Protein-nanoparticle Interactions Research Articles

In Situ Measurement of Nanoparticle-Blood Protein Adsorption and Its Heterogeneity with Single-Nanoparticle Resolution via Dual Fluorescence Quantification.

The formation of a protein corona gives nanomedicines a distinct biological identity, profoundly influencing their fate in the body. Nonspecific nanoparticle-protein interactions are typically highly heterogeneous, which can lead to unique biological behaviors and in vivo fates for individual nanoparticles that remain underexplored. To address this, we have established an in situ approach that allows quantitative examination of nanoparticle-protein adsorption at the individual nanoparticle level. This method integrates dual fluorescence quantification techniques, wherein the nanoparticles are first individually analyzed via nanoflow cytometry to detect fluorescent signals from adsorbed proteins. The obtained fluorescence intensity is then translated into protein quantities through calibration with microplate reader quantification. Consequently, this approach enables analysis of interparticle heterogeneity of nano-protein interactions, as well as in situ monitoring of protein adsorption kinetics and nanoparticle aggregation status in blood serum, preconditioning for a comprehensive understanding of nano-bio interactions, and predicting in vivo fate of nanomedicines.

Nanoparticle-assisted tubulin assembly is environment dependent

Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.

Binding effect of tyrosine-based chiral carbon dots on human serum albumin and lysozyme

Recently, chiral carbon dots (CCDs) have generated significant interest in biological fields such as biosensing, bioimaging, and drug delivery, due to the combination of inherent chirality and nanoscale size. Here, spectroscopic methods have been used to monitor the interaction between human serum albumin (HSA) and lysozyme (LYZ) with CCDs. Long-wavelength emission CCDs (LWE-CCDs) were synthesized using L/D-tyrosine (L/D-Tyr) and o-phenylenediamine (o-PDA) through a facile and one-step solvothermal approach. Two mentioned proteins interacted with L- and D-CDs and the results of secondary structure studies revealed that proteins preserve their native structure while having a small increase in their compactness. Furthermore, studies on the tertiary structure indicated more flexibility and the possibility of disorder in the hydrophobic regions of HSA, while LYZ appeared to become more rigid. Generally, the L- and D-CDs have more effect on the secondary and tertiary structures of HSA and LYZ, respectively. In addition, the interaction of L/D-Tyr (chiral precursor) with proteins was studied using molecular docking to explain the different effects of the CCDs on HSA and LYZ, and the obtained results are almost consistent with the results of experimental tests. This research offers new perspectives on the synthesis of LWE-CCD and the impact of their chirality on protein-nanoparticle interactions. Studying the action of CCDs at the molecular level will help researchers to choose suitable isomers to apply in biological applications.

Integrating Explicit and Implicit Fullerene Models into UNRES Force Field for Protein Interaction Studies.

Fullerenes, particularly C60, exhibit unique properties that make them promising candidates for various applications, including drug delivery and nanomedicine. However, their interactions with biomolecules, especially proteins, remain not fully understood. This study implements both explicit and implicit C60 models into the UNRES coarse-grained force field, enabling the investigation of fullerene-protein interactions without the need for restraints to stabilize protein structures. The UNRES force field offers computational efficiency, allowing for longer timescale simulations while maintaining accuracy. Five model proteins were studied: FK506 binding protein, HIV-1 protease, intestinal fatty acid binding protein, PCB-binding protein, and hen egg-white lysozyme. Molecular dynamics simulations were performed with and without C60 to assess protein stability and investigate the impact of fullerene interactions. Analysis of contact probabilities reveals distinct interaction patterns for each protein. FK506 binding protein (1FKF) shows specific binding sites, while intestinal fatty acid binding protein (1ICN) and uteroglobin (1UTR) exhibit more generalized interactions. The explicit C60 model shows good agreement with all-atom simulations in predicting protein flexibility, the position of C60 in the binding pocket, and the estimation of effective binding energies. The integration of explicit and implicit C60 models into the UNRES force field, coupled with recent advances in coarse-grained modeling and multiscale approaches, provides a powerful framework for investigating protein-nanoparticle interactions at biologically relevant scales without the need to use restraints stabilizing the protein, thus allowing for large conformational changes to occur. These computational tools, in synergy with experimental techniques, can aid in understanding the mechanisms and consequences of nanoparticle-biomolecule interactions, guiding the design of nanomaterials for biomedical applications.

