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Vitreous: a barrier to nonviral ocular gene therapy.

PURPOSE
Intravitreal injection of therapeutic DNA, complexed to nonviral carriers such as cationic liposomes, may be promising in the treatment of many severe retinal eye diseases. However, after intravitreal injection, such DNA/cationic liposome complexes-called lipoplexes (LPXs)-which are typically hundreds of nanometers in size, must first diffuse through the vitreous before they can reach the retina. The aim of this study was to elucidate whether vitreous is a barrier for the LPXs and to find strategies to overcome this barrier.


METHODS
Fluorescent polystyrene nanospheres and LPXs were mixed with vitreous, and their mobility was monitored by fluorescence recovery after photobleaching (FRAP), a microscopy-based technique. The stability of LPXs and naked plasmid DNA in vitreous was studied by gel electrophoresis.


RESULTS
We showed that polystyrene nanospheres, in our first experiments used as a model for the LPXs, do not diffuse freely into the vitreous but adhere to fibrillar structures in the vitreous, most likely to collagen fibers. Making the surfaces of the polystyrene nanospheres hydrophilic by attaching hydrophilic polyethylene glycol (PEG) chains at their surfaces circumvented the binding to fibrillar structures in the vitreous. FRAP revealed that "pegylated" polystyrene nanospheres, as long as they are smaller than 500 nm, are indeed mobile in the vitreous. It was further demonstrated that LPXs severely aggregate in vitreous and strongly bind to biopolymers in the vitreous, which immobilizes them completely. However, as observed for the polystyrene nanospheres, coating of the LPXs with PEG averted their aggregation in the vitreous and their binding to fibrillar structures.


CONCLUSIONS
Modifying the surfaces of LPXs with hydrophilic PEG chains prevents them from aggregating in vitreous. In this way, LPXs are obtained that can freely move in vitreous, an absolute criterion for reaching the retina after intravitreal injection.