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Hyun Jong Lee, Gabriella Fernandes-Cunha, Won-Gun Koh, Jeffrey L Goldberg, David Myung; Surface analytical studies of growth factor coupling to collagen by copper-free click chemistry. Invest. Ophthalmol. Vis. Sci. 2017;58(8):206. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Direct immobilization of growth factors onto the surface of ocular wounded tissue via biocompatible chemical reactions may improve their therapeutic potential. Given the continuous fluid turnover of the ocular environment, anchoring of growth factors would increase their residence time compared to topical application. To achieve direct immobilization, we utilized strain-promoted alkyne-azide cycloaddition (SPAAC) which is a bio-orthogonal form of copper-free click chemistry, a promising tool for binding growth factors onto a tissue surface due to its specificity, versatility, and biocompatibility.
N-Hydroxysuccinimide ester (NHS ester) reaction was used to conjugate azide groups onto epidermal growth factor (EGF) or fluorescein isothiocyanate (FITC)-labeled EGF, depending on the experiment being conducted. In all cases, EGF was conjugated with azide-NHS ester and then purified by dialysis. Collagen-coated polystyrene, glass, and gold were used depending on the surface analysis technique being used. Both bicyclononyne (BCN) and dibenzocyclooctyne (DBCO) moieties were applied to introduce alkyne groups to collagen. We attached the azide-EGF onto the alkyne-modified collagen surfaces via SPAAC and verified the binding via fluorescence microscopy, ellipsometry, surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA).
When the FITC conjugated Azide-EGFs (FITC-Azide-EGF) was applied, growth factor binding yielded surfaces with significantly higher green fluorescence compared to controls. EGF binding on collagen surfaces by physical adsorption and SPAAC were measured real-time by observation of increasing thicknesses and response units in ellipsometry and SPR, respectively. Chemical coupling of EGF via SPAAC yielded significantly greater ligand binding and resistance to dissociation compared to physisorption. ELISA assays showed that the EGF surface concentration could be modulated by varying the concentration of applied EGF as well as SPAAC reaction time.
Our results showed that SPAAC could be used to controllably and efficiently bind EGF to collagen surfaces. This method may provide a biocompatible way to immobilize therapeutic biomolecules to tissues in situ to promote ocular wound healing.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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