scholarly journals Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides

2015 ◽  
Vol 6 ◽  
Author(s):  
Andriy Bilichak ◽  
Justin Luu ◽  
François Eudes
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Delivery ◽  
Cationic Peptides

scholarly journals Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells

2020 ◽  
Vol 117 (20) ◽  
pp. 10976-10982 ◽  
Author(s):  
Jason N. Belling ◽  
Liv K. Heidenreich ◽  
Zhenhua Tian ◽  
Alexandra M. Mendoza ◽  
Tzu-Ting Chiou ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Acoustic Waves ◽  
Fluorescent Protein ◽  
Single Channel ◽  
Intracellular Delivery ◽  
Cell Types ◽  
Membrane Repair ◽  
Hematopoietic Stem ◽  
Cell Therapies ◽  
Membrane Rupture

Advances in gene editing are leading to new medical interventions where patients’ own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.

Assembly and intracellular delivery of quantum dot-fluorescent protein bioconjugates

2008 ◽  
Author(s):  
Igor L. Medintz ◽  
Thomas Pons ◽  
James B. Delehanty ◽  
Kimihiro Susumu ◽  
Philip E. Dawson ◽  
...  
Keyword(s):  
Quantum Dot ◽  
Fluorescent Protein ◽  
Intracellular Delivery

scholarly journals Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding

2018 ◽  
Author(s):  
Justin A. Jarrell ◽  
Amy A. Twite ◽  
Katherine H. W. J. Lau ◽  
Moein N. Kashani ◽  
Adrian A. Lievano ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Membrane ◽  
Vortex Shedding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Intracellular Delivery ◽  
Microfluidic Chips ◽  
Dna And Rna ◽  
Green Fluorescent ◽  
Membrane Poration

AbstractIntracellular delivery of functional macromolecules, such as DNA and RNA, across the cell membrane and into the cytosol, is a critical process in both biology and medicine. Herein, we develop and use microfluidic chips containing post arrays to induce microfluidic vortex shedding, or μVS, for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with μVS by delivery of a 996-nucleotide mRNA construct encoding enhanced green fluorescent protein (EGFP) and assessed transfection efficiencies by quantifying levels of EGFP protein expression. We achieved high transfection efficiency (63.6 ± 3.44% EGFP+ viable cells) with high cell viability (77.3 ± 0.58%) and recovery (88.7 ± 3.21%) in CD3+ T cells 19 hrs after μVS processing. Importantly, we show that processing cells via μVS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 × 106 cells s−1 at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of μVS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.

Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides

2020 ◽  
Vol 12 (45) ◽  
pp. 50222-50235
Author(s):  
Eric A. Dailing ◽  
Kameron V. Kilchrist ◽  
J. William Tierney ◽  
R. Brock Fletcher ◽  
Brian C. Evans ◽  
...  
Keyword(s):  
Cell Membranes ◽  
Intracellular Delivery ◽  
Cationic Peptides ◽  
Anionic Polymer

scholarly journals An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Brian C. Evans ◽  
R. Brock Fletcher ◽  
Kameron V. Kilchrist ◽  
Eric A. Dailing ◽  
Alvin J. Mukalel ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Small Molecules ◽  
Gene Editing ◽  
Intracellular Delivery ◽  
Cationic Polymer ◽  
Peptide Delivery ◽  
Cationic Lipids ◽  
Cationic Peptides ◽  
Superior Efficacy ◽  
Intracellular Targets

Abstract Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

Isolation and characterization of the genes encoding antimicrobial neutrophil cationic peptides, defensins.

Ensho ◽  
1995 ◽  
Vol 15 (1) ◽  
pp. 33-41
Author(s):  
Isao Nagaoka ◽  
Noriko Ishihara ◽  
Akimasa Someya ◽  
Kazuhisa Iwabuchi ◽  
Shin Yomogida ◽  
...  
Keyword(s):  
Cationic Peptides ◽  
Isolation And Characterization ◽  
Genes Encoding
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