Live cell integrated surface plasmon resonance biosensing approach to mimic the regulation of angiogenic switch upon anti-cancer drug exposure

Abstract

In this work, we report a novel surface plasmon resonance (SPR) based live-cell biosensing platform to measure and compare the binding affinity of vascular endothelial growth factor (VEGF) to vascular endothelial growth factor receptor (VEGFR) and VEGF to bevacizumab. Results have shown that bevacizumab binds VEGF with a higher association rate and affinity compared to VEGFR. Further, this platform has been employed to mimic the in vivo condition of the VEGF-VEGFR angiogenic switch. Competitive binding to VEGF between VEGFR and bevacizumab was monitored in real-time using this platform. Results demonstrated a significant blockage of VEGF-VEGFR binding by bevacizumab. From the results, it is evident that the proposed strategy is simple and highly sensitive for the direct and real-time measurements of bevacizumab drug efficacy to the VEGF-VEGFR angiogenic switch in living SKOV-3 cells.

Scheme 1. Schematic Illustration of the Biomimic System for Bevacizumab Drug Regulation Study

Figure 1

Merged fluorescent images (Hoechst channel & MitoTracker Red channel) of SKOV-3 cells on different type of substrates: Petri dishes (exposure time 400 ms), gelatin coated gaskets (exposure time 200 ms), and uncoated gaskets (exposure time 200 ms).

Figure 2

(A) SPR response corresponding to 3 μg/mL VEGF for each surface bound bevacizumab concentration: 5 μg/mL (red), 10 μg/mL (blue), 30 μg/mL (purple), 50 μg/mL (green); (B) SPR baseline shift induced by each different surface concentration of bevacizumab (n = 3): 8.5 ± 1.3 (5 μg/mL), 26.1 ± 3.6 (10 μg/mL), 96.1 ± 9.8 (30 μg/mL), 107.2 ± 9.3 (50 μg/mL), 111.6 ± 11.2 (70 μg/mL).

Figure 3

(A) SPR sensorgram of surface bound bevacizumab interacting with VEGF. Inset: enlarged sensorgram of the response upon VEGF binding. (B) SPR sensorgram of surface bound bevacizumab interacting with VEGFR. Inset: enlarged sensorgram of the response upon VEGFR binding.

Figure 4

(A) SPR sensorgram of VEGF binding response to bevacizumab (blue line) and VEGFR (red line). (B) Association rate constants (ka) of VEGF–bevacizumab binding (blue) and VEGF–VEGFR binding (red) calculated for each trial of experiment. (n = 3). (C) Binding affinity indicated by SPR baseline shift of VEGF–bevacizumab binding (blue) and VEGF–VEGFR binding (red) for each trial (n = 3).

Figure 5

(A) SPR response of VEGF (SKOV-3 cells released)–VEGFR interaction (red line) and VEGF (SKOV-3 cells released)–VEGFR interaction under bevacizumab regulation (blue line) in the biomimic system. (B) SPR baseline shift of VEGF–VEGFR interaction (red) and VEGF–VEGFR interaction under bevacizumab regulation (blue) for each trial. (n = 3).