Molecular Serum Albumin Unmask Nanobio Properties of Molecular Graphenes in Shungite Carbon Nanoparticles.

Serum albumin is a popular macromolecule for studying the effect of proteins on the colloidal stability of nanoparticle (NP) dispersions, as well as the protein-nanoparticle interaction and protein corona formation. In this work, we analyze the specific conformation-dependent phase, redox, and fatty acid delivery properties of bovine albumin in the presence of shungite carbon (ShC) molecular graphenes stabilized in aqueous dispersions in the form of NPs in order to reveal the features of NP bioactivity. The formation of NP complexes with proteins (protein corona around NP) affects the transport properties of albumin for the delivery of fatty acids. Being acceptors of electrons and ligands, ShC NPs are capable of exhibiting both their own biological activity and significantly affecting conformational and phase transformations in protein systems.

Impact of Protein Corona Formation on the Thermoresponsive Behavior of Acrylamide-Based Nanogels.

The ability to fine-tune the volume phase transition temperature (VPTT) of thermoresponsive nanoparticles is essential to their successful application in drug delivery. The rational design of these materials is limited by our understanding of the impact that nanoparticle-protein interactions have on their thermoresponsive behavior. In this work, we demonstrate how the formation of protein corona impacts the transition temperature values of acrylamide-based nanogels and their reversibility characteristics, in the presence of lysozyme, given its relevance for the ocular and intranasal administration route. Nanogels were synthesized with N-isopropylacrylamide or N-n-propylacrylamide as backbone monomers, methylenebis(acrylamide) (2.5-20 molar %) as a cross-linker, and functionalized with negatively charged monomers 2-acrylamido-2-methylpropanesulfonic acid, N-acryloyl-l-proline, or acrylic acid; characterization showed comparable particle diameter (c.a.10 nm), but formulation-dependent thermoresponsive properties, in the range 28-54 °C. Lysozyme was shown to form a complex with the negatively charged nanogels, lowering their VPTT values; the hydrophilic nature of the charged comonomer controlled the drop in VPTT upon complex formation, while matrix rigidity only had a small, yet significant effect. The cross-linker content was found to play a major role in determining the reversibility of the temperature-dependent transition of the complexes, with only 20 molar % cross-linked-nanogels displaying a fully reversible transition. These results demonstrate the importance of evaluating protein corona formation in the development of drug delivery systems based on thermoresponsive nanoparticles.

Structural and functional premise of transport protein transthyretin in the sight of silver and zinc oxide nanoparticles through atomistic simulations

Drug transport and distribution in excessive concentrations or at unspecified locations within the human body has a negative impact which leads to further complications. Multiple methodologies have been devised to facilitate precise drug delivery, ensuring the desired quantity reaches the intended target site. Nanoparticle-based drug delivery has emerged as a promising and advanced method. However, there remains a significant scope for deeper mechanistic understanding concerning the protein-nanoparticle corona, their interactions, and the diverse effects of nanoparticles at an atomistic scale. Through the analysis of protein-nanoparticle interactions, it is possible to gain insight into the drug delivery potential of nanoparticles. In this study, we have evaluated how Silver and Zinc Oxide Nanoparticles (AgNP/ ZnONP) affect the structure and function of Transthyretin, a serum transport protein with clinical significance. In the context of the structure–function relationship, our biophysical modeling indicates that Silver Nanoparticles (AgNP) interact with Transthyretin without causing changes in its secondary or tertiary structure. This interaction is projected to have no discernible impact on the protein's function. Yet, in the presence of Zinc Oxide Nanoparticles (ZnONP), Transthyretin experiences considerable changes in its secondary and tertiary structure, which shall lead to disruption of its normal function. This study suggests the potential preference for employing AgNP over ZnONP for transporting conjugated medicines or small molecules. Moreover, Transthyretin's capability to traverse the blood–brain barrier positions it as a promising candidate for nanoparticle-assisted drug delivery across this physiological barrier.

