UC Berkeley

Engineered nanoparticles are promising tools for delivery and sensing across diverse biological systems. As these nanoparticles function in vivo and traverse biological tissues, biomolecules such as proteins spontaneously interact with the nanoparticles, forming an associated protein corona. This protein corona interacts with cell membranes, cell surface receptors, and other biological components to ultimately determine the fate of the nanoparticles within living systems. As such, characterization of the composition of the protein corona and its influence on nanoparticle function is essential for the translation of nanotechnologies. Despite the significant impact of protein corona formation on nanoparticle function, a lack of standardization and robust methodologies has limited our ability to understand these interactions. In this dissertation, I provide perspective on considering protein corona formation across both plant and mammalian systems. I present a workflow for the robust characterization of protein corona formation on a type of soft nanoparticle and investigate the influence of identified enriched corona proteins on nanoparticle function in vitro.In agriculture, nanoparticles have the potential to improve crop yields through real-time monitoring of plant health signals. These advanced monitoring capabilities enable agricultural workers to adapt plant management strategies rapidly. As these technologies are used in agricultural fields, biomolecular interactions may hinder or alter the intended function of nanoparticles. In this work, we explore the status of nanoparticle-based sensors for agricultural development and the translational value of understanding protein corona formation for plant nanosensors. We highlight the unique challenges associated with nanoparticle-based proteomic studies in plant systems and the distinct biological barriers that plants present. By considering these plant biological features and their influence on biomolecular interactions with nanoparticles, nanoparticles for plant-focused applications can be developed and translated more efficiently. The challenges associated with protein corona formation on nanoparticles in agricultural applications are also present in clinical settings. Lipid nanoparticles (LNPs) are clinically advanced nonviral delivery vehicles for ribonucleic acid (RNA)-based therapeutics. The clinical success of these delivery vehicles has been demonstrated by the messenger ribonucleic acid (mRNA)-based LNP Moderna and Pfizer/BioNTech SARS-CoV-2 vaccines. Conventionally, LNP efficacy and tissue specificity are improved through screening of large combinatorial libraries that vary LNP composition. These screens have led to moderately improved LNP-mediated gene expression and tissue-specific delivery. However, we lack a mechanistic understanding of how these components interact with biological systems which could guide improved efficiency and the organ- and cell-type-specific biodistribution and expression. Protein corona formation on LNPs has been shown to contribute to enhanced delivery, yet protein corona formation on these lipid-based particles remains poorly understood. This protein corona is largely unexplored due to difficulties associated with isolating the protein-nanoparticle complex from endogenous biofluid nanoparticles. In this work, we develop a quantitative, label-free mass spectrometry-based proteomics workflow to explore the nano-bio interface of LNPs. We apply density gradient ultracentrifugation isolation and proteomic normalization to account for the presence of native nanoparticles in pooled human blood plasma. With this approach, we uncover proteins consistently enriched in the LNP corona.Next, we examine the influence of proteins consistently enriched in the LNP corona on LNP function: apolipoprotein E, vitronectin, alpha-2-macroglobulin, and C-reactive protein. We explore how LNPs pre-incubated with these enriched corona proteins influence LNPs during cargo delivery, including cell uptake, lysosomal trafficking, and mRNA expression in HepG2 liver cells. Counterintuitively, we find that increased levels of cell uptake, quantified through confocal microscopy image analysis and flow cytometry, do not correlate with increased mRNA expression. We find that LNPs pre-incubated with apolipoprotein E showed increased levels of cell uptake and lysosomal trafficking with no change in mRNA expression. We provide evidence to suggest that these differences observed between cell uptake and mRNA expression for LNPs pre-incubated with corona proteins may be due to protein corona-induced lysosomal trafficking of LNPs. In summary, these findings demonstrate the critical importance of characterizing protein corona formation to understand nanoparticle behavior in complex biological environments. The influence of protein interactions at key mechanistic steps, such as cellular uptake, trafficking, and delivery, can inform the rational design of next-generation nanoparticles. Future research should focus on integrating protein corona insights into nanoparticle engineering to develop more tunable, predictable, and targeted systems for both sensing and therapeutic delivery.