On Protein Corona Formation: Understanding Nano-Bio Interactions Toward Engineering Optical Nanomaterials
Engineered nanoparticles have emerged as a promising platform upon which to develop biological sensing, imaging, and delivery tools. Yet, a paramount challenge toward implementing such technologies is understanding how these engineered nanoparticles are affecting and being affected by the complex bioenvironments in which they are applied. Introducing a nanoparticle into a biological system rapidly establishes a nano-bio interface, as biomolecules, most notably proteins, adsorb to the nanoparticle surface to form the “corona”. The abruptness of protein adsorption on foreign nanoparticle surfaces causes proteins to interact in atypical modes, contrary to normal biomolecular interactions governed by precise genetic control, and often produces undesirable outcomes such as protein denaturation. Further, protein corona formation unpredictably changes the nanoparticle identity and fate, as the adsorbed proteins mask original surface characteristics and endow new biochemical properties to the nanoparticle. As a result, how the nanoparticle-corona complex interacts with biological machinery is impacted and in vivo circulation, bioaccumulation, and biocompatibility outcomes are drastically modified. Consequently, protein corona formation can disrupt the nanoparticle’s designated function by attenuating or eliminating nanoparticle efficacy relative to its in vitro performance. Conversely, the protein corona can be taken advantage of, with an engineered protein corona that facilitates new sensing modalities or stealth transport in targeted delivery applications, with improved nanoparticle functionality or therapeutic effect to follow. In both cases, the protein corona displayed on the nanoparticle surface is a principal design parameter for ensuring successful applications of nanotechnologies in biological systems.
In this dissertation, I develop a framework of analysis to quantitatively characterize protein corona formation that occurs on nanoparticle substrates in different biological environments. I adopt a primarily experimental approach, with complementary physics-based and statistical modeling, toward investigating these nano-bio interfacial interactions. I begin with a multimodal characterization of protein corona composition, driving forces of formation, and kinetics in relevant biological media. These studies primarily focus on protein adsorption to single-stranded DNA-functionalized single-walled carbon nanotube (ssDNA-SWCNT) probes. ssDNA-SWCNTs are an appealing tool for biological sensing and imaging because they operate at spatiotemporal scales necessary to capture information on complex biological systems, such as neurotransmitter signaling in the brain. However, it is of note that the methods of corona characterization and framework of understanding are generalizable to different nanoparticles in different bioenvironments. To probe the protein corona composition, I have optimized a platform to isolate nanoparticle-bound corona proteins and to determine abundance and differential enrichment vs. depletion of corona proteins by mass spectrometry-based proteomics. This analysis provides protein corona compositional maps for nanoparticles that underscore the selectivity of corona formation, reveal key proteins and protein functional roles implicated in corona formation, and enable analysis of protein physicochemical properties governing adsorption to nanoparticle surfaces. By varying incubation conditions of ssDNA-SWCNTs in biofluids, I investigate the role different interactions have on driving selective protein corona formation dependent on corona layer, including electrostatic and hydrophobic forces. These experimental studies are complemented by mathematical modeling, with a colloid thermodynamic framework to describe how the nanoparticle and protein interact in solution, and classification modeling, with a supervised learning approach to move toward predicting nano-bio interactions. To study the dynamic exchange of biomolecules on the SWCNT surface, I have developed a multiplexed fluorescence assay that enables real-time tracking of biomolecule adsorption and desorption events, with corresponding kinetic modeling of this exchange process. From these dynamic corona studies, I provide insight on the general translatability of whole-biofluid nano-bio interactions to single-protein experiments, while highlighting the role of cooperativity effects driving certain protein-nanoparticle interactions. Moreover, this corona exchange assay can be readily extended to examine various biomolecules binding on nanoparticles and enables study in solution rather than in a surface-immobilized, less biologically relevant, setting. Understanding the protein corona composition, driving forces of formation, and dynamics under relevant solution conditions informs design and synthesis of nanotechnology-based tools applied in protein-rich environments.
Although corona formation can impair nanobiotechnology efficacy, it also presents an opportunity to create improved protein-nanoparticle architectures by exploiting selective protein adsorption to the nanoparticle surface. Toward this end, I leverage the fundamental understanding of nano-bio interactions developed in the first portion of this dissertation to design and develop a novel nanosensor for viral protein detection. This nanosensor harnesses SWCNTs to provide the optical signal readout, together with proteins that possess intrinsic recognition ability for the analyte of interest: the ACE2 cell membrane protein is used to bind to the SARS-CoV-2 spike protein analyte. The resulting ACE2-SWCNT nanosensors are thus developed and validated in a proof-of-principle study to create hybrid nano-bio constructs for rapid, label-free protein detection. Notably, this sensing platform is intended to enable rapid detection of live virus (denoting current viral infection) with a near-infrared signal readout that is transmissible through biological media (detecting virus in unprocessed patient samples such as saliva and nasal fluid). As such, further development of this nanosensor construct is envisioned to yield an accessible, point-of-care testing platform.
In sum, this work develops techniques and analyses to characterize the protein corona and subsequently employs this knowledge toward rational design of nanobiotechnologies. The holistic approach developed herein represents a step toward generalized corona formation rules and understanding that will aid the translation of sensing, imaging, and delivery nanotechnologies into biological application.