Exploring Residue-Level Interactions between the Biofilm-Driving R2ab Protein and Polystyrene Nanoparticles.

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of significant interest because it impacts an organism's response to a nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. A residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Lysine methylation experiments reveal subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX rates become slower overall in the presence of PSNPs. However, some regions of the R2ab protein exhibit faster than average exchange rates in the presence of PSNPs, while others are slower than the average behavior, suggesting conformational changes upon binding. HDX rates and methylation ratios support a recently proposed "adsorbotope" model for PSNPs, wherein adsorbed proteins consist of unfolded anchor points interspersed with partially structured regions. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how R2ab adsorbs onto PSNP surfaces, this research emphasizes the need for advanced methods to comprehend residue-level interactions in the nanoparticle corona.

Tracking the effects of PLGA-based nanoparticles on protein expression in living cells through quantitative proteomics.

Mass spectrometry (MS)-based proteomics can identify and quantify the differential abundance of expressed proteins in parallel, and bottom-up proteomic approaches are even approaching comprehensive coverage of the complex eukaryotic proteome. Protein-nanoparticle (NP) interactions have been extensively studied owing to their importance in biological applications and nanotoxicology. However, the proteome-level effects of NPs on cells have received little attention, although changes in protein abundance can reflect the direct effects of nanocarriers on protein expression. Herein, we investigated the effect of PLGA-based NPs on protein expression in HepG2 cells using a label-free quantitative proteomics approach with data independent acquisition (DIA). The percentage of two-fold change in the protein expression of cells treated with PLGA-based NPs was less than 10.15% during a 6 hour observation period. Among the changed proteins, we found that dynamic proteins involved in cell division, localization, and transport are more likely to be more susceptible to PLGA-based NPs.

Functional magnetic nanoparticles for protein delivery applications: understanding protein-nanoparticle interactions.

Iron oxide nanoparticles (IONPs) surface functionalised with thermo-responsive polymers can encapsulate therapeutic proteins and release them upon heating with an alternating magnetic field above the lower critical solution temperature (LCST). In order to make this delivery system clinically-relevant, we prepared IONPs coated with poly-N-isopropylmethacrylamide (PNIPMAM), a polymer with LCST above human body temperature. The optimal polymer chain length and nanoparticle size to achieve LCST of ca. 45 °C were 19 kDa PNIPMAM and 16 nm IONPs. The PNIPMAM-coated IONPs could encapsulate a range of proteins which were released upon heating above LCST in the presence of a competitor protein or serum. A small amount of encapsulated protein leakage was observed below LCST. The efficiency of protein encapsulation and release was correlated with molecular weight and glycosylation state of the proteins. Magnetic heating resulted in a faster protein release as compared to conventional heating without significant temperature increase of the bulk solution.

A generic approach based on long-lifetime fluorophores for the assessment of protein binding to polymer nanoparticles by fluorescence anisotropy.

Quantitation of protein-nanoparticle interactions is essential for the investigation of the protein corona around NPs in vivo and when using synthetic polymer nanoparticles as affinity reagents for selective protein recognition in vitro. Here, a method based on steady-state fluorescence anisotropy measurement is presented as a novel, separation-free tool for the assessment of protein-nanoparticle interactions. For this purpose, a long-lifetime luminescent Ru-complex is used for protein labelling, which exhibits low anisotropy when conjugated to the protein but displays high anisotropy when the proteins are bound to the much larger polymer nanoparticles. As a proof of concept, the interaction of lysozyme with poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid) nanoparticles is studied, and fluorescence anisotropy measurements are used to establish the binding kinetics, binding isotherm and a competitive binding assay.

