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1. Thermodynamics of Protein-Surface Binding- the Model Makes all the Difference

   Nicholas C. Fitzkee, Kayla D ( February 2020 , Biophysical journal)

   null (Ed.)

   Gold nanoparticles (AuNPs) are now being used in such areas as diagnostics, drug delivery, and biological sensing. In these applications, AuNPs are frequently exposed to biological fluids. These fluids contain many different proteins, any of which may interfere with the intended function of the nanoparticle. In this work, we examine the thermodynamic consequences of proteinnanoparticle binding using a combined spectroscopic and calorimetric approach. We monitored binding using UV-Vis spectroscopy, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Six proteins were studied based on their differing chemical properties, and both 15 nm and 30 nm citrate-coated AuNPs were investigated. We interpreted the UV-Vis data using two different models: the commonly-used Langmuir isotherm model and a more complex mass transport model. Both models can be used to determine Kd values for the 30 nm AuNP data; however, the mass transport model is more appropriate for 15 nm AuNPs. This is because, when fitting the Langmuir model, it is commonly assumed that most proteins are not surface-associated, and this assumption fails for 15 nm AuNPs. The DSC thermograms show two transitions for a globular protein adsorbed to a 15 nm AuNP: one high-temperature transition that is similar to global protein unfolding (68 C), and one low-temperature transition that may correspond to unfolding at the surface (56 C). Conversely, ITC experiments show no net heat of adsorption for GB3, even at high protein/AuNP concentrations. Together, the spectroscopic and calorimetric data suggest a complex, multi-step process for protein-nanoparticle adsorption. Moreover, for the proteins studied, both AuNP curvature and protein chemistry contribute to protein adsorption, with proteins generally binding more weakly to the larger nanoparticles. In the future, this work may lead to principles for improving the design of AuNPbased therapeutics and sensors. 

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2. Predicting protein function and orientation on a gold nanoparticle surface using a residue-based affinity scale

   https://doi.org/10.1038/s41467-022-34749-w  

   Xu, Joanna Xiuzhu ; Alom, Md. Siddik ; Yadav, Rahul ; Fitzkee, Nicholas C. ( November 2022 , Nature Communications)

   The orientation adopted by proteins on nanoparticle surfaces determines the nanoparticle’s bioactivity and its interactions with living systems. Here, we present a residue-based affinity scale for predicting protein orientation on citrate-gold nanoparticles (AuNPs). Competitive binding between protein variants accounts for thermodynamic and kinetic aspects of adsorption in this scale. For hydrophobic residues, the steric considerations dominate, whereas electrostatic interactions are critical for hydrophilic residues. The scale rationalizes the well-defined binding orientation of the small GB3 protein, and it subsequently predicts the orientation and active site accessibility of two enzymes on AuNPs. Additionally, our approach accounts for the AuNP-bound activity of five out of six additional enzymes from the literature. The model developed here enables high-throughput predictions of protein behavior on nanoparticles, and it enhances our understanding of protein orientation in the biomolecular corona, which should greatly enhance the performance and safety of nanomedicines used in vivo.

    

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3. Size-Dependent Interactions of Lipid-Coated Gold Nanoparticles: Developing a Better Mechanistic Understanding Through Model Cell Membranes and in vivo Toxicity

   https://doi.org/10.2147/IJN.S249622  

   Engstrom AM, Faase RA ( June 2020 , International journal of nanomedicine)

   null (Ed.)

   Introduction: Humans are intentionally exposed to gold nanoparticles (AuNPs) where they are used in variety of biomedical applications as imaging and drug delivery agents as well as diagnostic and therapeutic agents currently in clinic and in a variety of upcoming clinical trials. Consequently, it is critical that we gain a better understanding of how physiochemical properties such as size, shape, and surface chemistry drive cellular uptake and AuNP toxicity in vivo. Understanding and being able to manipulate these physiochemical properties will allow for the production of safer and more efficacious use of AuNPs in biomedical applications. Methods and Materials: Here, AuNPs of three sizes, 5 nm, 10 nm, and 20 nm, were coated with a lipid bilayer composed of sodium oleate, hydrogenated phosphatidylcholine, and hexanethiol. To understand how the physical features of AuNPs influence uptake through cellular membranes, sum frequency generation (SFG) was utilized to assess the interactions of the AuNPs with a biomimetic lipid monolayer composed of a deuterated phospholipid 1.2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC). Results and Discussion: SFG measurements showed that 5 nm and 10 nm AuNPs are able to phase into the lipid monolayer with very little energetic cost, whereas, the 20 nm AuNPs warped the membrane conforming it to the curvature of hybrid lipid-coated AuNPs. Toxicity of the AuNPs were assessed in vivo to determine how AuNP curvature and uptake influence cell health. In contrast, in vivo toxicity tested in embryonic zebrafish showed rapid toxicity of the 5 nm AuNPs, with significant 24 hpf mortality occurring at concentrations ≥ 20 mg/L, whereas the 10 nm and 20 nm AuNPs showed no significant mortality throughout the five-day experiment. Conclusion: By combining information from membrane models using SFG spectroscopy with in vivo toxicity studies, a better mechanistic understanding of how nanoparticles (NPs) interact with membranes is developed to understand how the physiochemical features of AuNPs drive nanoparticle–membrane interactions, cellular uptake, and toxicity. 

