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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Nanoscale surface curvature modulates nanoparticle-protein interactions

Zehui Xiaa, Esteban Villarrealb, Hui Wangb, Boris L.T. Laua,*a Department of Civil & Environmental Engineering, University of Massachusetts Amherst, 130 Natural Resources Road, Amherst, MA 01003, USAbDepartment of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA

A R T I C L E I N F O

Keywords:Nanoscale surface curvatureSurface atomic coordinationProtein conformationBovine serum albuminGold nanoparticlesBio-nano interface

A B S T R A C T

Rational optimization of nanoparticle (NP) surfaces is essential for successful conjugation of proteins to NPs fornumerous applications. Using surface-roughened NPs (SRNPs) and quasi-spherical NPs (QSNPs) as two modelnanostructures, we examined the effects of local surface curvature on protein conformation and interfacialbehaviors by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy (FES), and isothermaltitration calorimetry (ITC). The surface of SRNPs consisted of a mixture of undercoordinated and close-packedsurface atoms at the highly curved and locally flat surface regions, respectively, whereas QSNPs were primarilyenclosed by {100} and {111} facets covered with close-packed surface atoms. Our findings demonstrated that: 1)SRNPs possess higher tendency to denature BSA and accommodate a higher number of BSA molecules on thesurface and 2) the aggregation of AuNP-BSA complexes, likely induced by either denatured BSA or reducedelectrostatic repulsion between complexes, is dependent on both the BSA concentration and the NP surfacecurvature. This study also indicated that NP local surface curvature could potentially be used as a design strategyto preserve the biological function of proteins.

1. Introduction

The adsorption of proteins may remarkably modify the surfacechemistry of a nanoparticle (NP) by creating a protein corona thatgreatly influences the NP’s cellular uptake and/or immune responses.[1–6] When proteins adsorb onto a particle surface, their conformationis likely to change [7–10]. Whether this change is desirable depends onthe specific context. Many studies have reported that such inducedconformational changes can result in protein misfolding, fibrillation/aggregation and malfunction [2,11–13]. This may significantly reducethe safety and efficacy of proteins specifically designed for therapeuticpharmaceuticals. Conversely, protein denaturation may serve to reducepotential health risks from pathogenic proteins such as prions, whichare resistant to inactivation by heat and some chemical agents [14]. Asa result, a deeper understanding of NP-protein interactions is critical,not only to the precise prediction of the NP behaviors in physiologicalenvironment, but also to the fine-tuning of the properties of NPs fortheir potential biomedical applications (e.g., drug delivery and ther-apeutics).

As proteins are very sensitive to small changes in the surroundingenvironment, the extent and ease of protein conformational changesupon adsorption onto NPs are determined by a complex interplay ofmany factors, including but not limited to, NP characteristics (e.g., size,

[15] charge [2,8], and functional groups [1,7,15]), protein character-istics (e.g., size [16] and geometry [17]) and solution chemistry (e.g.,pH [3] and ionic strength [18]). Many recent studies point to the keyrole of NP surface morphology on protein denaturation. For example,bovine serum albumin (BSA) retained various degrees of its secondarystructures depending on the shape of AuNPs (e.g., gold NPs (AuNPs)(e.g., nanorods, triangular nanoplates, and nanospheres). [9,19]Mandal et al. revealed that peptides lost more of its native structure onthe highly curved surfaces of 5 nm Au nanoparticles (AuNPs) due togreater number of Au-S bonds compared to relatively ‘planar’ surfaceswith reduced curvature [20]. A NP surface with nanoscale curvature isessentially composed of a variety of localized high-index facets and thesurface atomic coordinations are geometrically defined by the localsurface curvature of the NPs. Each type of crystallographic facets has itscharacteristic surface atom coordination. For example, high-index fa-cets possess a higher density of undercoordinated atoms (and hence,surface energy), in which the atomic-level surface structure of nano-crystals itself may influence the adsorption of proteins [21]. However,to minimize the surface energy, metal nanocrystals usually prefer togrow into shapes enclosed by low-index facets, such as {100} and{111}, with close-packed surface atomic configurations [22]. By judi-ciously tuning the nanocrystal growth kinetics, it becomes possible tosynthesize kinetically-favored, high-index faceting AuNPs that are rich

https://doi.org/10.1016/j.colsurfb.2020.110960Received 8 January 2020; Received in revised form 28 February 2020; Accepted 9 March 2020

⁎ Corresponding author.E-mail address: borislau@gmail.com (B.L.T. Lau).

