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BBA - Biomembranes

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Nanoparticles modulate membrane interactions of human Islet amyloidpolypeptide (hIAPP)☆

Yossef Peretza, Ravit Malisheva, Sofiya Kolushevab, Raz Jelineka,b,⁎

a Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israelb Ilse Katz Institute for Nanotechnology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel

A R T I C L E I N F O

Keywords:Human Islet amyloid polypeptideNanoparticlesAmyloid fibrilsNanoparticle-amyloid interactions

A B S T R A C T

The dramatic expansion of nanotechnology applications, particularly the advent of nanomaterials and nano-particles (NPs) into the consumer economy, have led to heightened awareness of their potential health risks. Thisstudy examines the impact of several NPs upon membrane-induced aggregation and bilayer interactions of thehuman Islet amyloid polypeptide (hIAPP). We report that several NPs – polymeric NPs, TiO2 NPs, and Au NPsdisplaying coating layers exhibiting different electrostatic charges - did not significantly interfere with the fi-brillation process and fibril morphology of hIAPP, both in buffer or in biomimetic DMPC:DMPG vesicle solutions.Spectroscopic and microscopic analyses suggest, in fact, that the NPs promoted membrane-induced fibrillation.Importantly, we find that all the NPs examined, regardless of composition or surface properties, gave rise tomore pronounced, synergistic bilayer interactions when co-incubated with hIAPP. NP-enhanced bilayer inter-actions of hIAPP might point to possible toxicity and pathogenicity risks of amyloidogenic peptides in thepresence of NPs.

1. Introduction

Misfolding and aggregation of soluble proteins are hallmarks indiverse, devastating disorders, including Alzheimer's disease (AD),Parkinson's disease, and type-II diabetes [1]. Though fibrillar ag-gregates appear to be a universal marker in amyloid diseases, the re-lationship between fibril assembly, disease initiation and progression,and pathogenicity remains unclear. Most amyloid peptides aggregateinto fibrils of cross β-sheet structure through different mechanismswhich generally consist of nucleation, oligomer/protofibril assembly,and ultimately formation of plaques comprising the mature fibrils [2,3].Membranes are believed to intimately participate in protein misfoldingand aggregation processes. Indeed, membranes and membrane inter-actions appear to mediate amyloidogenic proteins toxicity throughputative pore formation or other bilayer disruption events [4–8]. Fur-thermore, the membrane interface might constitute a platform for in-itiation and modulation of the fibrillation processes [5,8–10].

The human Islet amyloid polypeptide (hIAPP, also known asamylin) is a well-known amyloidogenic peptide, associated with type-IIdiabetes. hIAPP is a 37-residue hormone secreted in pancreatic β-cells[11,12]. For yet unknown reasons, in patients afflicted with type 2diabetes, hIAPP undergoes conformational transformations from a

random coil monomer, through an α-helix transient, and into cross-β-sheet fibrillar plaques which accumulate on the β-cells, severely dis-rupting their functionalities and ultimately affecting cell death [12].Membrane interactions of hIAPP, particularly involving peptide oligo-mers and proto-fibrils, have been shown to play important roles in theaggregation route of the peptide [12–17]. hIAPP pre-fibrillar specieshave been also shown to affect significant perturbations of bilayerstructure and dynamic properties [6,13,17–20]. hIAPP fibrillation andmembrane interactions have been shown to occur almost simulta-neously by different peptide domains [21].

hIAPP fibrillation, in particular, can be modulated by a wide varietyof compounds. A number of natural organic molecules, specificallypolyphenols such as resveratrol and epigallo-catechin gallate, haveshown inhibitory effects [22,23]. Short peptides, either de novo de-signed or derived from existing amyloidogenic proteins, were also de-monstrated to be effective amyloid inhibitors [24]. Insulin, for example,a peptide which is co-secreted with hIAPP in β-cells, was found to in-hibit fibril formation in vitro and block amyloid-related membrane in-teractions [25,26]. Metal ions such as calcium also affect hIAPP fi-brillation and hIAPP-bilayer interactions and [16,27]. Similarly, zincions modulate aggregation through binding to His-18 and concomitantdisruption of the peptide's secondary structure [28–30].

https://doi.org/10.1016/j.bbamem.2018.03.029Received 31 January 2018; Received in revised form 27 March 2018; Accepted 29 March 2018

☆ This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.⁎ Corresponding author at: Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.E-mail address: razj@bgu.ac.il (R. Jelinek).

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0005-2736/ © 2018 Elsevier B.V. All rights reserved.