The Development of an AF4 Characterization Pipeline for Polymeric Nanoparicle-Protein Interactions

The centrifugation-wash method is currently the standard technique used for the recovery of nanoparticle-protein complexes from protein-containing serum. However, this technique is disruptive, and leads to changes in nanoparticle parameters and the surface-bound protein composition. In this study, we explore the use of field flow fractionation (FFF) for the development of a robust pipeline for the characterization of nanoparticle parameters following protein corona formation under physiologically-relevant conditions. Model surface-modified polystyrene latex nanoparticles were treated with protein-containing medium mimicking cell-culture conditions under physiologically-relevant conditions. Following incubation, nanoparticle-protein samples were then fractionated using the FFF system and characterized using in-line detectors. Conventional nanoparticle-protein studies typically use the highly invasive centrifugation-wash method which leads to changes in measured particle parameters. Our studies show that AF4 allows for high resolution and characterization of in-situ nanoparticle-protein complexes. This work will allow for the development of a more harmonized approach for characterizing nanoparticle-protein interactions under biologically-relevant conditions.

Phase-Separated Lipid-Based Nanoparticles: Selective Behavior at the Nano-Bio Interface.

The membrane-protein interface on lipid-based nanoparticles influences their in vivo behavior. Better understanding may evolve current drug delivery methods toward effective targeted nanomedicine. Previously, the cell-selective accumulation of a liposome formulation in vivo is demonstrated, through the recognition of lipid phase-separation by triglyceride lipases. This exemplified how liposome morphology and composition can determine nanoparticle-protein interactions. Here, the lipase-induced compositional and morphological changes of phase-separated liposomes-which bear a lipid droplet in their bilayer- are investigated, and the mechanism upon whichlipases recognize and bind to the particles is unravelled. The selective lipolytic degradation of the phase-separated lipid droplet is observed, while nanoparticle integrity remains intact. Next, the Tryptophan-rich loop of the lipase is identified as the region with which the enzymes bind to the particles. This preferential binding is due to lipid packing defects induced on the liposome surface by phase separation. In parallel, the existing knowledge that phase separation leads to in vivo selectivity, is utilized to generate phase-separated mRNA-LNPs that target cell-subsets in zebrafish embryos, with subsequent mRNA delivery and protein expression. Together, these findings can expand the current knowledge on selective nanoparticle-protein communications and in vivo behavior, aspects that will assist to gain control of lipid-based nanoparticles.

Understanding protein-nanoparticle interactions leading to protein corona formation: In vitro - in vivo correlation study

Nanoparticles (NPs) in contact with biological fluids form a biomolecular corona through interactions with proteins, lipids, and sugars, acquiring new physicochemical properties. This work explores the interaction between selected proteins (hemoglobin and fetuin-A) that may alter NP circulation time and NPs of different surface charges (neutral, positive, and negative). The interaction with key proteins albumin and transferrin, the two of the most abundant proteins in plasma was also studied. Binding affinity was investigated using quartz crystal microbalance and fluorescence quenching, while circular dichroism assessed potential conformational changes. The data obtained from in vitro experiments were compared to in vivo protein corona data. The results indicate that electrostatic interactions primarily drive protein-NP interactions, and higher binding affinity does not necessarily translate into more significant structural changes. In vitro and single protein-NP studies provide valuable insights that can be correlated with in vivo observations, opening exciting possibilities for future protein corona studies.

How Does pH Affect the Adsorption of Human Serum Protein in the Presence of Hydrophobic and Hydrophilic Nanoparticles at Air-Water and Lipid-Water Interfaces?

