scholarly journals Investigation of Plasmid DNA Delivery and Cell Viability Dynamics for Optimal Cell Electrotransfection In Vitro

2020 ◽  
Vol 10 (17) ◽  
pp. 6070
Author(s):  
Sonam Chopra ◽  
Paulius Ruzgys ◽  
Martynas Maciulevičius ◽  
Milda Jakutavičiūtė ◽  
Saulius Šatkauskas
Keyword(s):  
Cell Membrane ◽  
Cell Viability ◽  
High Voltage ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Dna Delivery ◽  
Output Parameter ◽  
Gene Electrotransfer ◽  
Electric Pulses

Electroporation is an effective method for delivering plasmid DNA molecules into cells. The efficiency of gene electrotransfer depends on several factors. To achieve high transfection efficiency while maintaining cell viability is a tedious task in electroporation. Here, we present a combined study in which the dynamics of both evaluation types of transfection efficiency and the cell viability were evaluated in dependence of plasmid concentration as well as at the different number of high voltage (HV) electric pulses. The results of this study reveal a quantitative sigmoidal (R2 > 0.95) dependence of the transfection efficiency and cell viability on the distance between the cell membrane and the nearest plasmid. We propose this distance value as a new, more accurate output parameter that could be used in further optimization studies as a predictor and a measure of electrotransfection efficiency.

scholarly journals The Good and the Bad of Cell Membrane Electroporation

2021 ◽  
Vol 68 (4) ◽  
pp. 753-764
Author(s):  
Katja Balantič ◽  
Damijan Miklavčič ◽  
Igor Križaj ◽  
Peter Kramar
Keyword(s):  
Cell Membrane ◽  
Lipid Bilayer ◽  
High Voltage ◽  
Food Industry ◽  
Cell Membranes ◽  
Tissue Ablation ◽  
Gene Electrotransfer ◽  
Basic Principles ◽  
Electric Pulses ◽  
Hydrophobic Barrier

Electroporation is used to increase the permeability of the cell membrane through high-voltage electric pulses. Nowadays, it is widely used in different areas, such as medicine, biotechnology, and the food industry. Electroporation induces the formation of hydrophilic pores in the lipid bilayer of cell membranes, to allow the entry or exit of molecules that cannot otherwise cross this hydrophobic barrier. In this article, we critically review the basic principles of electroporation, along with the advantages and drawbacks of this method. We discuss the effects of electroporation on the key components of biological membranes, as well as the main applications of this procedure in medicine, such as electrochemotherapy, gene electrotransfer, and tissue ablation. Finally, we define the most relevant challenges of this romising area of research.

scholarly journals Analysis of DNA mobility and gene electrotransfer in 3D in vitro collagen gel model

2020 ◽  
Author(s):  
Saša Haberl Meglič ◽  
Mojca Pavlin
Keyword(s):  
3D Model ◽  
Transfection Efficiency ◽  
Collagen Gel ◽  
Gene Electrotransfer ◽  
Electric Pulses ◽  
Impaired Mobility ◽  
Efficient Gene ◽  
Dna Mobility

Abstract Background: Gene electrotransfer is an established method that enables transfer of DNA into cells with electric pulses. Several studies analyzed different parameters; however the question of the mechanisms involved in gene electrotransfer remains open. One of main obstacles toward efficient gene electrotransfer in vivo is relatively poor DNA mobility in tissues.Objective and method: In order to analyze the effect of impaired mobility on gene electrotransfer efficiency, we applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and compared gene electrotransfer efficiency and viability of cells.Results: We obtained the highest transfection of plated cells, while transfection efficiency of embedded cells in 3D model was lowest and similar as in in vivo. To further analyze poor DNA mobility in 3D model, we applied DNA in top of or injected it into 3D model and showed that former way increases gene electrotransfer efficiency as was shown in in vivo studies.Conclusion: We reported empirically and theoretically evidence that DNA has impaired mobility and diffusion in collagen environment. In addition our method provides resembling in vivo situation, where gene electrotransfer mechanisms can be studied.

