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

• Published in: Bioconjugate chemistry 2022-08, Vol.33 (9), p.1716-1728
• Main Authors: Zhang, Sandy, Ouyang, Tianhong, Reinhard, Björn M.
• Format: Article
• Language: English
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.2c00335

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-EGF 12 ) and 87 ± 6 EGF/NP (NP-EGF 87 )). In the EGFR overexpressing cell lines (MDA-MB-231 and MDA-MB-468), NP-EGF 87 achieved a measurable multivalent enhancement of ROS that peaked at concentrations c ROS max ≤ 25 pM and that was EGFR and nicotinamide adenine dinucleotide phosphate oxidase (NOX) dependent. NP-EGF 12 failed to generate comparable ROS levels as NP-EGF 87 in the investigated NP input concentration range (0-100 pM). In cells with nearly identical numbers of bound NP-EGF 87 and NP-EGF 12 , the ROS levels for NP-EGF 87 were systematically higher, indicating that the multivalent enhancement is not exclusively related to avidity but also to a stronger stimulation per NP. Importantly, the increase in EGF-induced ROS formation associated with EGF nanoconjugation at c ROS max resulted in a measurable gain in EGFR phosphorylation, confirming that ROS generation contributes to the multivalent enhancement of EGFR activation in response to NP-EGF 87 .