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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)

   Abstract 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. Mechanism of Cationic Peptide-Induced Assembly of Gold Nanoparticles: Modulation of Electrostatic Repulsion

   Lam, B; Ramji, R; Mullooly, M; Closser, K D; Pascal, T A; Jokerst, J V (April 2025, Aggregate)

   The aggregation of plasmonic nanoparticles can lead to new and controllable properties useful for numerous applications. We recently showed the reversible aggregation of gold nanoparticles (AuNPs) via a small, cationic di-arginine peptide; however, the mechanism underlying this aggregation is not yet comprehensively understood. Here, we seek insights into the intermolecular interactions of cationic peptide-induced assembly of citrate-capped AuNPs by empirically measuring how peptide identity impacts AuNP aggregation. We examined the nanoscale interactions between the peptides and the AuNPs via UV-vis spectroscopy to determine the structure-function relationship of peptide length and charge on AuNP aggregation. Careful tuning of the sequence of the di-arginine peptide demonstrated that the mechanism of assembly is driven by a reduction in electrostatic repulsion. We show that acetylated N-terminals and carboxylic acid C-terminals decrease the effectiveness of the peptide in inducing AuNP aggregation. The increase in peptide size through the addition of glycine or proline units hinders aggregation and leads to less redshift. Arginine-based peptides were also found to be more effective in assembling the AuNPs than cysteine-based peptides of equivalent length. We also illustrate that aggregation is independent of peptide stereochemistry. Finally, we demonstrate the modulation of peptide-AuNP behavior through changes to the pH, salt concentration, and temperature. Notably, histidine-based and tyrosine-based peptides could reversibly aggregate the AuNPs in response to the pH. 

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4. Mechanism of Cationic Peptide‐Induced Assembly of Gold Nanoparticles: Modulation of Electrostatic Repulsion

   https://doi.org/10.1002/agt2.70043  

   Lam, Benjamin; Ramji, Robert; Mullooly, Margaret; Closser, Kristina_D; Pascal, Tod_A; Jokerst, Jesse_V (April 2025, Aggregate)

   ABSTRACT The aggregation of plasmonic nanoparticles can lead to new and controllable properties useful for numerous applications. We recently showed the reversible aggregation of gold nanoparticles (AuNPs) via a small, cationic di‐arginine peptide; however, the mechanism underlying this aggregation is not yet comprehensively understood. Here, we seek insights into the intermolecular interactions of cationic peptide‐induced assembly of citrate‐capped AuNPs by empirically measuring how peptide identity impacts AuNP aggregation. We examined the nanoscale interactions between the peptides and the AuNPs via UV‐vis spectroscopy to determine the structure‐function relationship of peptide length and charge on AuNP aggregation. Careful tuning of the sequence of the di‐arginine peptide demonstrated that the mechanism of assembly is driven by a reduction in electrostatic repulsion. We show that acetylated N‐terminals and carboxylic acid C‐terminals decrease the effectiveness of the peptide in inducing AuNP aggregation. The increase in peptide size through the addition of glycine or proline units hinders aggregation and leads to less redshift. Arginine‐based peptides were also found to be more effective in assembling the AuNPs than cysteine‐based peptides of equivalent length. We also illustrate that aggregation is independent of peptide stereochemistry. Finally, we demonstrate the modulation of peptide‐AuNP behavior through changes to the pH, salt concentration, and temperature. Notably, histidine‐based and tyrosine‐based peptides could reversibly aggregate the AuNPs in response to the pH. 

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5. 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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