Intracellular delivery method based on a combination of electrokinetic forces and vibration-assisted cell membrane perforation

2014 ◽  
Author(s):  
Takayuki Shibata ◽  
Yasuharu Ito ◽  
Tatsuya Ozawa ◽  
Moeto Nagai ◽  
Takahiro Kawashima
Keyword(s):  
Cell Membrane ◽  
Intracellular Delivery ◽  
Delivery Method ◽  
Membrane Perforation

scholarly journals Minimally invasive intracellular delivery based on electrokinetic forces combined with vibration-assisted cell membrane perforation

2016 ◽  
Vol 56 (1) ◽  
pp. 017001 ◽  
Author(s):  
Takayuki Shibata ◽  
Tatsuya Ozawa ◽  
Yasuharu Ito ◽  
Keita Yamamoto ◽  
Moeto Nagai
Keyword(s):  
Cell Membrane ◽  
Minimally Invasive ◽  
Intracellular Delivery ◽  
Membrane Perforation

Electrokinetic intracellular delivery combined with vibration-assisted cell membrane perforation

MHS2013 ◽  
2013 ◽  
Author(s):  
Tatsuya Ozawa ◽  
Yasuharu Ito ◽  
Moeto Nagai ◽  
Takahiro Kawashima ◽  
Takayuki Shibata
Keyword(s):  
Cell Membrane ◽  
Intracellular Delivery ◽  
Membrane Perforation

scholarly journals Universal intracellular biomolecule delivery with precise dosage control

2018 ◽  
Vol 4 (10) ◽  
pp. eaat8131 ◽  
Author(s):  
Y. Cao ◽  
H. Chen ◽  
R. Qiu ◽  
M. Hanna ◽  
E. Ma ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Electric Fields ◽  
Low Voltage ◽  
Intracellular Delivery ◽  
Cell Types ◽  
Controlled Delivery ◽  
Biological Research ◽  
Biological Applications ◽  
Primary Cells ◽  
Cell Densities

Intracellular delivery of mRNA, DNA, and other large macromolecules into cells plays an essential role in an array of biological research and clinical therapies. However, current methods yield a wide variation in the amount of material delivered, as well as limitations on the cell types and cargoes possible. Here, we demonstrate quantitatively controlled delivery into a range of primary cells and cell lines with a tight dosage distribution using a nanostraw-electroporation system (NES). In NES, cells are cultured onto track-etched membranes with protruding nanostraws that connect to the fluidic environment beneath the membrane. The tight cell-nanostraw interface focuses applied electric fields to the cell membrane, enabling low-voltage and nondamaging local poration of the cell membrane. Concurrently, the field electrophoretically injects biomolecular cargoes through the nanostraws and into the cell at the same location. We show that the amount of material delivered is precisely controlled by the applied voltage, delivery duration, and reagent concentration. NES is highly effective even for primary cell types or different cell densities, is largely cargo agnostic, and can simultaneously deliver specific ratios of different molecules. Using a simple cell culture well format, the NES delivers into >100,000 cells within 20 s with >95% cell viability, enabling facile, dosage-controlled intracellular delivery for a wide variety of biological applications.

scholarly journals Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules

2005 ◽  
Vol 390 (2) ◽  
pp. 407-418 ◽  
Author(s):  
Catherine de Coupade ◽  
Antonio Fittipaldi ◽  
Vanessa Chagnas ◽  
Matthieu Michel ◽  
Sophie Carlier ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Mammalian Cells ◽  
Heparan Sulphate ◽  
Intracellular Delivery ◽  
Membrane Lipid ◽  
Cell Penetrating Peptides ◽  
Heparin Binding ◽  
Independent Manner ◽  
Cell Penetrating

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell® peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell® peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell® peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

An intracellular delivery method for siRNA by an arginine-rich peptide

2007 ◽  
Vol 70 (4) ◽  
pp. 579-586 ◽  
Author(s):  
Ya-Hui Wang ◽  
Yu-Wun Hou ◽  
Han-Jung Lee
Keyword(s):  
Intracellular Delivery ◽  
Delivery Method

