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Y. M. Khan, R. Angunawela, D. Bunka, P. Stockely, J. Marshall; Aptamer Based Selective Targeting of Keratocytes: Next Generation Therapeutics for Corneal Wound Management. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2945. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the specificity of ’tailor-made’ RNA aptamers for differentiating between active and passive keratocytes following corneal injury.
A library of aptamers with variable specificity for target cells (activated keratocytes) was developed using a counter selective molecular strategy (SELEX). Human corneas (n=10) were maintained under organ culture conditions in specially designed anterior chamber assemblies developed in the host institution. After stabilisation for a period of 3-6 days, defined wounds were created at 90 and 160 microns depth using femtosecond and Excimer- PRK platforms. Tissues were cultured for a further seven days to allow keratocyte activation and wound healing. Samples were then prepared for cryo-sectioning and aptamer localisation. Aptamers were conjugated with Cy 5 flurophore (Abs=649nm, EM= 670nm), and passive keratocytes were stained with TO-PRO-1 (Abs=514nm, EM=533nm). Treated corneas and naïve aptamer pools were used as controls.
On fluorescent confocal microscopy, individual aptamers demonstrated staining of activated keratocytes at wound interface with slight variation in specificity and affinity towards target cells. Aptamers that only localised strongly to activated keratocytes were designated as highly specific whilst others, with additional diffuse labelling of epithelial and endothelial cells were designated as being of reduced specificity. The highly specific aptamers did not show any labelling of passive keratocytes present in control tissues and cell culture preparations.
The results provide evidence for highly specific aptamer based recognition of activated keratocytes in the human cornea following tissue injury. Thus, aptamers coupled to anti-inflammatory agents provides a drug delivery vehicle to target a specific population of cells thus minimising harmful side effects during the wound healing process. These developments bring us a step closer to in-vivo cellular targeting in human cornea and exploiting the potential of aptamers as "therapeutic chaperones".
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