Evaluation of biocompatibility and efficiency of plasmid DNA delivery by gene-activated hydrogels in vitro

2019 ◽  
Vol XIV (2) ◽  
Author(s):  
I.Y. Bozo ◽  
A.A. Titova ◽  
M.N. Zhuravleva ◽  
A.I. Bilyalov ◽  
M.O. Mavlikeev ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Dna Delivery

Calcium influx determines the muscular response to electrotransfer

2012 ◽  
Vol 302 (4) ◽  
pp. R446-R453 ◽  
Author(s):  
Pernille Hojman ◽  
Camilla Brolin ◽  
Hanne Gissel
Keyword(s):  
Cell Membrane ◽  
High Voltage ◽  
Voltage Pulse ◽  
Low Voltage ◽  
Calcium Influx ◽  
High Voltage Pulse ◽  
Electric Pulses ◽  
Tnf Α ◽  
Cell Membrane Permeabilization ◽  
The Times

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca2+-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca2+ was assessed using 45Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca2+ influx and muscular response. Moderate Ca2+ influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca2+ influx. Larger Ca2+ influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca2+ influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca2+ may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.

scholarly journals In Vitro Gene Delivery Mediated by Asialofetuin-Appended Cationic Liposomes Associated with γ-Cyclodextrin into Hepatocytes

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Keiichi Motoyama ◽  
Yoshihiro Nakashima ◽  
Yukihiko Aramaki ◽  
Fumitoshi Hirayama ◽  
Kaneto Uekama ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Gene Delivery ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Cationic Liposomes ◽  
Nonviral Vector ◽  
Liposomal Membrane ◽  
Transfer Activity ◽  
Potential Use

The purpose of this study is to evaluate in vitro gene delivery mediated by asialofetuin-appended cationic liposomes (AF-liposomes) associating cyclodextrins (CyD/AF-liposomes) as a hepatocyte-selective nonviral vector. Of various CyDs, AF-liposomes associated with plasmid DNA (pDNA) and γ-cyclodextrin (γ-CyD) (pDNA/γ-CyD/AF-liposomes) showed the highest gene transfer activity in HepG2 cells without any significant cytotoxicity. In addition, γ-CyD enhanced the encapsulation ratio of pDNA with AF-liposomes, and also increased gene transfer activity as the entrapment ratio of pDNA into AF-liposomes was increased. γ-CyD stabilized the liposomal membrane of AF-liposomes and inhibited the release of calcein from AF-liposomes. The stabilizing effect of γ-CyD may be, at least in part, involved in the enhancing gene transfer activity of pDNA/γ-CyD/AF-liposomes. Therefore, these results suggest the potential use of γ-CyD for an enhancer of transfection efficiency of AF-liposomes.

scholarly journals Paclitaxel-loaded Folate-targeted Albumin-Alginate Nanoparticles Crosslinked with Ethylenediamine. Synthesis and In Vitro Characterization.

2021 ◽  
Author(s):  
A.M. Martínez ◽  
M. Benito ◽  
E. Pérez ◽  
C. Teijón ◽  
R.M. Olmo ◽  
...  
Keyword(s):  
Folic Acid ◽  
Cell Membrane ◽  
Cell Viability ◽  
Folate Receptor ◽  
Antineoplastic Drugs ◽  
Secondary Effects ◽  
In Vitro Characterization ◽  
Nanometric Range ◽  
Mcf 7

Among the different ways to reduce the secondary effects of antineoplastic drugs in cancer treatment, the use of nanoparticles has demonstrated good results due to the protection of the drug and the possibility of releasing compounds to a specific therapeutic target. The α-isoform of folate receptor (FR) is overexpressed on a significant number of human cancers; therefore, folate-targeted crosslinked nanoparticles based on BSA and alginate mixtures and loaded with paclitaxel (PTX) have been prepared to maximizing the proven antineoplastic activity of the drug against solid tumors. Nanometric-range sized particles (169 ± 28nm - 296 ± 57nm), with negative Z-potential values (between -0.12 ± 0.04 and -94.1± 0.4), were synthesized. The loaded PTX (2.63±0.19 - 3.56 ±0.13 µg PTX/mg Np) was sustainably released along 23 and 27h. Three cell lines (MCF-7, MDA-MB-231 and HeLa) were selected to test the efficacy of the folate-targeted PTX-loaded BSA/ALG nanocarriers. The presence of FR on cell membrane led to a significant larger uptake of BSA/ALG-Fol nanoparticles regarding to the equivalent nanoparticles without folic acid on its surface. The cell viability results demonstrated a cytocompatibility of unloaded nanoparticle-Fol and a gradual decrease in cell viability after treatment with PTX-loaded nanoparticles-Fol due to the sustainable PTX release.

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