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4. Ad aurum: tunable transfer of N-heterocyclic carbene complexes to gold surfaces

   https://doi.org/10.1039/d2qi01941h  

   Dominique, Nathaniel L. ; Chen, Ran ; Santos, Alyssa V. ; Strausser, Shelby L. ; Rauch, Theodore ; Kotseos, Chandler Q. ; Boggess, William C. ; Jensen, Lasse ; Jenkins, David M. ; Camden, Jon P. ( November 2022 , Inorganic Chemistry Frontiers)

   The exceptional stability of N-heterocyclic carbene (NHC) monolayers on gold surfaces and nanoparticles (AuNPs) is enabling new and diverse applications from catalysis to biomedicine. Our understanding of NHC reactivity at surfaces; however, is quite nascent when compared to the long and rich history of NHC ligands in organometallic chemistry. In this work, well-established transmetalation reactions, previously developed for NHC transfer in homogeneous organometallic systems, are explored to determine how they can be used to create carbene functionalized gold surfaces. Two classes of NHCs, based on imidazole and benzimidazole scaffolds, were tested. The resulting AuNP surfaces were analyzed using X-ray photoelectron spectroscopy (XPS), laser desorption ionization mass spectrometry (LDI-MS), and surface-enhanced Raman spectroscopy (SERS). Reaction of either a Au( i ) or Ag( i ) isopropyl benzimidazole NHC complex with citrate-capped AuNPs yields, in both cases, a chemisorbed NHC that is bound through a Au adatom. Theoretical calculations additionally illustrate that binding through the Au adatom is favored by more than 10 kcal mol −1 , in good agreement with experiments. Surprisingly, reaction of Au( i ), Ag( i ), and Cu( i ) diisopropylphenyl imidazole NHCs do not follow the same pattern. The Cu complex undergoes transmetalation with very little deposition of Cu; whereas, unexpectedly, the Ag complex foregoes transmetalation and instead adducts to the AuNP with retention of the Ag–C bond. Theoretical calculations illustrate that the imidazole ligand affords significant dispersion interactions with the gold surface, which may stabilize binding through the Ag adatom motif, despite its less favorable bonding energies. Taken together these results suggest a unique ability to tune the reactivity by changing the carbene structure and raise critical questions about how established transmetalation reactions in organometallic chemistry can be applied to form NHC functionalized surfaces. 

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5. The Landscape of Gold Nanocrystal Surface Chemistry

   https://doi.org/10.1021/acs.accounts.3c00109  

   Greskovich, Katherine M. ; Powderly, Kelly M. ; Kincanon, Maegen M. ; Forney, Nathan B. ; Jalomo, Catherine A. ; Wo, Anita ; Murphy, Catherine J. ( January 2023 , Accounts of Chemical Research)

   ConspectusGold nanoparticles (AuNPs) exhibit unique size- and shape-dependent properties not obtainable at the macroscale. Gold nanorods (AuNRs), with their morphology-dependent optical properties, ability to convert light to heat, and high surface-to-volume ratios, are of great interest for biosensing, medicine, and catalysis. While the gold core provides many fascinating properties, this Account focuses on AuNP soft surface coatings, which govern the interactions of nanoparticles with the local environments. Postmodification of AuNP surface chemistry can greatly alter NP colloidal stability, nano-bio interactions, and functionality. Polyelectrolyte coatings provide controllable surface-coating thickness and charge, which impact the composition of the acquired corona in biological settings. Covalent modification, in which covalently bound ligands replace the original capping layer, is often performed with thiols and disulfides due to their ability to replace native coatings. N-heterocyclic carbenes and looped peptides expand the possible functionalities of the ligand layer.The characterization of surface ligands bound to AuNPs, in terms of ligand density and dynamics, remains a challenge. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for understanding molecular structures and dynamics. Our recent NMR work on AuNPs demonstrated that NMR data were obtainable for ligands on NPs with diameters up to 25 nm for the first time. This was facilitated by the strong proton NMR signals of the trimethylammonium headgroup, which are present in a distinct regime from other ligand protons’ signals. Ligand density analyses showed that the smallest AuNPs (below 4 nm) had the largest ligand densities, yet spin–spin T2 measurements revealed that these smallest NPs also had the most mobile ligand headgroups. Molecular dynamics simulations were able to reconcile these seemingly contradictory results.While NMR spectroscopy provides ligand information averaged over many NPs, the ligand distribution on individual particles’ surfaces must also be probed to fully understand the surface coating. Taking advantage of improvements in electron energy loss spectroscopy (EELS) detectors employed with scanning transmission electron microscopy (STEM), a single-layer graphene substrate was used to calibrate the carbon K-edge EELS signal, allowing quantitative imaging of the carbon atom densities on AuNRs with sub-nanometer spatial resolution. In collaboration with others, we revealed that the mean value for surfactant-bilayer-coated AuNRs had 10–30% reduced ligand density at the ends of the rods compared to the sides, confirming prior indirect evidence for spatially distinct ligand densities.Recent work has found that surface ligands on nanoparticles can, somewhat surprisingly, enhance the selectivity and efficiency of the electrocatalytic reduction of CO2 by controlling access to the active site, tuning its electronic and chemical environment, or denying entry to impurities that poison the nanoparticle surface to facilitate reduction. Looking to the future, while NMR and EELS are powerful and complementary techniques for investigating surface coatings on AuNPs, the frontier of this field includes the development of methods to probe the surface ligands of individual NPs in a high-throughput manner, to monitor nano-bio interactions within complex matrices, and to study structure–property relationships of AuNPs in biological systems. 

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