Real-time Monitoring of Protein Corona Formation on Polymer Dots Using Fluorescence Resonance Energy Transfer Spectroscopy

Luminescent polymer dots (Pdots) have emerged as versatile luminescent polymer nanoparticles for biological applications due to their outstanding luminescent properties, high colloidal stability in biological solutions, and ease of synthesis and surface modifications. Pdots doped with polystyrene maleic anhydride are colloidally stable in aqueous solutions due to their high negative zeta potential of about −40 mV. When suspended in model biological solutions, our fluorescence resonance energy transfer (FRET) spectroscopy studies reveal protein corona formation on the surface of Pdots. The protein corona formation is driven by electrostatic interactions with positively charged proteins and nonelectrostatic forces, which play an increasing role as the solution pH increases. Protein corona formation on Pdots could significantly reduce their targeting capabilities by masking targeting surface functionalities such as antibodies or other cell recognition components. This study explores the use of FRET spectroscopy to study the binding dynamics of a single protein to Pdots. The selectivity of FRET to the Pdots surface enables real-time monitoring of protein adsorption and desorption processes under varying concentrations and environmental conditions without requiring washing steps. FRET spectroscopy data show that protein adsorption and corona formation increase with increasing protein concentrations and at lower pH due to the increased attractive electrostatic interactions. In contrast, the elevated ionic strength of the solution reduces protein adsorption and corona formation due to ion shielding. Additionally, protein adsorption and corona formation decrease with increasing anionic charge density on the Pdots surface due to increased repulsive electrostatic interactions. FRET measurements were also used to monitor the displacement of loosely bound proteins from the Pdot surface. The study provides insights into optimizing the surface functionalization of Pdots for enhanced stability and targeting efficiency in biological solutions.