Colloids and Surfaces B: Biointerfaces 190 (2020) 110960

0927-7765/ © 2020 Elsevier B.V. All rights reserved.

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in undercoordinated surface atoms [23–25]. These high-index facetingAuNPs have been shown to 1) exhibit enhanced catalytic activities andsurface plasmonic properties, relative to low-index faceting AuNPs and2) emerge as potential new agents for biocatalyst, biosensors and na-nomedicine. Therefore, it is essential to investigate the effects of na-noscale surface curvature and surface atomic coordination on theconformations and interfacial behaviors of the proteins adsorbed on NPsurfaces to fully evaluate the suitability of protein-coated NPs for spe-cific applications.

In this study, BSA was used as the model protein and Au quasi-spherical nanoparticles (QSNPs) and surface-roughened nanoparticles(SRNPs) of similar overall diameters (∼140 nm) were chosen as twomodel nanostructures representing locally flat low-index faceting andlocally curved high-index faceting surfaces, respectively, for systematiccomparative studies. The surfaces of QSNPs are dominated by {100}and {111} facets covered with close-packed Au atoms, whereas thesurfaces of SRNPs are rich of undercoordinated surface atoms at thehighly curved surface sites. [23]. Unlike previous studies in whichsurface curvature is caused by different nanoparticle size, [20,26] thenanoscale curvature of SRNPs is essentially induced by the enrichmentof undercoordinated Au surface atoms. Complementary tools, includingcircular dichroism (CD) spectroscopy, fluorescence emission spectro-scopy (FES), and isothermal titration calorimetry (ITC) were used tocharacterize the changes in BSA conformations and the thermo-dynamics of BSA-NP interactions. This study provided new mechanisticinsights into the role of nanoscale surface curvature in conformationsand interfacial behaviors of proteins adsorbed onto NP surfaces.

2. Experimental Section

2.1. Preparations of AuNPs and proteins

QSNPs and SRNPs were synthesized using a kinetically controlled,seed-mediated nanocrystal growth method described by Villarreal et al.[23]. Both types of AuNPs were stabilized by a positively-charged,surface-capping ligand, cetyltrimethylammonium chloride (CTAC). Theas-obtained Au SRNPs were washed with water 3 times through cen-trifugation/redispersion cycles, and finally redispersed in water. BovineSerum Albumin (BSA) was purchased from Sigma Aldrich and usedwithout further purification. All chemicals were prepared using Milli-Q(Millipore, Billerica, MA) water with a resistivity of 18.2 MΩ cm. Syr-inge filters of 0.2 μm pore size (Millipore, Billerica, MA) were used tofilter buffer solutions. All experiments were conducted in 1mM HEPESbuffer of pH 7.5. All stock solutions were stored at 4 °C and used withinone week.

2.2. Stability of AuNP-BSA complexes

The adsorption of BSA onto AuNPs was monitored by dynamic lightscattering (DLS, Zetasizer Nano, Malvern Panalytical Inc.,Worcestershire, UK). A disposable polystyrene 1 cm cuvette (SarstedtAG&Co., Nümbrecht, Germany) filled with 1ml sample of 0.125 nMAuNPs sample was placed in the instrument sample holder. Then analiquot of BSA was added into the system to reach the desired con-centrations. The DLS measurement was then collected immediately.Scattering of the incident light (563 nm wavelength) was measured at173°. The average hydrodynamic diameter, DH was calculated ap-proximately by averaging 10–15 individual 10 s measurements. Allmeasurements were conducted at 25 °C. Zeta (ζ)-potential values wereestimated from the electrophoretic mobility of 1ml aliquot solution in afolded capillary cell (DTS 1061, Malvern Panalytical Ltd.,Worcestershire, UK). Nine measurements of ζ-potentials were con-ducted for each sample.

