Purchase this article with an account.
Ana Vanessa V. Oliveira, Andreia P. Silva, Ana M. Costa, Gabriela A. Silva; Modified Chitosan Carriers for Ocular Gene Therapy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1411.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Our aim is to produce and optimize chitosan-based carriers as vectors for ocular gene therapy. To improve their transfection efficiency, disulfide bonds were covalently added to chitosan, which can be cleaved by cytoplasmatic gluthatione, thus releasing the DNA load more efficiently. These optimized carriers will i) improve nucleic acid incorporation; ii) protect their load from endossomal/lysossomal degradation; and iii) enhance nuclear penetration, hence gene expression.
Chitosan-DNA and thiolated chitosan-DNA carriers were prepared using a NH3:PO4 ratio of 5:1. These nanoparticles were used for pDNA encapsulation and release studies as well as cytotoxicity and transfection studies on retinal pigment epithelial cells.
H1 RMN confirmed that chitosan was successfully modified to incorporate cleavable dissulfide bonds. Nanoparticles with diameter of approximately 200 nm were measured by dynamic light scattering. Plasmid DNA containing GFP under the control of the CMV promoter was incorporated by both types of chitosan, forming complexes that protected the DNA from DNAse degradation. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, but neither showed cell toxicity. Thiolation did not seem to improve significantly the transfection efficiency. We are currently evaluating long-term GFP expression and the time course action of glutathione. We have found that the nanoparticles’ administration method has a marked influence on the transfection efficiency.
Size and stability of a carrier are crucial characteristics for its success as a gene therapy vector. The apparent lack of improvement in the transfection efficiency by chitosan’s thiolation is being evaluated, namely by fluorophore-tracking of the nanoparticles’ intracellular path and by developing alternative methods to administer the nanoparticles.
This PDF is available to Subscribers Only