Real-Time Probing of Integrin αVβ3 Signal Pathway Involved in Wound Healing using Biofunctionalized Quantum Dots

Abstract

Wound healing involves the stringent spatial and temporal coordination of several processes including directional cell migration, the extracellular matrix remodeling, and the angiogenesis. Recently, migration stimulating factor (MSF) has been reported to stimulate the migration of fibroblasts, epithelial and endothelial cells, where the corresponding cell response is mediated by the binding of the MSF to integrin αVβ3 receptor. However, wound healing can be delayed when patients are unable to produce wound healing cytokines, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and MSF. In this study, we propose a nanotechnology-based approach to monitoring the real-time integrin αVβ3 signal pathway involved in wound healing and its relationship to cytoskeleton. We apply a MSF-like fluorescent small molecule probe MM-IGD-Fl-2 to stain and identify the distribution of integrin αVβ3 in living human dermal fibroblast cells CCD-996SK. We then utilize the biofunctionalized quntum dots (QDs) conjugated with MSF-like small molecule MM-IGD-Az-2 to provide a more direct access to probing the spatiotemporal distribution of QD-MM-IGD-Az-2-integrin αVβ3 complexes. Accordingly, the signal pathway activated after binding of MM-IGD-Az-2 to integrin αVβ3 receptor could be traced. On the other hand, to synchronize cellular responses and to eliminate the possibility of receptor-mediated internalization, CCD-996SK cells are incubated with QD-MM-IGD-Az-2 at 4°C, followed by further incubation at 37 °C in a temperature controlled flow chamber. We anticipate the proposed approach to be a starting point for more sophisticated in the mechanisms of wound healing at the cellular level.