2.3. Circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy was used to characterize thechanges in the secondary structure of BSA upon their adsorption ontoAuNPs. This technique has been frequently used to monitor the con-formational changes of proteins interacting with NPs [7–10,27–30]. CDmeasurements were undertaken by a Jasco J-1500 spectrophotometer(Jasco Inc., Easton, MD) with a 10mm path length quartz cell at roomtemperature. The CD spectra were recorded from 200 to 250 nm andeach spectrum was an average of 3 scans. The test solutions for CD wereprepared by mixing BSA and AuNPs and the spectra were collectedimmediately. The concentration of BSA was fixed at 0.25 μM, and theAuNP concentration ranged from 0 to 1.25 nM. Data were fit using theK2D method run through DichroWeb to determine the secondarystructure contents including alpha-helix, beta-sheet and random coil. Afit to the experimental data was accepted when the normalized root-mean-square deviation was<0.22 as suggested.

2.4. Fluorescence emission spectroscopy

Fluorescence emission spectroscopy (FES) is another importanttechnique used to unravel the NP-BSA interactions as it directly mea-sures the change of intrinsic fluorescence of the tryptophan (Trp) re-sidues upon adsorption of BSA onto NPs, providing information on thetertiary structure of BSA [9,30–32]. BSA has two Trp residues, Trp 134located on the surface and Trp 212 buried inside the hydrophobicpocket, both of which are sensitive to changes in the local micro-environment. Therefore, any change of fluorescence could provideimportant information that maybe invisible to the techniques discussedabove, including the polar or nonpolar microenvironment surroundingthe NPs and BSA. At pH 7.4 and in HEPES buffer, Trp in BSA usuallyexhibits fluorescence emission at about 346 nm when excited at 280 nm[33]. The emission fluorescence intensity was recorded at room tem-perature using the Jasco J-1500 spectrophotometer between 285 and401 nm after excitation at 280 nm. Samples were handled in the sameway as in the CD spectroscopy experiments. The data collected werefrom an average of three accumulations and the background signalsfrom the AuNPs were subtracted from the overall readings.

2.5. Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) has been widely used tocharacterize the thermodynamics behind the NP-protein interactions[1,7,16]. Measurements were performed using a MicroCal Auto-iTC200system (Malvern Panalytical Inc., Westborough, MA). The AuNP sus-pensions were dialyzed overnight against 1 mM HEPES buffer (renewedtwo times) at pH 7.5, and BSA stock solutions were prepared in the lastdialysate. The titration experiment involved 40 injections (1 μl per in-jection) of proteins ([BSA]= 0.25 and 25 μM for QSNPs and SRNPs,respectively) at 120 s intervals into the sample cell (volume =0.4ml)containing the AuNP solution ([QSNP]=1.25 nM and[SRNP]=0.75 nM). The reference cell was filled with 1mM HEPESbuffer. During the experiment, the sample cell was stirred continuouslyat 1000 rpm. The heat profile of protein dilution in the buffer alone wassubtracted from the titration data (both normalized to 0) for each ex-periment. The data were analyzed to determine the thermodynamics ofthe reaction using the coupled Origin software (Origin Inc., North-ampton, MA).

2.6. Octanol-water partition coefficient measurements

Octanol-water partition coefficients (KOW) of AuNPs were measuredto determine their relative hydrophobicity. We adapted the shake-flaskmethod described by Xiao et al. [34] which was analogous to KOW

measurements performed for organic compounds. 5 ml octanol and 5ml1mM HEPES buffer solution were premixed and shaken at 200 rpm on

Z. Xia, et al. Colloids and Surfaces B: Biointerfaces 190 (2020) 110960

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an orbital shaker overnight before use. 0.5 ml of 1 nM AuNP suspen-sions were added to the aqueous phase carefully, after which the mix-ture was shaken for another 24 h at room temperature. The mixture wasthen allowed to stand and equilibrate for approximately 3 h. The aqu-eous phase was collected for Au concentration determination by in-ductively coupled plasma mass spectroscopy (PerkinElmer Sciex ELANDRC-e, PerkinElmer Inc., Waltham, MA). The KOW was calculated bycomparing initial NPs concentration and aqueous one. Measurementswere performed in triplicates.

3. Results and discussion

The effect of nanoscale surface curvature of AuNPs on protein ad-sorption was characterized by mixing BSA with QSNP or SRNP sus-pensions. Details on the synthesis and characterization of AuNPs can befound in previous studies [23–25]. Both types of AuNPs have a similardiameter of ∼ 140 nm (see scanning electron microscopy images inFigure S1 in Supporting Information) and a hydrodynamic diameter of∼150 nm (Table 1) to help attributing their effects on BSA conforma-tional changes to difference in local surface curvatures and surfaceatomic coordination.

