Abstract
It has been shown that extracellular glycosaminoglycans (GAGs) limit the gene transfer by cationic lipids and polymers. The purpose of this study was to clarify how interactions with anionic GAGs (hyaluronic acid and heparan sulfate) modify the cellular uptake and distribution of lipoplexes and polyplexes. Experiments on cellular DNA uptake and GFP reporter gene expression showed that decreased gene expression can rarely be explained by lower cellular uptake. In most cases, the cellular uptake is not changed by GAG binding to the lipoplexes or polyplexes. Reporter gene expression is decreased or blocked by heparan sulfate, but it is increased by hyaluronic acid; this suggests that intracellular factors are involved. Confocal microscopy experiments demonstrated that extracellular heparan sulfate and hyaluronic acid are taken into cells both with free and DNA-associated carriers. We conclude that extracellular GAGs may alter both the cellular uptake and the intracellular behavior of the DNA complexes.
Highlights
Gene therapy holds great promise in the treatment of genetic and acquired diseases
Confocal microscopy experiments demonstrated that extracellular heparan sulfate and hyaluronic acid are taken into cells both with free and DNA-associated carriers
By confocal microscopy we found that extracellular GAGs are taken into cells by the cationic carriers and the cationic complexes and that they may alter the intracellular behavior of the complexes
Summary
Gene therapy holds great promise in the treatment of genetic and acquired diseases. The majority of gene transfer protocols utilizes viral delivery systems. Many promising results have been achieved, the safety concerns and the difficulty of production on a large scale limit the usefulness of the recombinant viral vectors This has prompted further development of viral vectors and the search for efficient, nonimmunogenic, and easy-to-prepare nonviral vector systems. The delivery of naked plasmid DNA to the target cells is not efficient because of the large size and multiple negative charges of the DNA molecule [1]. The complexes have a positively charged surface, which facilitates the delivery of DNA into the target cells due to electrostatic. GAGs may bind to the cationic carrier or to the surface of positively charged complexes, and in some cases, GAG may eventually replace DNA in the complex resulting in the uptake of GAG into the cells instead of DNA. By confocal microscopy we found that extracellular GAGs are taken into cells by the cationic carriers and the cationic complexes and that they may alter the intracellular behavior of the complexes
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