scholarly journals Size effect of complexed plasmid DNA to gene transfection efficiency of microbubble-mediated sonoporation

2013 ◽  
Author(s):  
Yoichiro Matsumoto ◽  
Yiwei Zhang ◽  
Takashi Azuma ◽  
Kiyoshi Yoshinaka ◽  
Kensuke Osada ◽  
...  
Keyword(s):  
Size Effect ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection

Size effect of complexed plasmid DNA to gene transfection efficiency of microbubble-mediated sonoporation

2013 ◽  
Vol 133 (5) ◽  
pp. 3495-3495
Author(s):  
Yoichiro Matsumoto ◽  
Yiwei Zhang ◽  
Takashi Azuma ◽  
Kiyoshi Yoshinaka ◽  
Kensuke Osada ◽  
...  
Keyword(s):  
Size Effect ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection

scholarly journals Dual delivery of nucleic acids and PEGylated-bisphosphonates via calcium phosphate nanoparticles

2019 ◽  
Author(s):  
Sofia Bisso ◽  
Simona Mura ◽  
Bastien Castagner ◽  
Patrick Couvreur ◽  
Jean-Christophe Leroux
Keyword(s):  
Calcium Phosphate ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Physical Stability ◽  
Entrapment Efficiency ◽  
Endosomal Escape ◽  
Particle Uptake ◽  
Success Stories

AbstractDespite many years of research and a few success stories with gene therapeutics, efficient and safe DNA delivery remains a major bottleneck for the clinical translation of gene-based therapies. Gene transfection with calcium phosphate (CaP) nanoparticles brings the advantages of low toxicity, high DNA entrapment efficiency and good endosomal escape properties. The macroscale aggregation of CaP nanoparticles can be easily prevented through surface coating with bisphosphonate conjugates. Bisphosphonates, such as alendronate, recently showed promising anticancer effects. However, their poor cellular permeability and preferential bone accumulation hamper their full application in chemotherapy. Here, we investigated the dual delivery of plasmid DNA and alendronate using CaP nanoparticles, with the goal to facilitate cellular internalization of both compounds and potentially achieve a combined pharmacological effect on the same or different cell lines. A pH-sensitive poly(ethylene glycol)-alendronate conjugate was synthetized and used to formulate stable plasmid DNA-loaded CaP nanoparticles. These particles displayed good transfection efficiency in cancer cells and a strong cytotoxic effect on macrophages. The in vivo transfection efficiency, however, remained low, calling for an improvement of the system, possibly with respect to the extent of particle uptake and their physical stability.Graphical abstract

scholarly journals Cellular Injury of Cardiomyocytes during Hepatocyte Growth Factor Gene Transfection with Ultrasound-Triggered Bubble Liposome Destruction

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Kazuo Komamura ◽  
Rie Tatsumi ◽  
Yuko Tsujita-Kuroda ◽  
Takatoshi Onoe ◽  
Kunio Matsumoto ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cellular Injury ◽  
Factor Gene ◽  
Trade Offs ◽  
Growth Factor Gene ◽  
Low Concentrations ◽  
Hepatocyte Growth ◽  
Cultured Cardiomyocytes

We transfected naked HGF plasmid DNA into cultured cardiomyocytes using a sonoporation method consisting of ultrasound-triggered bubble liposome destruction. We examined the effects on transfection efficiency of three concentrations of bubble liposome (1×106, 1×107, 1×108/mL), three concentrations of HGF DNA (60, 120, 180 μg/mL), two insonification times (30, 60 sec), and three incubation times (15, 60, 120 min). We found that low concentrations of bubble liposome and low concentrations of DNA provided the largest amount of the HGF protein expression by the sonoporated cardiomyocytes. Variation of insonification and incubation times did not affect the amount of product. Following insonification, cardiomyocytes showed cellular injury, as determined by a dye exclusion test. The extent of injury was most severe with the highest concentration of bubble liposome. In conclusion, there are some trade-offs between gene transfection efficiency and cellular injury using ultrasound-triggered bubble liposome destruction as a method for gene transfection.

scholarly journals Preparation and Characterization of Folate-Targeted Fe3O4 Nanoparticle Codelivering Cisplatin and TFPI-2 Plasmid DNA for Nasopharyngeal Carcinoma Therapy

2017 ◽  
Vol 2017 ◽  
pp. 1-10
Author(s):  
Juan Zhang ◽  
Huanhuan Weng ◽  
Xiangwan Miao ◽  
Quanming Li ◽  
Siqi Wang ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Fluorescence Analysis ◽  
Average Diameter ◽  
Transmission Electron Microscopy Image ◽  
Proton Nuclear Magnetic Resonance ◽  
Targeting Therapy ◽  
The Core