This study investigates interaction between hydrophilic (11-mercaptoundecanoic acid (MUA)) and hydrophobic (1-undecanethiol (UDT)) gold nanoparticles (GNPs) with human serum albumin (HSA) protein on air-water and lipid-water interfaces at pH 3 and 7. Vibrational sum frequency generation (VSFG) spectroscopy is used to analyze changes in the intensity of interfacial water molecules and the C-H group of the protein. At the air-water interface, the hydrophobic interaction between the HSA protein and hydrophobic GNPs at pH 3 leads to their accumulation at the interface, resulting in an increased C-H intensity of the protein with a slight decrease in water intensity. Whereas, at pH 7, where the negative charge of the protein results in the reduced surface activity of the HSA compared to pH 3, the interaction between alkyl chain of the hydrophobic GNPs and alkyl group of the protein results in the adsorption of the protein-capped GNPs at the interface. This leads to an increased intensity of the C-H group of protein and water molecules. However, negatively charged hydrophilic GNPs do not induce significant changes in the interfacial water structure or the C-H group of the protein due to the electrostatic force of repulsion with the negatively charged HSA at pH 7. In contrast, at the lipid-water interface, both hydrophobic and hydrophilic GNPs interact with HSA protein, causing disordering of interfacial water molecules at pH 3 and ordering at pH 7. Interestingly, similar behavior of the protein with both types of GNPs results in comparable ordering/disordering at the interface depending on the pH of solution. Furthermore, the VSFG results obtained with the deuterated lipid suggest that changes in ordering and disorder occur due to increased protein adsorption in the presence of GNPs, causing alterations in the membrane structure. These findings give a better understanding of the mechanisms that govern protein-nanoparticle interaction and their consequential effects on the structure, function, and behavior of molecules at the biological membrane interface, which is crucial for developing safe and effective nanoparticle-based therapeutics.

Predicting Protein Corona Formation on Carbon Nanotubes

Engineered nanoparticles are poised to transform sensing, imaging, and delivery in biological systems. In particular, single-walled carbon nanotubes (SWCNTs) are uniquely suited for biological sensing and imaging due to their tissue-transparent and photostable near-infrared fluorescence. SWCNTs have been functionalized with various biomolecules, including synthetic peptides and proteins, to construct fluorescence-based nanosensors. Optimizing these -nanoparticle interactions is key in enhancing nanotechnology function. Yet, functionalized SWCNTs and other nanotechnologies more broadly suffer from as-of-yet unpredictable interactions with the biological environments in which they are applied: when nanoparticles are introduced into biological systems, endogenous proteins rapidly bind and often lead to decreased ability of the nanoparticles to perform their intended functions. As such, testing compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where this unpredictable protein corona formation often prevents effective implementation.In this work, we develop a classifier that identifies proteins binding to nanotubes based solely on the protein’s amino acid sequence. We build and validate our classifier using mass spectrometry-based quantitative proteomic data that characterizes proteins bound to SWCNT-based nanosensors. Distribution changes among protein properties allow us to determine protein features associated with increased likelihood of SWCNT binding, including high content of solvent-exposed glycines and non-secondary structure-associated amino acids. Understanding these key protein features that drive protein-nanotube binding informs the design of physics-based modeling approaches of such interfacial nano-bio interactions. Lastly, we apply the classifier to rapidly identify candidate proteins with high binding affinity to SWCNTs and experimentally validate adsorption of these proteins with a kinetic exchange assay to evaluate the predictive power of our model. In sum, this classifier provides a step toward undertaking the otherwise intractable problem of predicting protein-nanoparticle interactions.

Time Evolution of Ultrasmall Gold Nanoparticle-Protein Interactions.

To date, much effort has been devoted toward the study of protein corona formation onto large gold nanoparticles (GNPs). However, the protein corona concept breaks down for GNPs in the ultrasmall size regime (<3 nm), and, as a result, our understanding of ultrasmall GNP (usGNP)-protein interactions remains incomplete. Herein, we used anionic usGNPs and six different proteins as model systems to systematically investigate usGNP-protein interactions, with particular focus on the time evolution and long-term behavior of complex formation. The different proteins comprised chymotrypsin (Cht), trypsin (Try), thrombin (Thr), serum albumin (HSA), cytochrome c (Cyt c), and factor XII (FXII). We used a range of biochemical and biophysical methods to estimate binding affinities, determine the effects of usGNPs on protein structure and function, assess the reversibility of any protein structural and functional changes, and evaluate usGNP-protein complex stability. Among the main findings, we observed that prolonged (24 h)─but not short-term (10 min)─interactions between proteins and usGNPs permanently altered protein function, including enzyme activities (Try, Thr, and FXIIa), peroxidase-like activity (Cyt c), and ligand-binding properties (HSA). Remarkably, this occurred without any large-scale loss of the native global conformation, implying time-dependent effects of usGNPs on local protein conformation or dynamics. We also found that both short-(10 min) and long-term (24 h) interactions between proteins and usGNPs yielded short-lived complexes, i.e., there was no time-dependent "hardening" of the interactions at the binding interface as usually seen with large GNPs. The present study increases our fundamental understanding of nano-bio interactions in the ultrasmall size regime, which may assist the safe and effective translation of usGNPs into the clinic.