Please cite this article as: Peretz, Y., BBA - Biomembranes (2018), https://doi.org/10.1016/j.bbamem.2018.03.029

The relationship between nanomaterials and amyloidogenic pro-teins has attracted significant research interest due to the increasing useof such materials in technologies and products. Nanoparticles (NPs), inparticular, are a cause of concern since their small dimensions facilitatepenetration through physiological barriers and cellular membranes[31–34]. NPs might affect amyloid cytotoxicity through interference indifferent stages of amyloid fibrillation pathways [35]. NPs exhibit highsurface area that can adsorb amyloid protein monomers, thereby re-ducing their concentration in solution. Also, binding onto NPs mightaffect structural transformations of soluble proteins, thereby interferingwith misfolding pathways [36–38]. On the other hand, NPs might serveas nucleation sites, leading to acceleration of amyloid fibril formation.Au nanoparticles both inhibited and accelerated fibril formation, de-pending on the type of aggregating peptide and coating layer [39–43].For example, fullerenes [44], inorganic quantum dots [45], silver na-noparticles [46] and others [47–51], inhibited amyloid formation. Onthe other hand, TiO2 NPs [52] or polymeric NPs comprising polystyreneor N-isopropylacrylamide/N-tert-butylacrylamide copolymer wereshown to promote fibril formation by reducing the aggregation lag timethereby increasing fibril abundance [36,47].

It should be emphasized that almost all previous studies addressingNP effects upon protein misfolding and aggregation have not con-sidered the role of the membrane in affecting NP-amyloid peptide in-teractions and the aggregation processes. This issue is of paramountimportance in light of the insidious role of the cell membrane in variedprotein fibrillation routes. Accordingly, this work examines the mutualinteractions between hIAPP, biomimetic membrane vesicles, and dif-ferent types of nanoparticles. The NPs selected represent varied com-positions and particle surface properties, particularly electrostaticcharge. The experiments carried out demonstrate that almost all NPsremodeled membrane-associated fibrillation of hIAPP. Furthermore, weshow that the NPs contributed to enhanced bilayer interactions of thepeptide, regardless of composition or surface properties, illuminating asignificant and largely overlooked potential biological risk factor.

2. Materials and methods

2.1. Materials

Human IAPP (amylin, human) was purchased from Anaspec (UnitedStates), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-di-myristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG), were pur-chased from Avanti Polar Lipids (AL). Thioflavin T (ThT), 1,1,1,3,3,3-hexafluoro-2-propanol, sodium hydrosulfite, sodium phosphate mono-basic, and TiO2 nanopowder (20 nm mean diameter) were purchasedfrom Sigma-Aldrich (Rehovot, Israel). Gold nanoparticles (30 nm dia-meter) coated with Thiol Polyethyleneglycol Carboxylic acid(Mw=5 k), Thiol Polyethyleneglycol Amine (Mw=5 k) and ThiolPolyethyleneglycol Carboxylic acid:Amine (Mw=5 k) 1:1mol ratiowere obtained from Nanopartz Inc. (United States), amine modifiedpolystyrene (60 nm diameter) was bought from Weiss Scientific, Ltd.(Timrot, Israel). 1-(4-Trimethylam- moniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was obtained from Molecular Probes, Inc.(United States).

2.2. Peptide sample preparation

hIAPP was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) ata concentration of 0.5 mg/mL and stored at −20 °C until use to preventaggregation. For each experiment, the solution was thawed, and therequired amount was dried by evaporation for 6–7 h to remove theHFIP. The dried peptide sample was dissolved in 10mM phosphatebuffer (pH 7.4).

2.3. Vesicle preparation

Vesicles consisting of DMPC:DMPG were prepared by dissolving thelipid components in chloroform/ethanol (1:1, v/v) and drying togetherin vacuo. Small unilamellar vesicles (SUVs; DMPC:DMPG 0.5:0.5 molratio) were prepared in 10mM phosphate buffer (pH 7.4) by probe-sonication of the aqueous lipid mixtures at room temperature for10min. Vesicle suspensions were incubated for 1 h at room temperatureprior to usage.

2.4. Thioflavin T (ThT) fluorescence assay

ThT fluorescence measurements were taken at 25 °C using 96-wellblack plate on a Biotek Synergy H1 plate reader. Measurements weremade on a samples containing 15 μM IAPP in the absence or presence ofPS (7.3 nM), TiO2 (1 μg/mL) and Au(−), Au(± ), Au(+) (1.5 nM), andin the absence or presence of lipid vesicles (final concentration0.466mM). A 150-μL aliquot of the aggregation reaction was mixedwith 10 μM ThT in 10mM phosphate buffer (pH 7.4). The fluorescenceintensity was measured every 5min for 24 h at λex=440 andλem=485 nm. Results are presented as means ± SD.

