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Proceedings ArticleDOI

Nanofocused electric field for localized single cell nanoelectroporation with membrane reversibility

07 Apr 2013-pp 961-964
TL;DR: This report presents a new type localized single cell membrane electroporation (LSCMEP), at specific region of the single cell with the application of 800 μs electric pulse, which generates well-controlled nano-pores allowing rapid recovery of cell membrane and provides a clear optical path potentially tracking of drugs to deliver inside single cell.
Abstract: Despite the significant research in electroporation, high electric field was applied to the whole cells resulted in permeabilizing the membrane of millions of cells without reversibility [1]. To deliver biomolecules through the specific region of the cell membrane with high cell viability and high transfection rate is important for various biological and therapeutic applications.This report presents a new type localized single cell membrane electroporation (LSCMEP), at specific region of the single cell with the application of 800 μs electric pulse. The ITO nano-electrodes with 100nm thickness and 500 nm gap between two electrodes can generate an intense electric field to track biomolecules inside HeLa cell in our studies. This small gap between two nano-electrodes can neglect thermal effect on cell membrane and permit reversible electroporation with high cell viability (90%) and minimum effected electroporation region (0.48 μm). Our approach successfully delivers biomolecules through a specific region of single cell with high transfection rate (82%) and high cell viability. This process, not only generates well-controlled nano-pores allowing rapid recovery of cell membrane, but also it provides a clear optical path potentially tracking of drugs to deliver inside single cell.
Citations
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Journal ArticleDOI
TL;DR: This review article will emphasize the basic concept and working mechanism associated with electroporation, single cell Electroporation and biomolecular delivery using micro/nanofluidic devices, their fabrication, working principles and cellular analysis with their advantages, limitations, potential applications and future prospects.
Abstract: © 2018 IOP Publishing Ltd. The ability to deliver foreign molecules into a single living cell with high transfection efficiency and high cell viability is of great interest in cell biology for applications in therapeutic development, diagnostics and drug delivery towards personalized medicine. Many chemical and physical methods have been developed for cellular delivery, however most of these techniques are bulk approach, which are cell-specific and have low throughput delivery. On the other hand, electroporation is an efficient and fast method to deliver exogenous biomolecules such as DNA, RNA and oligonucleotides into target living cells with the advantages of easy operation, controllable electrical parameters and avoidance of toxicity. The rapid development of micro/nanofluidic technologies in the last two decades, enables us to focus an intense electric field on the targeted cell membrane to perform single cell micro-nano-electroporation with high throughput intracellular delivery, high transfection efficiency and cell viability. This review article will emphasize the basic concept and working mechanism associated with electroporation, single cell electroporation and biomolecular delivery using micro/nanoscale electroporation devices, their fabrication, working principles and cellular analysis with their advantages, limitations, potential applications and future prospects.

41 citations

Book ChapterDOI
01 Jan 2020
TL;DR: This chapter mainly focuses on different physical drug-delivery techniques such as electroporation, optoporation, mechanopsoration, magnetoporation and hybrid techniques along with their working mechanisms, advantages, disadvantages, and limitations.
Abstract: Delivery of exogenous materials or cargo such as drugs, proteins, peptides, and nucleic acids into cells is a vital segment in molecular and cellular biology for potential cellular therapy and drug-discovery applications contributing toward personalization of medicine. Over the years, drug-delivery techniques have been developed in order to gain more control over the drug dosage, targeted delivery, and to minimize side effects. The major drug-delivery techniques can be classified as viral, chemical, and physical methods. Viral vectors are prominently used for gene therapy; however, they are cell-specific and have an immune response with high toxicity. Chemical methods are often limited by the low efficiency of plasmid delivery into different cell types due to plasmid degradation and toxicity. Considering these limitations, different physical methods such as photoporation, gene gun, hydrodynamic injection, electroporation, and mechanoporation, etc., are being widely developed for highly efficient cargo delivery with low toxicity. These methods are able to create transient hydrophilic membrane pores to deliver cargos into cells using different physical energies. Currently, ex vivo cargo delivery is widely studied while few in vivo applications have been developed. Concerning several obstacles to cargo delivery into cells, this chapter mainly focuses on different physical drug-delivery techniques such as electroporation, optoporation, mechanoporation, magnetoporation, and hybrid techniques along with their working mechanisms, advantages, disadvantages, and limitations. An insight into the future prospects and real-time applications of these techniques is also discussed.

