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Abstract Purpose  Intravitreal injection of nonviral gene complexes may be promising in the treatment of retinal diseases. This study investigates the permeation of lipoplexes and polystyrene nanospheres through the neural retina and their uptake by the retinal pigment epithelium (RPE) either with or without ultrasound application. Materials and Methods  Anterior parts and vitreous of bovine eyes were removed. The neural retina was left intact or peeled away from the RPE. (Non)pegylated lipoplexes and pegylated nanospheres were applied. After 2 h incubation, the RPE cells were detached and analyzed for particle uptake by flow cytometry and confocal microscopy. Results  The neural retina is a significant transport barrier for pegylated nanospheres and (non)pegylated lipoplexes. Applying ultrasound improved the permeation of the nanoparticles up to 130 nm. Conclusions  Delivery of liposomal DNA complexes to the RPE cells is strongly limited by the neural retina. Ultrasound energy may be a useful tool to improve the neural retina permeability, given the nucleic acid carriers are small enough. Our results underline the importance to design and develop very small carriers for the delivery of nucleic acids to the neural retina and the RPE after intravitreal injection. [ABSTRACT FROM AUTHOR]