3.1. SRNPs induced greater conformational changes of BSA than QSNPs

The CD spectrum of BSA typically has negative peaks at 222 and208 nm, characteristic of the alpha-helix structure (Figs. 1A and C). Thestructure contents (i.e., alpha-helix, beta-sheet and random coil) weredetermined by fitting the CD spectra (Figs. 1B and D). The addition ofAuNPs led to a decrease in intensity of the two negative peaks (Figs. 1Aand C), which suggests a disruption of the secondary structure of BSA.At low concentrations (0.0125 and 0.125 nM) of AuNPs (i.e., whenhigher surface loadings of BSA), the secondary structure was onlyslightly affected by the addition of AuNPs, potentially due to less AuNP-BSA interaction as the surface of AuNPs became saturated [35]. Thedisruption was most apparent at the highest AuNP concentration of1.25 nM. In this scenario, both QSNP-BSA and SRNP-BSA complexesshowed suppressed alpha-helix content with enhanced beta-sheet andrandom coil contents. At the highest concentration of AuNPs (1.25 nM),the secondary structure of SRNP-BSA (Fig. 1C) deviated more from theBSA controls than QSNP-BSA (Fig. 1A), indicating a higher amount ofdenatured BSA induced by the adsorption onto SRNPs. The differencemay stem from a higher density of surface undercoordinated atoms andstepped structures on SRNP surfaces [23]. Although AuNPs were sta-bilized by CTAC, BSA may still be capable of interacting with the bareAu surfaces, in which case surface gold atoms would exert an influenceon the BSA structure. The undercoordinated Au atoms on the highly-curved surfaces (e.g., kinks and steps) are usually of high energy (andhence, more reactive) and would form more covalent bonds (e.g., Au-S)than the ones on ‘planar’ surfaces [20], subsequently altering theirstructure. Furthermore, using the density functional theory approach,Fajin et al. reported that the cysteine, one of the major residues in BSA,displayed more favorable adsorption energy onto stepped gold surfacesthan on flat surfaces [36]. In light of those findings, SRNPs were likelyto induce greater conformational changes to BSA than QSNPs.

Table 1Physicochemical properties of AuNPs and BSA at 1mM HEPES, pH 7.5.

Hydrodynamic diameter, nm ζ-potential, mV Calculated log KOW

SRNPs 153.40 ± 1.00 45.60 ± 2.96 3.56 ± 0.09QSNPs 146.90 ± 1.06 38.30 ± 2.88 2.79 ± 0.04BSA 9.47 ± 0.15 −24.80 ± 0.77 N/A

Fig. 1. Circular dichroism spectra (A, C) and fitted secondary structure calculations (B, D) for 0.25 μM BSA under different QSNPs (A and B) and SRNPs (C and D)concentrations at pH 7.5.

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As BSA molecules adsorb to the nanoparticle surface and lose sec-ondary structure, their hydrophobic cores can be exposed, leading tothe aggregation of the complexes induced by hydrophobic protein-protein interactions [10,29]. In the next series of measurements, wemonitored the colloidal stability of AuNP-BSA complexes by measuringtheir ΔDH and ζ-potentials to further understand the interaction.

3.2. Aggregation of AuNP-BSA complexes is concentration-dependent

The interaction of AuNPs with BSA was characterized by DLS in BSAconcentrations ranging from 0.025 to 25 μM. ΔDH and ζ-potential ofAuNP-BSA complexes are presented in Fig. 2. It was found that theaggregation of AuNP-BSA complexes occurred only at specific con-centration ranges of BSA. For QSNPs, aggregation was observed at0.025 to 0.25 μM BSA. Over that range, the addition of BSA resulted inan increase of ΔDH to over 100 nm which continued to increase atlonger times (Figure S2 in Supporting Information). The aggregationcan be explained by 1) the denaturation of BSA and/or 2) decreasedelectrostatic repulsion. The disrupted BSA could interact with othercomplexes and thus initiate the aggregation. This is supported by a lossof secondary structure as observed at a similar surface loading of BSA(Fig. 1A, at [QSNP]= 1.25 nM). Further increase of BSA concentrationsdid not lead to a substantial ΔDH. At 25 μM BSA, ΔDH and the ζ-po-tential of QSNP-BSA complexes were approximately 8 nm and-24.8 ± 0.77mV, respectively, similar to those of unbound BSA(Table 1), indicating QSNPs were stabilized by a BSA layer.

