scholarly journals Carrier-Free Cellular Transport of CRISPR/Cas9 Ribonucleoprotein for Genome Editing by Cold Atmospheric Plasma

Biology ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1038
Author(s):  
Haodong Cui ◽  
Min Jiang ◽  
Wenhua Zhou ◽  
Ming Gao ◽  
Rui He ◽  
...  

A carrier-free CRISPR/Cas9 ribonucleoprotein delivery strategy for genome editing mediated by a cold atmospheric plasma (CAP) is described. The CAP is promising in many biomedical applications due to efficient production of bioactive ionized species. The MCF-7 cancer cells after CAP exposure exhibit increased extracellular reactive oxygen and nitrogen species (RONS) and altered membrane potential and permeability. Hence, transmembrane transport of Ca2+ into the cells increases and accelerates ATP hydrolysis, resulting in enhanced ATP-dependent endocytosis. Afterwards, the increased Ca2+ and ATP contents promote the release of cargo into cytoplasm due to the enhanced endosomal escape. The increased membrane permeability also facilitates passive diffusion of foreign species across the membrane into the cytosol. After CAP exposure, the MCF-7 cells incubated with Cas9 ribonucleoprotein (Cas9-sgRNA complex, Cas9sg) with a size of about 15 nm show 88.9% uptake efficiency and 65.9% nuclear import efficiency via passive diffusion and ATP-dependent endocytosis pathways. The efficient transportation of active Cas9sg after the CAP treatment leads to 21.7% and 30.2% indel efficiencies in HEK293T and MCF-7 cells, respectively. This CAP-mediated transportation process provides a simple and robust alternative for the delivery of active CRISPR/Cas9 ribonucleoprotein. Additionally, the technique can be extended to other macro-biomolecules and nanomaterials to cater to different biomedical applications.

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 249
Author(s):  
Zhitong Chen ◽  
Richard Obenchain ◽  
Richard E. Wirz

Conventional plasma jets for biomedical applications tend to have several drawbacks, such as high voltages, high gas delivery, large plasma probe volume, and the formation of discharge within the organ. Therefore, it is challenging to employ these jets inside a living organism’s body. Thus, we developed a single-electrode tiny plasma jet and evaluated its use for clinical biomedical applications. We investigated the effect of voltage input and flow rate on the jet length and studied the physical parameters of the plasma jet, including discharge voltage, average gas and subject temperature, and optical emissions via spectroscopy (OES). The interactions between the tiny plasma jet and five subjects (de-ionized (DI) water, metal, cardboard, pork belly, and pork muscle) were studied at distances of 10 mm and 15 mm from the jet nozzle. The results showed that the tiny plasma jet caused no damage or burning of tissues, and the ROS/RNS (reactive oxygen/nitrogen species) intensity increased when the distance was lowered from 15 mm to 10 mm. These initial observations establish the tiny plasma jet device as a potentially useful tool in clinical biomedical applications.


Plasma ◽  
2018 ◽  
Vol 1 (1) ◽  
pp. 119-125 ◽  
Author(s):  
Jamoliddin Razzokov ◽  
Maksudbek Yusupov ◽  
Annemie Bogaerts

Cold atmospheric plasma (CAP) has shown its potential in biomedical applications, such as wound healing, cancer treatment and bacterial disinfection. Recent experiments have provided evidence that CAP can also enhance the intracellular uptake of glucose molecules which is important in diabetes therapy. In this respect, it is essential to understand the underlying mechanisms of intracellular glucose uptake induced by CAP, which is still unclear. Hence, in this study we try to elucidate the possible mechanism of glucose uptake by cells by performing computer simulations. Specifically, we study the transport of glucose molecules through native and oxidized membranes. Our simulation results show that the free energy barrier for the permeation of glucose molecules across the membrane decreases upon increasing the degree of oxidized lipids in the membrane. This indicates that the glucose permeation rate into cells increases when the CAP oxidation level in the cell membrane is increased.