2.5. Transmission electron microscopy (TEM)

5 μL aliquots from samples used in the ThT experiments (after 24 hincubation) were placed on 400-mesh copper grids covered with acarbon-stabilized Formvar film. Excess solutions were removed fol-lowing 2min of incubation, and the grids were negatively stained for30 s with a 1% uranyl acetate solution. Samples were viewed in an FEITecnai 12 TWIN TEM operating at 120 kV.

2.6. Circular dichroism spectroscopy

CD spectra were recorded in the range of 190–260 nm at roomtemperature on a Jasco J-715 spectropo-larimeter, using 1-mm quartzcuvettes. Solutions composed of 350 μL contained 15 μM IAPP in theabsence or presence of PS (7.3 nM), TiO2 (1 μg/mL) and Au(−), Au(± ), Au(+) (1.5 nM), and in the absence or presence of lipid vesicles(final concentration 0.466mM). Spectra were recorded at t= 10min.Spectra were collected at a temperature of 25 °C. Cuvette path lengthwas 1mm. CD signals resulting from vesicles, buffer and the nano-particles were subtracted from the corresponding spectra.

2.7. Fluorescence anisotropy

The fluorescence probe TMA-DPH was incorporated into the SUVsDMPC:DMPG (0.5:0.5 mol ratio) by adding the dye dissolved in THF(1mM) to vesicles up to a final concentration of 0.6 μM. After 30min ofincubation at 28 °C of TMA-DPH, fluorescence anisotropy was measuredat λex=360 nm and λem=430 nm using a Flourolog spectro-fluorimeter. Anisotropy values were collected before and after the ad-dition of the IAPP, nanoparticles, or their mixtures solution. Anisotropyvalues were automatically calculated by the spectrofluorimeter soft-ware using the equation: r= (IVV−GIVV)/(IVV+ 2GIVH). G= IVH/IHH,in which IVV is the intensity with excitation and emission polarizersmounted vertically; IHH corresponds to the excitation and emissionpolarizers mounted horizontally; IHV is the excitation polarizer hor-izontal and the emission polarizer vertical; IVH pertains to the excitationpolarizer vertical and emission polarizer horizontal. All measurementswere at a temperature of 25 °C. Results are presented as means ± SEM.

3. Results and discussion

3.1. Experimental approach

Fig. 1 outlines the objective of this study and experimental design.

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hIAPP undergoes enhanced fibrillation in the presence of negatively-charged lipid bilayers [6,53] (Fig. 1A); furthermore, hIAPP-membraneinteractions, particularly involving pre-fibrillary and oligomeric spe-cies, have been implicated in the cytotoxicity of the peptide and itspathophysiological profile [13]. The scheme in Fig. 1B depicts the ex-periments carried out. Different nanoparticles (NPs) were pre-incubatedwith hIAPP monomers, and the NP/hIAPP mixtures were added to ve-sicles comprising zwitterionic phospholipids (dimyristoylpho-sphatidylcholine, DMPC) and anionic phospholipids (dimyristoylpho-sphatidylglycerol, DMPG), designed to mimic the environment of thecell membrane [54–57]. The experiments carried out (Figs. 2–5) both

assessed the effects of the NPs upon the bilayer-associated aggregationprocess of hIAPP, as well as interrogated membrane interactions of thehIAPP species in the peptide/NP mixtures. Evaluation of the impact ofNPs upon the reciprocal processes of peptide aggregation and mem-brane disruption is important, since the mutual interactions of NPs,membranes, and peptides constitute the actual scenario in physiologicalenvironments.

The NPs employed in this study are outlined in Fig. 1C, representingdifferent NP families and surface properties (electron microscopyimages of the NPs are presented in Fig. 1, SI). Specifically, we examinedAmine-modified polystyrene NPs (PS NPs) exhibiting approximately

Fig. 1. Experimental scheme. A. Fibrillation of hIAPP is enhanced in the presence of lipid bilayers mimicking cellular membranes; B. the presented experiments aredesigned to probe the effect of different NPs upon aggregation of hIAPP and its membrane interactions. C. Schematic representation of nanoparticles employed in thisstudy: Amine-modified polystyrene (PS) NPs, TiO2 NPs, and Au NPs functionalized with negative, positive and zwitterionic coating layers, respectively.