For SRNPs, aggregation did not occur until BSA reached a con-centration of 2.5 μM or higher. Below that, there were only minorchanges to ΔDH and ζ-potential of SRNP-BSA (1.60 ± 2.86 and4.00 ± 0.21 nm, 41.70 ± 3.25 and 37.70 ± 2.02mV at BSA of 0.025and 0.25 μM, respectively, Fig. 2). This observation suggested that BSAdenaturation was not required for the aggregation of SRNP-BSA com-plex, despite SRNP-BSA exhibited greater structure changes than QSNP-BSA (Figs. 1A and C, at [AuNP]= 1.25 nM). When BSA reached 25 μM,ΔDH was increased to over 600 nm, with ζ-potential being slightly ne-gatively-charged of approximately −5mV, indicating the aggregationof SRNP-BSA complexes.

Our data suggested that the aggregation of QSNP-BSA and SRNP-BSA complexes was dependent on BSA concentration. SRNPs werecapable of accommodating a higher amount of BSA before the initiationof aggregation. This can potentially be a consequence of their greatersurface curvature and thus larger surface area available for BSA ad-sorption, which was estimated to be 4.5 time of a QSNP of similardiameter (Table S1 in Supporting Information) [23,25]. In addition, thegreater surface coverage of BSA on SRNPs than QSNPs may be related

to the difference in their surface hydrophobicity. Based on octanol-water distribution coefficient (KOW) measurements, SRNPs appeared tobe more hydrophobic than QSNPs (Table 1). The stronger hydrophobicinteractions may be responsible for the greater adsorption of BSA ontothe more hydrophobic SRNPs. This is in agreement with a previousstudy in which a considerably higher amount of human serum albumin(HSA) was required to saturate the 200 nm hydrophobic polymeric NPsthan hydrophilic ones [26]. Furthermore, NP hydrophobicity can besubstantially enhanced by increasing nanoscale surface curvature andsurface energy [37–43], both of which were characteristic of SRNPs.The aggregation of SRNP-BSA could be driven by decreased electro-static repulsion between the complexes rather than the denatured BSA.The positive surface charge of SRNPs was gradually neutralized by theadsorption of more negatively-charged BSA (45.60 ± 2.96 vs-4.87 ± 0.48mV in the absence and presence of 25 μM BSA, Fig. 2),inducing the aggregation of SRNP-BSA complexes.

Our data from the previous two sections indicated that the ag-gregation of AuNP-BSA complexes is concentration-dependent, which isconsistent with the literature [8,10,28,29,44]. More importantly, thisstudy set itself apart by examining the effect of nanoparticle surfacecurvature on BSA structure and complex stability. The enrichment ofundercoordinated Au atoms and highly curved surfaces of SRNPs in-duced more conformational changes of BSA, when compared to theatomically close-packed and less curved surfaces of QSNPs. On theother hand, each SRNP is capable of accommodating a greater numberof BSA due to their higher specific surface area till the aggregation ofSRNP-BSA complexes occurs.

3.3. SRNPs induced less quenching of BSA fluorescence than QSNPs

Various concentrations of AuNPs were added into a BSA solutionwith a fixed concentration of 0.25 μM. Significant decrease in fluores-cence intensity was observed upon the addition of both types of AuNPs(Figs. 3A and B). No fluorescence signal was observed in samplescontaining AuNPs alone. A blue shift from 345 to 320 nm in the emis-sion peak was observed in the presence of increasing concentrations ofAuNPs (Fig. 3D). This potentially suggests a reduction of polarity andan increase of hydrophobicity of the local microenvironment aroundTrp [9]. This is anticipated as both SRNPs and QSNPs were coated byCTAC ligands with long hydrocarbon chains. Upon adsorption, the localhydrophobic microenvironment of BSA would be enhanced upon theirclose proximity to the hydrophobic CTAC ligands. In addition, a greaterblue-shift was observed for SRNP-BSA at the lowest concentration ofSRNPS (0.01 nM), which could be explained by the higher surface hy-drophobicity of SRNPs than QSNPs.