Determining the impact of gold nanoparticles on amyloid aggregation with 2D IR spectroscopy.

As nanomaterials become more prevalent in both industry and medicine, it is crucial to fully understand their health risks. One area of concern is the interaction of nanoparticles with proteins, including their ability to modulate the uncontrolled aggregation of amyloid proteins associated with diseases, such as Alzheimer's disease and type II diabetes, and potentially extend the lifetime of cytotoxic soluble oligomers. This work demonstrates that two-dimensional infrared spectroscopy and 13C18O isotope labeling can be used to follow the aggregation of human islet amyloid polypeptide (hIAPP) in the presence of gold nanoparticles (AuNPs) with single-residue structural resolution. 60nm AuNPs were found to inhibit hIAPP, tripling the aggregation time. Furthermore, calculating the actual transition dipole strength of the backbone amide I' mode reveals that hIAPP forms a more ordered aggregate structure in the presence of AuNPs. Ultimately, such studies can provide insight into how mechanisms of amyloid aggregation are altered in the presence of nanoparticles, furthering our understanding of protein-nanoparticle interactions.

Promotion of protein assembly by nanoparticles: From in vitro to in-cell.

Engineered nanomaterials are increasingly being studied for biomedical applications such as in diagnosis and therapeutics, due to their similarity in size to subcellular scales, and their tunable physico-chemical properties. Nanomaterials acquire a biomolecular corona upon introduction to biological media, which can influence their biological identity and subsequently, their interactions within the cell. Nanoparticle-protein interactions at the nano-bio interface also affect proteins in the corona, leading to biological transformations such as a change in protein function, unmasking new epitopes, and fibrilization. Silica nanoparticles - one of the most widely studied nanomaterials in biomedical applications - were reported to show a dose-dependent or surface ligand-dependent effect on the kinetics and morphology of protein self-assembly in systems like amyloid-β fibrils, α-synuclein and lysozyme. The interaction of silica nanoparticles with polymerizing cytoskeletal proteins, specifically tubulin proteins, has not been investigated quantitatively. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains, acquired by bacteria from eukaryotic cells via horizontal gene transfer. BtubAB dimers are known to self-assemble like eukaryotic tubulin, forming four-stranded microtubules in vitro as well as five-stranded microtubules in vivo. FRET-labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure the binding interactions of BtubAB dimers in the presence of silica nanoparticles in vitro and in cell using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in vitro, whereas they appear to have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment.

Predicting the interaction of nanoparticles with proteins using lab automation and machine learning.

Nanoparticles in biological fluids interact with proteins forming a “corona” of proteins adsorbed on the nanoparticle surface. Subsequent biological interactions and responses are determined by the protein corona rather than the bare nanoparticle. The development of more effective nanomedicines and nanosensors requires a better understanding of the initial protein-nanoparticle interaction. We have developed an automated workflow, using a liquid handling robot coupled with low-cost proteomics, to generate and characterize protein coronas in a 96-well format. We have used this approach to characterize the concentration and composition of the protein corona formed on carboxylate-modified iron oxide nanoparticles (200 nm) as a function of incubation time and serum concentration. We measure the concentration of the resulting protein corona with a colorimetric assay and the composition of the corona with proteomics, reporting both abundance and enrichment relative to the fetal bovine serum (FBS) proteins used to form the corona. Incubation time was found to be an important parameter for corona concentration and composition at high (100% FBS) incubation concentrations, with only a slight effect at low (10%) FBS concentrations. Current research is aimed at using random forest regression and a suite of nanoparticles (50 nm, 150 nm, 300 nm; −50 mV to +40 mV) to determine what nanoparticle surface properties lead to the adsorption of specific proteins.