June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Growth factor immobilization in situ by copper-free click chemistry: in vitro binding, cytocompatibility, and cell proliferation studies

Author Affiliations & Notes
  • Gabriella Maria Fernandes Cunha
    Ophthalmology , Stanford University, Palo Alto , California, United States
  • Hyun Jong Lee
    Ophthalmology , Stanford University, Palo Alto , California, United States
  • Alisha Kumar
    Ophthalmology , Stanford University, Palo Alto , California, United States
  • Alexander Kreymerman
    Ophthalmology , Stanford University, Palo Alto , California, United States
  • Jeffrey L Goldberg
    Ophthalmology , Stanford University, Palo Alto , California, United States
  • David Myung
    Ophthalmology , Stanford University, Palo Alto , California, United States
  • Footnotes
    Commercial Relationships   Gabriella Maria Fernandes Cunha, None; Hyun Jong Lee, None; Alisha Kumar, None; Alexander Kreymerman, None; Jeffrey Goldberg, None; David Myung, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2620. doi:
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      Gabriella Maria Fernandes Cunha, Hyun Jong Lee, Alisha Kumar, Alexander Kreymerman, Jeffrey L Goldberg, David Myung; Growth factor immobilization in situ by copper-free click chemistry: in vitro binding, cytocompatibility, and cell proliferation studies

      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):2620.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Corneal epithelial wound healing remains a major clinical challenge. Topically applied growth factors are limited in effect because of their local depletion via endocytosis of growth factor-receptor complexes and substantial volumetric losses into the lacrimal system. We hypothesized that growth factors bound to stromal collagen through strain-promoted azide-alkyne cycloaddition (SPAAC), a form of copper-free click chemistry, would enhance the corneal epithelialization without impacting corneal cell viability.

Methods : The epithelial layer from freshly enucleated bovine eyes was scraped and the exposed stromal layer was treated with an aqueous solution of dibenzocyclooctyne (DBCO)-sulfo-N-hydroxysuccinimide (NHS). The cornea was washed with phosphate buffer-saline (PBS) and then azide-conjugated, FITC-labeled epidermal growth factor (EGF-azide) was applied the alkyne-functionalized corneal stroma to yield chemically bound EGF. The eyes were washed and the stromal surface was scraped for western blot analysis. In vitro cell proliferation was performed on polystyrene tissue culture plates. The plates were coated with collagen and treated with DBCO-sulfo-NHS followed EGF-azide. After washing, senescent primary corneal epithelial cells were seeded on the modified surfaces. Cell proliferation was followed for 5 days. The cytocompatibility of the click reaction was assessed by live/dead experiments 1 and 72 hours after direct exposure primary corneal keratocytes to DBCO-sulfo-NHS followed by EGF-azide.

Results : Western blot analysis confirmed chemical attachment of EGF to corneal stroma by click chemistry. EGF-bound collagen surfaces yielded greater corneal epithelial cell proliferation over 5 days compared to surfaces without chemically bound EGF. Cell viability of corneal keratocytes was greater than 90% after direct application of the click chemistry reaction.

Conclusions : Our results were consistent with our hypothesis that binding of growth factors to collagen through copper-free click chemistry in situ is cytocompatible and can improve cell proliferation in vitro.

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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