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

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.

Download Full-text

Enhanced Gene Transfection in Macrophages Using Mannosylated Cationic Liposome-Polyethylenimine-Plasmid DNA Complexes

Journal of Drug Targeting ◽

10.3109/10611860108997928 ◽

2001 ◽

Vol 9 (3) ◽

pp. 201-207 ◽

Cited By ~ 28

Author(s):

Ayumi Sato ◽

Shigeru Kawakami ◽

Makiko Yamada ◽

Fumiyoshi Yamashita ◽

Mitsuru Hashida

Keyword(s):

Plasmid Dna ◽

Gene Transfection ◽

Cationic Liposome ◽

Dna Complexes

Download Full-text

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

10.1101/621102 ◽

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

Download Full-text

Interaction of skin tissue with nanosecond pulsed laser-induced stress wave used for gene transfection

LEOS 2007 - IEEE Lasers and Electro-Optics Society Annual Meeting Conference Proceedings ◽

10.1109/leos.2007.4382323 ◽

2007 ◽

Author(s):

Shunichi Sato ◽

Mitsuhiro Terakawa ◽

Shinta Takano ◽

Hitoshi Tsuda ◽

Hiroshi Ashida ◽

...

Keyword(s):

Stress Wave ◽

Gene Transfection ◽

Pulsed Laser ◽

Skin Tissue ◽

Induced Stress ◽

Nanosecond Pulsed Laser

Download Full-text

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

Journal of Drug Delivery ◽

10.1155/2011/453619 ◽

2011 ◽

Vol 2011 ◽

pp. 1-8 ◽

Cited By ~ 2

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.

Download Full-text