Multivalent Ligand-Nanoparticle Conjugates Amplify Reactive Oxygen Species Second Messenger Generation and Enhance Epidermal Growth Factor Receptor Phosphorylation

Abstract

The epidermal growth factor (EGF) receptor (EGFR) is heterogeneously distributed on the cellular surface and enriched in clusters with diameters of tens of nanometers. Multivalent presentation of EGF ligand on nanoparticles (NPs) provides an approach for controlling and amplifying the local activation of EGFR in these clusters. Reactive oxygen species (ROS) have been indicated to play a role in the regulation of EGFR activation as second messengers, but the effect of nanoconjugation on EGF-mediated ROS formation and ROS-induced EGFR activation is not well established. The goal of this manuscript is to characterize the multivalent enhancement of EGF-induced ROS formation and to test its effect on EGFR phosphorylation in breast cancer cell models using gold (Au) NPs with a diameter of 81 ± 1 nm functionalized with two different EGF ligand densities (12 ± 7 EGF/NP (NP-EGF12) and 87 ± 6 EGF/NP (NP-EGF87)). In the EGFR overexpressing cell lines MDA-MB-231 and MDA-MB-468, NP-EGF87 achieved a measurable multivalent enhancement of ROS that peaked at concentrations c ROSmax ≤ 25 pM and that were EGFR and nicotinamide adenine dinucleotide phosphate oxidase (NOX) dependent. NP-EGF12 failed to generate comparable ROS levels as NP-EGF87 in the investigated NP input concentration range (0–100 pM). In cells with nearly identical numbers of bound NP-EGF87 and NP-EGF12, the ROS levels for NP-EGF87 were systematically higher, indicating that the multivalent enhancement is exclusively related not only to avidity but also to a stronger stimulation per NP. Importantly, the increase in EGF-induced ROS formation associated with EGF nanoconjugation at c ROSmax resulted in a measurable gain in EGFR phosphorylation, confirming that ROS generation contributes to the multivalent enhancement of EGFR activation in response to NP-EGF87.

Multivalent Display of Erythropoietin on Quantum Dots Enhances Aquaporin-4 Expression and Water Transport in Human Astrocytes In Vitro

“…Yamamoto et al showed that epidermal growth factor (EGF)-gold NP (AuNP) conjugates modulate EGF signaling by altering the nature of EGF-EGF receptor (EGFR) endocytosis and downstream kinase activity . More recently, Zhang et al demonstrated that the multivalent presentation of EGF on AuNPs augmented the phosphorylation of EGFR and the formation of reactive oxygen species …”

“…9 More recently, Zhang et al demonstrated that the multivalent presentation of EGF on AuNPs augmented the phosphorylation of EGFR and the formation of reactive oxygen species. 41 In this study, we have presented an NP-based strategy for the controlled induction of AQPN-4 expression in HA cells that is built upon the self-assembly and multivalent display of EPO on the surface of a 5 nm QD scaffold. Given that EPOR is known to cluster upon binding of EPO, our working hypothesis was that the multivalent presentation of EPO to EPOR would facilitate enhanced JAK/STAT signaling and synthesis of AQPN-4 membrane channels.…”