High Affinity Targeting of Activated Corneal Keratocytes in vitro: Subtractive SELEX; New Frontiers for in vivo Cell Labelling and Targeted Drug Delivery

Abstract

Abstract: : Purpose: To determine if the SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique could be used to evolve high affinity ssRNA aptamers, capable to differentiating activated, from quiescent corneal keratocyte in–vitro. Methods: Cells were liberated by collagenase digestion of fresh bovine corneas. Quiescent and activated bovine keratocytes were cultured in serum free and serum containing medium respectively. The highly random ssRNA starting pool was incubated with quiescent cells, to eliminate aptamers binding to non specific shared cellular components of both cell types. The remaining ssRNA pool was then exposed to the activated cell phenotype, and binding RNA’s isolated (product). These were amplified and this iterative process was repeated with the selectively reduced ssRNA pool (product) of the previous round. After 10 rounds, the final product was coupled to a fluorophore for in–vitro detection by fluorescent microscopy of cultured cells. Both cell phenotypes were incubated with either the final high affinity aptamer or with the random fluorophore coupled non specific ssRNA pool. As a further control we also used bovine skin fibroblasts to determine the specificity of the final product. Results: In the high affinity aptamer group, fluorescence was only detected with the activated corneal keratocyte phenotype. There was no fluorescence of either the quiescent cell type or of skin fibroblasts. In contrast, fluorescence was detected with all cells incubated with the random fluorophore coupled ssRNA pool. These results indicate that this subtractive SELEX technique yields highly specific ligands, that can distinguish between cells of homologous origin, and hence differentiate activated, from quiescent corneal keratocytes in–vitro Conclusions: Through a subtractive SELEX selection method, we have developed a ssRNA aptamer with high affinity for activated bovine corneal keratocytes. Applying a similar protocol, we have now begun the synthesis of an aptamer for activated human corneal keratocytes. The novel properties of aptamers have enormous potential for in–vivo application; in the critical appraisal of new therapeutic products, corneal wound healing events and significantly, for specific, and targeted drug delivery in the human cornea.

Keywords: cornea: stroma and keratocytes • cornea: basic science