A novel folate (FA) receptor-targeted superparamagnetic Fe3O4 nanoparticles (SPIONs) codelivering cisplatin (CDDP) and tissue factor pathway inhibitor-2 (TFPI-2) plasmid DNA (pDNA) was constructed. The core shell nanocomposites (FA-PEG-PEI@SPION-CDDP-TFPI-2) were composed of superparamagnetic Fe3O4 core that binds CDDP and TFPI-2 shell that combines with folate-polyethylene glycol-polyethyleneimine (FA-PEG-PEI) via electrostatic interaction. The shell containing FA-PEG-PEI and TFPI-2 plasmid was synthesized through amidation reaction and electrostatic adsorption and the core containing SPION-CDDP was modified by aldehyde sodium alginate. Proton nuclear magnetic resonance and Fourier transform infrared spectra on FA-PEG-PEI polymers showed characteristic peaks of various metabolites in corresponding frequency. Transmission electron microscopy image of FA-PEG-PEI@SPION-CDDP-TFPI-2 nanoparticles demonstrated a near-monodisperse spherical morphology, while dynamic light scattering studies indicated an intensity-average diameter of 149.5 nm. Zeta potential was 14.89 ± 1.83 mv and the final concentration of loaded CDDP was 100 ug/ml. Gel electrophoresis data showed that the nanocomposite would protect TFPI-2 pDNA from being digested by DNases. Compared with CNE-2 cells, the good targetability and better gene transfection efficiency (57.9%) were detected by Prussian blue iron stain and fluorescence analysis in HNE-1 cells in vitro. The results suggested the potential application of FA-PEG-PEI@SPION-CDDP-TFPI-2 as a multifunctional anticancer nanomedicine on targeting therapy for FR positive NPC.

Free radical-mediated transgene inactivation of macrophages by endotoxin

2000 ◽  
Vol 279 (5) ◽  
pp. L878-L883 ◽  
Author(s):  
Sujatha Dokka ◽  
David Toledo ◽  
Liying Wang ◽  
Xianglin Shi ◽  
Chuanshu Huang ◽  
...  
Keyword(s):  
Free Radical ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cationic Liposome ◽  
Sodium Formate ◽  
Cell Types ◽  
Polymyxin B ◽  
Free Radical Scavengers ◽  
Metal Catalyzed

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N- t-butyl-α-phenylnitrone, the H2O2 scavenger catalase, and the ·OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O2 − scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that ·OH formed by H2O2-dependent, metal-catalyzed Fenton reaction play a major role in this process.

scholarly journals 46 EFFICIENT TRANSFECTION OF PLASMID DNA INTO CELLS FOR USE AS NUCLEAR DONORS

2005 ◽  
Vol 17 (2) ◽  
pp. 172
Author(s):  
S.-L. Lee ◽  
S.-A. Ock ◽  
H.-J. Song ◽  
B. Mohana Kumar ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
South Korea ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cumulus Cells ◽  
Cell Types ◽  
Development Project ◽  
Exogenous Dna ◽  
Transfected Cells ◽  
Fetal Fibroblasts

Somatic cell nuclear transfer (SCNT) has the potential to significantly improve the production of valuable livestock that produce recombinant proteins, such as pharmaceutical proteins for human disease or biomaterials for medical use. The success of this potential depends on efficient and optimized protocols for introducing exogenous DNA into cells. In this study, we compared two methods of transfection, Effectene (Qiagen, Inc., Valencia, CA, USA) and electroporation. Plasmid DNA (pEGFF-N1, Clontech, Seoul, Korea) was transfected into fetal fibroblasts (FFB), cumulus cells (CUC), and adult ear skin cells (ESC). Transfection efficiency, chromosome normality, gene expression, and apoptosis were assessed. Cells cultured in α-modified Eagle's medium (α-MEM; BioWhittaker, Walkersville, MD, USA) + 10% FBS were transfected with pEGFP-N1. For electroporation, cells (5 × 106 cells/mL) were mixed in 300 μL perrim buffer (75% Cytosalts with 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 5 mM MgCl2, and 25% α-MEM) + 15 μg pEGFP-N1, and subjected to two pulses of 0.38 kV and 400 μF delivered by Gene Pulser (Bio-Rad; BMS, Ltd., Seoul, South Korea). For Effectene transfection, the procedure suggested by the manufacture was followed. Transfected cells were selected with 600 μg/mL G418 (Gibco; KDR Biotech Co., Ltd., Seoul, South Korea) and cultured at 39°C, 5% CO2 in air. Assessments of EGFP transfected cells by green fluorescence was carried out under an inverted epifluorescence microscope (Nicon, Kanagawa, Japan) equipped with a filter for FITC (excitation maximum = 488 nm; emission maximum = 507 nm). Differences among the groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Most cells (>80%) in confluence were at G0/G1 phase, and transfection of the gene into all three cell types did not affect the incidence of chromosomal abnormality or change morphology. In addition, the rates of apoptosis assessed by TUNEL did not differ in all three cell types by either method of transfection at different cell passages. However, the efficiency of gene transfection into FFB by Effectene reagent (14.2 ± 1.7%) was significantly (P < 0.05) higher than that by electroporation (5.1 ± 1.0%). Among the three type cells, the efficiency of gene transfection by Effectene and electroporation of FFB (14.2 ± 1.7 and 5.1 ± 1.0%, respectively) was significantly (P < 0.05) higher than those of CUC and ESC (9.4 ± 1.5 and 3.3 ± 0.8; 8.8 ± 0.7 and 2.1 ± 0.4%, respectively). In conclusion, although there were no differences in the alteration of chromosomes, cell morphology, and apoptosis among three cell types transfected with or without plasmid DNA, FFB is the most effective cell type to be transfected. Effectene is superior to other currently available methods for introducing plasmid DNA into a variety of cells. The high level of transfection achieved by Effectene will encourage its use as a tool for producing transgenic embryos and animals by SCNT. This work was supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010.

Laser-induced stress wave-assisted gene transfection: improved transfection efficiency with cationic liposome-modified plasmid DNA

2007 ◽  
Author(s):  
Risa Otsuka ◽  
Mitsuhiro Terakawa ◽  
Shunichi Sato ◽  
Yasushi Satoh ◽  
Kunio Takishima ◽  
...  
Keyword(s):  
Stress Wave ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cationic Liposome ◽  
Induced Stress

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

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