Journal of Orthopaedic Translation (Jul 2014)
A novel in vitro system for intracellular delivery of nonviral DNA
Abstract
Articular chondrocytes are the main cell population in cartilage, and damage to them is a key step in osteoarthritis. There are chondrocyte-based strategies to treat osteoarthritis, among which includes nonviral gene therapy. However, so far there is no ideal way to achieve this because chondrocyte cells are very difficult to transfect. Also, an effective tracking system to evaluate exogenous DNA delivery in chondrocytes is a necessary part of this strategy. Here, we show our development of a novel tracking system by labelling cell membranes, nuclei, and plasmids, without disturbing their expression, to view the intracellular behaviour of plasmids before, after, and during the entire transfection process. We applied this system to compare the intracellular behaviour of exogenous DNA in chondrocytes and cancer cells. We also used this system to compare the intracellular behaviour of exogenous DNA which is transfected by liposomes or polymers. Finally, we discovered that when transfected by liposomes, exogenous DNA has a quicker cell entry and nucleus entry in cancer cells than in chondrocytes, and the transfection efficiency is higher in cancer cells than in chondrocytes. When the cells are transfected by polymers, exogenous DNA has the same quick cell entry and nucleus entry in both types of cells. However, the transfection efficiency was higher in cancer cells than in chondrocytes. Applying this system has proved to be simple, easy to operate, repeatable, and stable and it enables tracking of the behaviour of plasmid DNA before and after expression. Meanwhile, the rate-limiting zones of exogenous plasmid DNA in living cells and a comprehensive overview of the expression and transfection efficiency of DNA can also be obtained through this system, making it ideal for the research and development of nonviral DNA delivery systems.
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