarker for liver function. Furthermore, serum miR-122 was assessed in patients with hepatitis B and hepatocarcinoma, resulting in distinct miR-122 profiles in these two closely related diseases. In addition to miR-122, another small RNA, U6 small nuclear RNA, was downregulated in hepatocarcinoma patients, suggesting its prognostic significance in this disease. Taken together, these lines of evidence indicate that serum miR-122 may provide a biomarker for diverse liver diseases and, more importantly, suggest that a combination of nucleic acid biomarkers may be used as a sensitive and specific index for discriminating closely related diseases.

Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.

A Laminar Flow Electroporation System for Efficient DNA and siRNA Delivery

By introducing a hydrodynamic mechanism into a microfluidics-based electroporation system, we developed a novel laminar flow electroporation system with high performance. The laminar buffer flow implemented in the system separated the cell suspension flow from the electrodes, thereby excluding many unfavorable effects due to electrode reaction during electroporation, such as hydrolysis, bubble formation, pH change, and heating. Compared to conventional microfluidic electroporation systems, these improvements significantly enhanced transfection efficiency and cell viability. Furthermore, successful electrotransfection of plasmid DNA and, more importantly, synthetic siRNA, was demonstrated in several hard-to-transfect cell types using this system.

Flow-through cell electroporation microchip integrating dielectrophoretic viable cell sorting.

Microfluidics based continuous cell electroporation is an appealing approach for high-throughput cell transfection, but cell viability of existing methods is usually compromised by adverse electrical or hydrodynamic effects. Here we present the validation of a flow-through cell electroporation microchip, in which dielectrophoretic force was employed to sort viable cells. By integrating parallel electroporation electrodes and dielectrophoresis sorting electrodes together in a simple straight microfluidic channel, sufficient electrical pulses were applied for efficient electroporation, and a proper sinusoidal electrical field was subsequently utilized to exclude damaged cells by dielectrophoresis. Thus, the difficulties for seeking the fine balance between electrotransfection efficiency and cell viability were steered clear. After careful investigation and optimization of the DEP behaviors of electroporated cells, efficient electrotransfection of plasmid DNA was demonstrated in vulnerable neuron cells and several hard-to-transfect primary cell types with excellent cell viability. This microchip constitutes a novel way of continuous cell transfection to significantly improve the cell viability of existing methodologies.

Electroporation on microchips: the harmful effects of pH changes and scaling down

Electroporation has been widely used in delivering foreign biomolecules into cells, but there is still much room for improvement, such as cell viability and integrity. In this manuscript, we investigate the distribution and the toxicity of pH changes during electroporation, which significantly decreases cell viability. A localized pH gradient forms between anode and cathode leading to a localized distribution of cell death near the electrodes, especially cathodes. The toxicity of hydroxyl ions is severe and acute due to their effect in the decomposition of phospholipid bilayer membrane. On the other hand, the electric field used for electroporation aggravates the toxicity of hydroxyl because the electropermeabilization of cell membrane makes bilayer structure more loosen and vulnerable. We also investigate the side effects during scaling down the size of electrodes in electroporation microchips. Higher percentage of cells is damaged when the size of electrodes is smaller. At last, we propose an effective strategy to constrain the change of pH by modifying the composition of electroporation buffer. The modified buffer decreases the changes of pH, thus enables high cell viability even when the electric pulse duration exceeds several milliseconds. This ability has potential advantage in some applications that require long-time electric pulse stimulation.

A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo

Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices.

A Pliable Electroporation Patch (ep-Patch) for Efficient Delivery of Nucleic Acid Molecules into Animal Tissues with Irregular Surface Shapes

Delivery of nucleic acids into animal tissues by electroporation is an appealing approach for various types of gene therapy, but efficiency of existing methodsis not satisfactory. Here we present the validation of novel electroporation patch (ep-Patch) for efficient delivery of DNA and siRNA into mouse tissues. Using micromachining technology, closely spaced gold electrodes were made on the pliable parylene substrate to form a patch-like electroporation metrics. It enabled large coverage of the target tissues and close surface contact between the tissues and electrodes, thus providing a uniform electric field to deliver nucleic acids into tissues, even beneath intact skin. Using this ep-Patch for efficiently delivery of both DNA and siRNA, non-invasive electroporation of healthy mouse muscle tissue was successfully achieved. Delivery of these nucleic acids was performed to intact tumors with satisfactory results. Silencing of tumor genes using the ep-Patch was also demonstrated on mice. This pliable electroporation patch method constitutes a novel way of in vivo delivery of siRNA and DNA to certain tissues or organs to circumvent the disadvantages of existing methodologies for in vivo delivery of nucleic acid molecules.

High-density distributed electrode network, a multi-functional electroporation method for delivery of molecules of different sizes

We present a multi-functional electroporation method for delivery of biomolecule utilizing a high-density distributed electrode network (HDEN) under tri-phase electric stimulation. The HDEN device, with which drastic pH change during the electroporation was avoided,was demonstrated to be highly effective for transfection of not only DNA plasmids and small interfering RNAs (siRNA), but also a small molecular anti-cancer drug, into cells in adjustable volumes of cell suspension. The method constitutes a very flexible electroporation approach in a wide range of in vitro or ex vivo scenarios in various tubes, standard multi-well plates as well as flow chambers.

Method for Electric Parametric Characterization and Optimization of Electroporation on a Chip

We have developed a rapid method to optimize the electric parameters of cell electroporation. In our design, a pair of ring-dot formatted electrodes was used to generate a radial distribution of electric field from the center to the periphery. Varied electric field intensity was acquired in different annulus when an electric pulse was applied. Cells were cultured on the microchips for adherent cell electroporation and in situ observation. The electroporation parameters of electric field intensity were explored and evaluated in terms of cell viability and transfection efficiency. The optimization was performed in consideration of both cell viability, which was investigated to decrease as electric field increases, and the transfection rate, which normally increases at stronger electric field. The electroporation characteristics HEK-293A and Hela cells were investigated, and the optimum parameters were obtained. Verified by a commercial electroporation system as well as self-made microchips endowed the optimization with wider meaning. At last, as applications, we acquired the optimal electroporation pulse intensity of Neuro-2A cells and a type of primary cell (human umbilical vein endothelial cell, HUVEC) by one time electroporation using the proposed method.

A parylene-based flexible electroporation chip applicable for in vivo gene and siRNA delivery

We report a parylene-based flexible electroporation chip for in vivo nucleic acid delivery. Benefited from the flexibility of parylene film, our chip fits the natural shape of the electroporated objects, thereby provides a uniform electric field over a large area. In addition, the subject is less likely to be harmed owing to the chip features, including good biocompatibility, less invasive electroporation and lower applied voltage because of smaller space between electrodes. These characteristics offer vast potential for applications of implantable electroporation devices. Using the proposed chip, we achieved successful plasmid DNA expression and siRNA transfection in both healthy mouse tissue and living tumor with excellent efficacy (90%).