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Isabel Arranz-Valsero, Jenny E. Párraga, Laura Contreras-Ruiz, G K. Zorzi, Antonio López-García, Begoña Seijo, Alejandro Sánchez, Yolanda Diebold; In Vitro Silencing of TGFß1 and its Receptor 2 in a Human Corneal Epithelial Cell Line Using Cationized Gelatin/Chondroitin Sulphate Nanoparticles. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1429.
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To silence in vitro the expression of the identified in ocular surface inflammatory disorders pro-inflammatory molecule TGFß1 and its Receptor 2 (TGFBR2), using cationized gelatine (CG)/chondroitin sulphate (CS) nanoparticles (NPs) as vehicle to deliver small interfering RNA (siRNA).
NPs were prepared from CG and CS, using either spermine or ethylenediamine as cationizing agents (WO2010/049562A1), and loaded with specific or with mismatching siRNAs, at different concentrations (150 or 300nM). Cells from a Human Corneal Epithelial cell line (HCE) were transfected with siTGFß1- or siTGFBR2-loaded CG/CS NPs. Naked siRNAs were used as controls, and Lipofectamine was used as control transfection reagent. siRNA-loaded NPs internalization was evaluated using a Cy3-conjugated siGAPDH. Potential toxicity was evaluated by the XTT test. Levels of mRNA were determined by Real Time RT-PCR (RT²-PCR) and protein expression by ELISA or Western blot.
CG/CS NPs with the siRNA incorporated in their matrix had a diameter around 300nm. They were able to deliver siRNA into cell cytosol, as determined by fluorescence microscopy, while maintaining high cell viability. Decreased levels of TGFß1 and TGFBR2 mRNA were observed in HCE cells transfected with siRNA-NPs but not with blank NPs or with naked siRNAs, as determined by RT²-PCR. The silencing was also observed at the protein level for both molecules after siRNA-NPs transfection by ELISA or Western blotting.
It is possible to silence TGFß1 and TGFBR2 expression in HCE cells by conventional techniques obtaining acceptable silencing levels while maintaining high cell viability. CG/CS NPs are promising new vehicles to deliver siRNA to human cells from the ocular surface as potential therapeutic option to treat inflammatory diseases.
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