Continuous-flow, electrically-triggered, single cell-level electroporation

TECHNOLOGY ◽  
2017 ◽  
Vol 05 (01) ◽  
pp. 31-41 ◽  
Author(s):  
Mingde Zheng ◽  
Joseph J. Sherba ◽  
Jerry W. Shan ◽  
Hao Lin ◽  
David I. Shreiber ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Field Strength ◽  
Electric Field ◽  
Cell Viability ◽  
Electric Field Strength ◽  
Continuous Flow ◽  
Intracellular Delivery ◽  
Membrane Permeabilization ◽  
Flow Environment ◽  
Cell Impedance

Electroporation creates transient openings in the cell membrane, allowing for intracellular delivery of diagnostic and therapeutic substances. The degree of cell membrane permeability during electroporation plays a key role in regulating the size of the delivery payload as well as the overall cell viability. A microfluidic platform offers the ability to electroporate single cells with impedance detection of membrane permeabilization in a high-throughput, continuous-flow manner. We have developed a flow-based electroporation microdevice that automatically detects, electroporates, and monitors individual cells for changes in permeability and delivery. We are able to achieve the advantages of electrical monitoring of cell permeabilization, heretofore only achieved with trapped or static cells, while processing the cells in a continuous-flow environment. We demonstrate the analysis of membrane permeabilization on individual cells before and after electroporation in a continuous-flow environment, which dramatically increases throughput. We have confirmed cell membrane permeabilization by electrically measuring the changes in cell impedance from electroporation and by optically measuring the intracellular delivery of a fluorescent probe after systematically varying the electric field strength and duration and correlating the pulse parameters to cell viability. We find a dramatic change in cell impedance and propidium iodide (PI) uptake at a pulse strength threshold of 0.87 kV/cm applied for a duration of 1 ms or longer. The overall cell viability was found to vary in a dose dependent manner with lower viability observed with increasing electric field strength and pulse duration. Cell viability was greater than 83% for all cases except for the most aggressive pulse condition (1[Formula: see text]kV/cm for 5[Formula: see text]ms), where the viability dropped to 67.1%. These studies can assist in determining critical permeabilization and molecular delivery parameters while preserving viability.

scholarly journals Photocatalytic Nanofabrication and Intracellular Raman Imaging of Living Cells with Functionalized AFM Probes

Micromachines ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 495
Author(s):  
Takayuki Shibata ◽  
Hiromi Furukawa ◽  
Yasuharu Ito ◽  
Masahiro Nagahama ◽  
Terutake Hayashi ◽  
...  
Keyword(s):  
Biological Sciences ◽  
Cell Membrane ◽  
Living Cell ◽  
Living Cells ◽  
Raman Imaging ◽  
Photochemical Oxidation ◽  
Membrane Perforation ◽  
Afm Probe ◽  
Tip Enhanced Raman Spectroscopy

Atomic force microscopy (AFM) is an effective platform for in vitro manipulation and analysis of living cells in medical and biological sciences. To introduce additional new features and functionalities into a conventional AFM system, we investigated the photocatalytic nanofabrication and intracellular Raman imaging of living cells by employing functionalized AFM probes. Herein, we investigated the effect of indentation speed on the cell membrane perforation of living HeLa cells based on highly localized photochemical oxidation with a catalytic titanium dioxide (TiO2)-functionalized AFM probe. On the basis of force–distance curves obtained during the indentation process, the probability of cell membrane perforation, penetration force, and cell viability was determined quantitatively. Moreover, we explored the possibility of intracellular tip-enhanced Raman spectroscopy (TERS) imaging of molecular dynamics in living cells via an AFM probe functionalized with silver nanoparticles in a homemade Raman system integrated with an inverted microscope. We successfully demonstrated that the intracellular TERS imaging has the potential to visualize distinctly different features in Raman spectra between the nucleus and the cytoplasm of a single living cell and to analyze the dynamic behavior of biomolecules inside a living cell.

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