2008 ◽  
Vol 71 (2) ◽  
pp. 302-308 ◽  
Author(s):  
STEFANO PERNI ◽  
DAVID W. LIU ◽  
GILBERT SHAMA ◽  
MICHAEL G. KONG

This investigation describes the inactivation by cold atmospheric plasmas of one pathogenic and three spoilage organisms on the pericarps of mangoes and melons. The operating voltage necessary for efficient microbial decontamination of fruit pericarps was first established using Escherichia coli at a concentration of 107 CFU/cm2 on the surface of mango. It was found that, when the plasma was sustained slightly above its breakdown voltage of 12 kV (peak to peak), no inactivation was detected when cells were plated onto tryptone soya extract agar (TSA). However, when plated onto eosin methylene blue agar, sublethal injury corresponding to approximately 1 log reduction was achieved, whereas on TSA supplemented with 4% NaCl a greater reduction of 1.5 log was revealed. When the voltage was increased by 33% to 16 kV, a reduction in cell counts of 3 log was achieved on all three plating media. Further investigations at these new operating conditions were conducted using a range of spoilage microorganisms (Saccharomyces cerevisae, Pantoea agglomerans, and Gluconacetobacter liquefaciens) all at a surface concentration of 106 CFU/cm2 on the pericarps of mango and melon. P. agglomerans and G. liquefaciens were reduced below the detection limit (corresponding to 3 log) after only 2.5 s on both fruits, whereas E. coli required 5 s to reach the same level of inactivation. S. cerevisae was the most resistant organism studied and was reduced in numbers below the detection limit after 10 s on mango and 30 s on melon. The optical emission spectra generated by the cold atmospheric plasma at both high and low operating voltages were compared in order to identify putative lethal species. It was shown that an increase in the applied voltage led to more efficient production of reactive plasma species, particularly oxygen atoms, and the production of oxygen atoms was related to the level of bacterial inactivation achieved. Production of atomic oxygen could be used as an indicator of inactivation efficiency for scaling up cold plasma systems for whole fruit.


RSC Advances ◽  
2016 ◽  
Vol 6 (82) ◽  
pp. 78457-78467 ◽  
Author(s):  
Fanny Girard ◽  
Vasilica Badets ◽  
Sylvie Blanc ◽  
Kristaq Gazeli ◽  
Laurent Marlin ◽  
...  

Cold Atmospheric Plasmas (CAPs) are increasingly used for biomedical applications, their various reactive components must be then better determined. We demonstrate that peroxynitrite (ONOO−) is effectively a major reactive species generated by CAPs.


2020 ◽  
Vol 53 (12) ◽  
pp. 125202
Author(s):  
Hao Zhang ◽  
Jishen Zhang ◽  
Jie Ma ◽  
Jie Shen ◽  
Yan Lan ◽  
...  

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 2011 ◽  
Author(s):  
Sungbin Park ◽  
Heejoo Kim ◽  
Hwee Won Ji ◽  
Hyeon Woo Kim ◽  
Sung Hwan Yun ◽  
...  

Paclitaxel (Tx) is a widely used therapeutic chemical for breast cancer treatment; however, cancer recurrence remains an obstacle for improved prognosis of cancer patients. In this study, cold atmospheric plasma (CAP) was tested for its potential to overcome the drug resistance. After developing Tx-resistant MCF-7 (MCF-7/TxR) breast cancer cells, CAP was applied to the cells, and its effect on the recovery of drug sensitivity was assessed in both cellular and molecular aspects. Sensitivity to Tx in the MCF-7/TxR cells was restored up to 73% by CAP. A comparison of genome-wide expression profiles between the TxR cells and the CAP-treated cells identified 49 genes that commonly appeared with significant changes. Notably, 20 genes, such as KIF13B, GOLM1, and TLE4, showed opposite expression profiles. The protein expression levels of selected genes, DAGLA and CEACAM1, were recovered to those of their parental cells by CAP. Taken together, CAP inhibited the growth of MCF-7/TxR cancer cells and recovered Tx sensitivity by resetting the expression of multiple drug resistance–related genes. These findings may contribute to extending the application of CAP to the treatment of TxR cancer.


2017 ◽  
Vol 110 ◽  
pp. 280-290 ◽  
Author(s):  
Seungyeon Lee ◽  
Hyunkyung Lee ◽  
Dawoon Jeong ◽  
Juyeon Ham ◽  
Sungbin Park ◽  
...  

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