Furthermore, once the BSA adsorbed onto AuNPs, the intrinsicfluorescence of Trp accessible to the NP surfaces were constantlyquenched (Fig. 3C). At a concentration of AuNPs higher than ∼0.05 nM when the surface loading of BSA was lower and less crowded,the quenching effect was less significant for SRNPs than QSNPs(Fig. 3C). This is especially apparent at the highest concentration ofAuNPs (1.25 nM) when the BSA adsorbed onto QSNPs were almostquenched completely (Fig. 3A). Another notable phenomenon hap-pened at 0.31, 0.625, and 1.25 nM, BSA adsorbed onto SRNPs alsoexhibit a second peak at its intrinsic peak wavelength of 346 nm, in-dicating that the fluorescence of some of the BSA molecules might havenot been quenched (Figs. 3B and D). The efficiency of fluorescencequenching usually depends on the distance between the quencher andfluorophore [9,45,46]. To obtain a stable conformation, BSA (with aheart shape morphology, Figure S3 in Supporting Information) mayapproach NP surface with different domains and orientations [9]. Usingtheoretical simulations, Nandi et al. reported that human serum al-bumin (HSA) are capable of adjusting their orientations to geome-trically fit various NP curvatures [47]. It is likely that BSA adsorbedonto SRNPs in an orientation where both tryptophan residues weremoving farther away from NP surface and thus less quenched, when

Fig. 2. The change of hydrodynamic diameters (ΔDH, shown as column graphs)and ζ-potentials (shown as plots) of 0.125 nM AuNPs in the absence and pre-sence of BSA of various concentrations at pH 7.5. Error bars represent thevariations between triplicate experiments.

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compared to the ones adsorbed onto QSNPs.

3.4. Binding isotherm was nanoparticle-dependent

The heat profiles of titrating BSA with QSNPs and SRNPs are pre-sented in Fig. 4. In concert with the DLS results, more BSA was neededto saturate the surface of SRNPs than QSNPs (Table S1 in SupportingInformation). The trend of ΔH showed that the adsorption of BSA ontoQSNPs featured a negative ΔH (Fig. 4A) while the interaction of BSAwith SRNPs displayed a positive ΔH (Fig. 4B). Previous studies by De et.al provided a way to understand the thermodynamics between proteinsand NPs, in which the overall complexation process was proposed astwo simultaneous processes [16]. The first process, involving non-covalent complexation (e.g., electrostatic forces, van der Waals attrac-tion and hydrogen bonding) between NPs and BSA, is exothermic withnegative ΔH, whereas the second process of interfacial water re-organization and release is endothermic with positive ΔH [16].Therefore, whether the binding between AuNPs and BSA is exothermic(ΔH<0) or endothermic (ΔH>0) depends on which of the processmentioned above dominates over the interaction.

In this study, the adsorption of BSA onto QSNPs seemed to becontrolled by a concerted interplay of noncovalent forces. In contrast,the adsorption of BSA onto SRNPs appears to be an endothermic pro-cess, suggesting the re-organization of solvation shells upon the for-mation of SRNP-BSA complexes. The positive ΔH was partially offset bythe noncovalent forces. More importantly, the endothermic heat changewas associated with the displacement of ordered interfacial water

molecules surrounding the SRNPs and subsequent adsorption of BSA.The difference in the thermodynamics of AuNP-BSA interaction can berationalized by the greater surface hydrophobicity of SRNPs (Table 1).The adsorption of proteins onto more hydrophobic NPs usually releasedhigher amount of interfacial water and featured a more endothermicbinding process [16–18,48]. Thus, the greater hydrophobic interactionbetween SRNPs and BSA could be the driving force for the endothermicheat changes measured. The results here provided valuable thermo-dynamics clues required for various applications (e.g., effective pho-tothermal heating to induce drug release from NPs) [49–52].

4. Conclusion

Our findings indicated that the atomic coordination on AuNP sur-faces, which is intimately tied to the local surface curvature, greatlyinfluenced the conformation of adsorbed BSA. SRNPs with high abun-dance of undercoordinated Au atoms on their highly-curved surfacespossess higher tendency to denature BSA than the thermodynamicallystable, multifaceted QSNPs. We found that the aggregation of AuNP-BSA complexes to be concentration-dependent and the aggregationappears to be caused by denatured BSA and/or reduced electrostaticrepulsion between complexes. SRNPs are capable of accommodating agreater amount of BSA due to their greater specific surface areas andsurface hydrophobicity. Along with the blue-shifted peak, a less sig-nificant intrinsic fluorescence quenching effect was observed for BSA-SRNPs complexes than BSA-QSNPs ones. This can potentially be at-tributed to differences in binding orientation when BSA approaches the

Fig. 3. Fluorescence emission spectra of QSNP-BSA (A) and SRNP-BSA (B) and plots describing fluorescence quenching effect, F0/F (C) and peak positions (D) withincreasing concentrations of AuNPs at pH 7.5.

