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Composites of malonic acid diamides and phospholipids — Impact of lipoplex stability on transfection efficiency

Author:
Janich, Christopher, Wölk, Christian, Erdmann, Frank, Groth, Thomas, Brezesinski, Gerald, Dobner, Bodo, Langner, Andreas
Source:
Journal of Controlled Release 2015 v.220 pp. 295-307
ISSN:
0168-3659
Subject:
DNA, cell culture, clinical trials, confocal laser scanning microscopy, deoxyribonuclease I, ethidium, gel electrophoresis, gene therapy, heparin, light scattering, malonic acid, phospholipids, polymerase chain reaction, proteoglycans, sorption isotherms, spectroscopy, transfection
Abstract:
The use of cationic lipids as gene delivery systems is a basic method in gene therapy. Through ongoing research, lipofection is currently the leader of non-viral vectors in clinical trials. However, in order to unleash the full potential of lipofection further intensive investigations are indispensable.In this study, various lipoplex formulations were compared regarding their ability to bind DNA. To obtain information about a possible premature release of DNA at the cell surface, heparin and chondroitin dependent lipoplex destabilization experiments were carried out. Complementary investigations in cell culture were performed to quantify DNA outside the cell. Additionally, DNase I stability was investigated. In this regard a multitude of methods, namely confocal laser scanning microscopy (CLSM), polymerase chain reaction (PCR), cell culture experiments, ethidium bromide assay, gel electrophoresis, Langmuir-isotherm experiments, infrared reflection absorption spectroscopy (IRRAS), Brewster angle microscopy (BAM), zeta-(ζ)-potential measurements, and dynamic light scattering (DLS), were applied. Although the complexation of DNA is a fundamental step, we show that the DNA release by biological agents (proteoglycans) and an unsuccessful cell attachment are major transfection limiting parameters.
Agid:
5830245