Nanoscience and Nanotechnology Commons^™

High Throughput And Highly Controllable Methods For In Vitro Intracellular Delivery, Justin Brooks, Grayson Minnick, Prithvijit Mukherjee, Arian Jaberi, Lingqian Chang, Horacio D. Espinosa, Ruiguo Yang

Department of Mechanical and Materials Engineering: Faculty Publications

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a …

Molecular Dynamics Simulations Of Ion Transport Through Electrically Stressed Biological Membranes, Federica Castellani

Biomedical Engineering Theses & Dissertations

The cell membrane is a selectively permeable barrier that controls the transport of ions, molecules, and other materials into and out of a cell. The manipulation of the cell membrane permeability is the basis for several biotechnological and biomedical applications, including electroporation. Electroporation (or electropermeabilization) occurs when the application of an external electric pulse causes water intrusion into the membrane interior and the formation of conductive transmembrane electropores. These electropores allow drugs, genetic material, and other normally impermeant molecules to enter a cell. Despite years of study, the complex mechanisms underlying this process are still not well understood. Molecular dynamics …

Probing Nanoelectroporation And Resealing Of The Cell Membrane By The Entry Of Ca2+ And Ba2+ Ions, Wenfei Bo, Mantas Silkunas, Uma Mangalanathan, Vitalij Novickij, Maura Casciola, Iurii Semenov, Shu Xiao, Olga N. Pakhomova, Andrei G. Pakhomov

Bioelectrics Publications

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca²⁺, Cd²⁺, Zn²⁺, and Ba²⁺ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca²⁺ and Ba²⁺ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than >7 …

Fabrication And Characterization Of A Mcro/Nanofluidic Platform For Electroporation., Hanwen Yuan

Electronic Theses and Dissertations

For traditional electroporation devices, there are a number of problems associated with these devices such as insufficient understanding of its theoretical mechanism, low cell viability, inadequate electroporation efficiency, excess voltage applied to generate required electric field due to the large size of these devices and sample contamination. Although newly developed microfluidic electroporation devices have solved most of the above existing problems in traditional bulk electroporation devices, they appear to lack the ability to control the precise dose of biomolecules or genes transfecting into cells and, from a manufacturing perspective, the fabrication methods do not enable repeatable production of such devices …

Size Specific Transfection To Mammalian Cells By Micropillar Array Electroporation, Yingbo Zu

Doctoral Dissertations

Electroporation serves as a promising non-viral gene delivery approach, while its current configurations carry drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here, we developed a new micropillar array electroporation (MAE) platform to advance the delivery of plasmid DNA and RNA to mammalian cells. By introducing well-patterned micropillar array on the electrode surface, the number of pillars each cell faces varies with its cell membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently done and contributed to a 2.5~3 fold increase on plasmid DNA transfection and …

Comparison Between Direct And Reverse Electroporation Of Cells In Situ: A Simulation Study, Leila Towhidi, Delaram Khodadadi, Nataly Maimari, Ryan M. Pedrigi, Henry Ip, Zoltan Kis, Brenda R. Kwak, Tatiana W. Petrova, Mauro Delorenzi, Rob Krams

Department of Mechanical and Materials Engineering: Faculty Publications

The discovery of the human genome has unveiled new fields of genomics, transcriptomics, and proteomics, which has produced paradigm shifts on how to study disease mechanisms, wherein a current central focus is the understanding of how gene signatures and gene networks interact within cells. These gene function studies require manipulating genes either through activation or inhibition, which can be achieved by temporarily permeabilizing the cell membrane through transfection to deliver cDNA or RNAi. An efficient transfection technique is electroporation, which applies an optimized electric pulse to permeabilize the cells of interest. When the molecules are applied on top of seeded …

Cnt Membrane Platforms For Transdermal Drug Delivery And Aptamer Modulated Transport, Tao Chen

Theses and Dissertations--Chemical and Materials Engineering

CNT membrane platforms are biomimetic polymeric membranes imbedded with carbon nanotubes which show fast fluid flow, electric conductivity, and the ability to be grafted with chemistry. A novel micro-dialysis probe nicotine concentration sampling technique was proposed and proved in vitro, which could greatly improve the efficiency and accuracy of future animal transdermal studies. To enhance the scope of transdermal drug delivery which was limited to passive diffusion of small, potent lipophilic drugs, a wire mesh lateral electroporation design was also proposed which could periodically disrupt the skin barrier and enhance drug flux.

It was shown that AMP binding aptamer …

Primary Pathways Of Intracellular Ca2+ Mobilization By Nanosecond Pulsed Electric Field, Iurii Semenov, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which determines multifarious downstream effects. By using fast ratiometric Ca²⁺ imaging with Fura-2, we quantified the external Ca²⁺ uptake, compared it with Ca²⁺ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca²⁺ channels, so that the nsPEF-induced [Ca²⁺]_i changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2+]_i increase …