Real-Time Probing of the Spatiotemporal Distributions of Integrin αvβ3 in Single Living Cells using Micro-Scale Surface Enhanced Raman Spectroscopy System

Abstract

Wound healing involves multiple processes including stimulating cell migration, remodelling of the tissue matrix and angiogenesis. Migration stimulating factor (MSF), a natural protein present in normal foetal tissues, known to stimulate the migration of fibroblasts at very low concentrations, where the corresponding cell response is mediated by the binding of the MSF to integrin αvβ3 receptor. However, patients unable to produce wound healing cytokines will result in delayed healing; therefore, it is required to develop improved healing strategies. Here, we apply a synthesized MSF-like small molecule MM-IGD-Az-2 to evaluate the effect of MM-IGD-Az-2 on wound healing behavior at the cellular level. We will propose a design of micro-scale surface enhanced Raman spectroscopy (μSERS) system to probe spatiotemporal distributions of integrin αvβ3 and the corresponding cellular response in living human dermal fibroblast (CCD-996SK) cells due to the presence of MM-IGD-Az-2. We then utilize the biofunctionalized gold nanoparticles (AuNPs) conjugated with MM-IGD-Az-2 that work as a SERS probe to provide a more direct access to probing the spatiotemporal distribution of integrin αvβ3 complexes. Accordingly, the Raman signals of integrin αvβ3 in the vicinity of the AuNPs can be amplified after binding of AuNP-MM-IGD-Az-2 to integrin αvβ3 receptor. Furthermore, to synchronize cellular responses and to eliminate the possibility of receptor-mediated internalization, CCD-996SK cells are incubated with AuNP-MM-IGD-Az-2 at 4°C, followed by further incubation at 37 °C in a temperature controlled flow chamber. Therefore, the Raman spectra of AuNP-MM-IGD-Az-2-integrin αvβ3 complexes within the single living cell could be traced. We anticipate the proposed approach could be applied to build a methodology for wound healing behavior at the cellular level and practical support about bioactivity and dosimetry of MSF-like small molecules for further animal-model studies.