18 citations

Book ChapterDOI
01 Jan 2016
TL;DR: Electroporation technique opens up the new window for the manipulation of genomics, proteomics, trascriptomics, metabolomics, or fluxomics at single cell level for biological research and therapeutic applications.
Abstract: Single-cell analysis is a powerful technique to understand cell to cell or cell to environment behaviors. It can provide detailed information of cell proliferation, differentiation, and different responses to external stimuli and intracellular reactions. For single cell analysis, electroporation or electropermeabilization is an efficient and fast method, where high external electric field is applied on cell membrane to form transient membrane pores to deliver ions or molecules in or out of the cell. Conventional electroporation or bulk electroporation (BEP) is performed in a batch mode with millions of cells in suspension, which can only provide an average value. Since the last decade, microfabricated devices have been developed to perform single cell electroporation, where electric field is only intense on a single cell, resulting in high transfection efficiency with high cell viability compared to BEP. Single cell analysis using electroporation technique can be performed with microfabricated electrode arrays, carbon fiber microelectrodes, micropipettes or electrolyte-filled capillaries-based devices. Recently developed nanofabricated electrodes can realize localized single cell electroporation for delivery of different molecules with high transfection efficiency and high cell viability. Thus, single cell electroporation technique opens up the new window for the manipulation of genomics, proteomics, trascriptomics, metabolomics, or fluxomics at single cell level. This chapter emphasizes the recent advancement of electroporation technique for cellular delivery and the analysis method which might be potentially applicable for biological research and therapeutic applications.

10 citations

References
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Journal ArticleDOI
TL;DR: A simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed, according to which the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.
Abstract: Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 12 micrograms DNA Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport

2,496 citations


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  • ...entry into cells and electroporation[5-11]....

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Journal ArticleDOI
TL;DR: Electroporation has found applications in introduction of plasmids or foreign DNA into living cells for gene transfections, fusion of cells to prepare heterokaryons, hybridoma, hybrid embryos, and constructing animal model by fusing human cells with animal tissues.

1,053 citations

Journal ArticleDOI
TL;DR: This review provides a summary of the current knowledge of the interactions between the immune system adeno-associated virus, adenoviral and lentiviral vectors, and their transgene products.
Abstract: Viral vectors are potent gene delivery platforms used for the treatment of genetic and acquired diseases. However, just as viruses have evolved to infect cells efficiently, the immune system has evolved to fight off what it perceives as invading pathogens. Therefore, innate immunity and antigen-specific adaptive immune responses against vector-derived antigens reduce the efficacy and stability of in vivo gene transfer. In addition, a number of vectors are derived from parent viruses that humans encounter through natural infection, resulting in preexisting antibodies and possibly in memory responses against vector antigens. Similarly, antibody and T-cell responses may be directed against therapeutic gene products that often differ from the endogenous nonfunctional or absent protein that is being replaced. As details and mechanisms of such immune reactions are uncovered, novel strategies are being developed, and vectors are being specifically engineered to avoid, suppress or manipulate the response, ideally resulting in sustained expression and immune tolerance to the transgene product. This review provides a summary of our current knowledge of the interactions between the immune system adeno-associated virus, adenoviral and lentiviral vectors, and their transgene products.

585 citations


"Nanofocused electric field for loca..." refers methods in this paper

  • ...Many viral and nonviral transfection techniques has been developed day to day including viral vectors [2-4], chemical methods such as...

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Journal ArticleDOI
TL;DR: Over the last 5 years, physical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, in some cases reaching the efficiencies of viral vectors.
Abstract: Over the last 5 years, physical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, in some cases reaching the efficiencies of viral vectors. In vivo electroporation dramatically increases transfection efficiency for a variety of tissues. Other methods with clinical precedent, pressure-perfusion and ultrasound, also improve plasmid gene transfer. Alternatives such as focused laser, magnetic fields and ballistic (gene gun) approaches can also enhance delivery. As plasmid DNA appears to be a safe gene vector system, it seems likely that plasmid with physically enhanced delivery will be used increasingly in clinical trials.

387 citations


Additional excerpts

  • ...entry into cells and electroporation[5-11]....

    [...]

Journal ArticleDOI
01 Sep 2006-Methods
TL;DR: The immunogenicity and protective efficacy of PMED DNA vaccines in nonhuman primates and swine and studies that have directly compared the effectiveness of PM ED in these large animal models to existing licensed vaccines and intramuscular or intradermal delivery of DNA vaccines with a needle are reviewed.

154 citations