Oligonucleotide-Polyethylenimine Complexes Targeting Retinal Cells: Structural Analysis and Application to Anti-TGFβ-2 Therapy
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The aim of this study was to characterize oligonucleotide–polyethylenimine (ODN/PEI) complex preparation for potential transfection of retinal cells in vitro and in vivo.
The effect of medium preparation [HEPES-buffered saline (HBS), water] on particle size and morphology was evaluated. Cultured Lewis rat retinal Müller glial (RMG) cells were transfected using fluorescein isothiocyanate (FITC)–ODN/PEI complexes specifically directed at transforming growth factor beta (TGFβ)-2. Efficacy of transfection was evaluated using confocal microscopy, and regulation of gene expression was assayed using quantitative real-time RT-PCR and ELISA assay. One, 24, and 72 h after injection of FITC–ODN/PEI complexes into the vitreous of rat eyes, their distribution was analyzed on eye sections.
Complexes prepared in HBS were smaller than complexes prepared in pure water and presented a core–shell structure. These particles showed a high cellular internalization efficacy, along with a significant and specific down-regulation of TGFβ-2 expression and production in RMG cells, correlating with specific inhibition of cell growth at 72 h. In vivo, complexes efficiently transfect retinal cells and follow a transretinal migration at 24 h. After 72 h, ODN seems to preferentially target RMG cells without inducing any detectable toxicity.
Specific down-regulation of TGFβ-2 expression using ODN/PEI complexes may have potential interest for the treatment of retinal diseases associated with glial proliferation.
Key Wordsantisense oligonucleotides polyethylenimine retinal Müller glial cells TGFβ-2
We gratefully acknowledge Dr. C. Deloménie from Plate-forme Transcriptome (INSERM IFR-75 ISIT), Dr. D. Jaillard (UMR CNRS 8080, CCME, Université Paris-Sud), Dr. H. Alphandary (UMR CNRS 8612), Dr. F. Garnier (Ecole Centrale Paris), G. Frebourg. and Dr. J.-P. Lechaire (UMR CNRS 7622, Université Pierre et Marie Curie) for their technical assistance. Ana L. G. Santos was supported by a fellowship from Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES (Brazil). We also acknowledge the “Fondation pour l'Avenir” and EviGenoRet LSHG-CT-2005-512036 for funding of this work.
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