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NP surfaces. The interaction between QSNPs and BSA featured anexothermic process involving the interplay of noncovalent forces. Incontrast, the adsorption of BSA onto SRNPs exhibited an endothermicprocess, possibly by their greater surface hydrophobicity. Our studydemonstrated the importance of surface atomic coordination and sur-face curvature in determining NP-BSA interactions. It also providesinsights to the design and implementation of novel nanomaterials withtunable impacts on protein adsorption in biomedical applications.

CRediT authorship contribution statement

Zehui Xia: Methodology, Investigation, Data curation, Formalanalysis, Writing - original draft, Visualization. Hui Wang: Writing -review & editing, Supervision, Funding acquisition. Boris L.T. Lau:Conceptualization, Writing - review & editing, Supervision, Projectadministration, Funding acquisition.

Declaration of Competing Interest

There are no conflicts to declare.

Acknowledgements

This research was financially supported by the US National ScienceFoundation (CBET 1454443 to B.L.T. L.) and (DMR-1253231 to H.W.).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.colsurfb.2020.110960.

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Supporting Information 1 

2 

Nanoscale Surface Curvature Modulates Nanoparticle-Protein Interactions 3 

4 

Zehui Xiaa, Esteban Villarrealb, Hui Wangb, and Boris L. T. Laua,* 5 

6 

aDepartment of Civil & Environmental Engineering, University of Massachusetts Amherst, 130 7 

Natural Resources Road, Amherst, MA 01003, USA 8 

bDepartment of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, 9 

Columbia, SC 29208, USA 10 

11 

* Correspondence: Boris L. T. Lau 12 

E-mail: borislau@umass.edu 13 

   14 

 15 

Figure S1. SEM images of QSNPs (A) and SRNPs (B). 16 

17 

18 

Figure S2. Time-resolved hydrodynamic diameter changes, ∆DH, of 0.125 nM QSNPs (A) and 19 

SRNPs (B) under different BSA concentrations at pH 7.5. The data shown in Figure 2 are related 20 

to the ∆DH at approximately t=10 minutes. 21 

22 

23 

Figure S3. Three-dimensional structure of BSA (Adapted from Prasanth,  S.;    Rithesh  Raj,  D.;  24 Vineeshkumar, T. V.;  Thomas, R. K.; Sudarsanakumar, C., Exploring the interaction of l‐cysteine capped 25 CuS nanoparticles with bovine serum albumin (BSA): a spectroscopic study. RSC Advances 2016, 6 (63), 26 

58288‐58295). Different domains and two tryptophan residues are shown in the figure. 27  28 

29 

Estimation of BSA surface coverage on a single AuNP surface 30 

Both theoretical estimate and experimental data suggested that SRNPs can accommodate more 31 

BSA molecules per NP than QSNPs. To obtain a stable conformation, BSA (with a heart shape 32 

morphology and dimensions of ~ 4×4×9 nm, Figure S3 in Supporting Information) may approach 33 

NP surface with different domains and orientations (e.g., end-on and/or side-on orientations). 34 

Table S1. Comparison of number of BSA molecules per AuNP as determined by different 35 

methods 36 

QSNP SRNP

Surface Area 66,392 nm2 335,107 nm2

BSA Surface Coverage (theoretical, side-on orientation) 1,844 9,308

BSA Surface Coverage (theoretical, end-on orientation)η 4,149 20,944

BSA Surface Coverage (experimental) 10 1,500

This estimation is based on a spherical morphology and a particle diameter of 146.9nm. 37 

The surface area of a SRNP was estimated to be 4.5 times of that of a spherical NP with a 38 

diameter of 153.4 nm.1 39 

For a side-on orientation, a BSA molecule occupies 36 nm2 cross-sectional surface area. 40 

η For an end-on orientation, a BSA molecule occupies 16 nm2 cross-sectional surface area. 41 

Based on ITC results. 42 

 43 

Reference 44 

1. E. Villarreal, G. G. Li, Q. Zhang, X. Fu and H. Wang, Nano. Lett., 2017, 17, 4443-4452. 45 

46