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60 nm diameter, TiO2 NPs (20 nm diameter), and Au NPs coated with anegatively-charged carboxyl-functionalized surfactant [Au(−) NPs], apositively-charged amine-functionalized surfactant [Au(+) NPs], orzwitterionic Au NPs functionalized with both carboxyl and amine sur-factants in a 1:1mol ratio [Au(± ) NPs]. The three Au NP species ex-hibited similar sizes, approximately 30 nm in diameter (Fig. 1, SI). In-teractions of Au NPs exhibiting different surface properties with lipidbilayers have been studied [31,34,58]; interestingly, distinct effects ofAu NPs coated with different surface functionalization upon variety of

amyloid peptides were discerned [40,41,59,60]. PS NPs and TiO2 NPshave been also shown to modulate amyloid fibrillation [36,52]. Themorphologies and surface properties of the NPs were characterized byTEM and ζ-potential, respectively. ζ-potential measurements, summar-ized in Table 1, SI, reveal that the surface of PS NPs is highly positivelycharged. TiO2 NPs and Au(+) NPs, on the other hand, exhibited ne-gative ζ-potential which is likely due to a solvation layer within thebuffer. The Au(−) and Au(± ) NPs both had close to neutral surfacecharges, which is consistent with their surface modifications.

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Fig. 3. Transmission electron microscopy (TEM) images of hIAPP and NPs in buffer (A), and in the presence of DMPC:DMPG vesicles (B). hIAPP concentration was15 μM; NP concentrations were 7.24 nM (PS); 1 μg/mL (TiO2); 1.5 nM (the three Au NPs). Scale bars correspond to 100 nm.

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3.2. Effects of NPs upon hIAPP aggregation

Fig. 2 presents thioflavin-T (ThT) fluorescence curves illuminatingthe aggregation kinetics of hIAPP, and the effects of lipid bilayers andNPs upon peptide aggregation. ThT fluorescence has been widely usedfor monitoring protein aggregation, as the fluorescence emission of theThT dye increases upon incorporation into β-sheet protein fibril as-semblies [61]. The ThT fluorescence curves in Fig. 2 demonstrate thatboth DMPC:DMPG vesicles (size approximately 60 nm, Fig. 2, SI) aswell as the NPs significantly modulated hIAPP aggregation kinetics.Specifically, while hIAPP exhibited little aggregation in buffer (Fig. 2A,black curve), PS NPs, TiO2 NPs, and the zwitterionic Au NPs [Au(± )NPs], gave rise to higher and faster increase of the ThT fluorescence,likely reflecting more pronounced hIAPP fibrillation (Fig. 2A). Bothnegatively-charged Au(−) NPs and positively-charged Au(+) NPs ap-pear to have effectively inhibited hIAPP aggregation, accounting for thenegligible ThT fluorescence recorded over time (Fig. 2A, purple andorange curves, respectively).

Modulation of hIAPP aggregation as reflected in the ThT fluores-cence curves was apparent in the presence of DMPC:DMPG vesicles(Fig. 2B). hIAPP alone gave rise to higher ThT fluorescence in thepresence of lipid vesicles compared to the buffer solution (Fig. 2B, blackcurve), corresponding to bilayer-promoted fibrillation [62,63]. Echoingthe ThT fluorescence data in buffer, both TiO2 NPs and PS NPs ac-celerated hIAPP fibril assembly in the DMPC:DMPG vesicle solutionscompared to the peptide alone (Fig. 2B). Notably, the zwitterionic Au

NPs appear to have inhibited hIAPP aggregation in the presence ofDMPC:DMPG vesicles (Fig. 2B, blue curve) – different than the apparentfibrillation enhancement recorded in buffer (Fig. 2A, blue curve). Fig. 2Balso reveals that both positive and zwitterionic Au NPs seem to havereduced hIAPP fibrillation in the presence of lipid vesicles, giving rise tolower ThT fluorescence compared to the peptide alone (Fig. 2B).

The intriguing “switch” from acceleration to inhibition of hIAPPfibrillation in lipid bilayer environments likely reflect Au(± ) interac-tions with the negatively charged headgroups of DMPG. Indeed, in thepresence of lipid bilayers, AuNPs exhibited increasing inhibition ac-tivity as the degree of positive charges on the NP surface was morepronounced, suggesting that Au NP/bilayer binding “screens” hIAPPinteractions with the bilayer. A subtler change of fibril abundance isobserved for PS NPs which possess much higher positive ζ-potential(Table 1, SI). These data might indicate NP-Membrane interactionswhich reflect a balance between van der Waals (vdW) interactions andelectrostatic attraction. Overall, the ThT fluorescence data in Fig. 2point to significant modulation of hIAPP fibrillation pathways by theNPs, both in buffer solutions and in the presence of the biomimeticmembranes.

The transmission electron microscopy (TEM) images in Fig. 3complement the ThT analysis and provide a visual depiction of the ef-fects of the NPs upon hIAPP fibril morphology. In particular, the TEMimages recorded for hIAPP fibrils in DMPC:DMPG vesicles (Fig. 3B)attest to the enhanced fibril formation affected by the biomimeticmembranes. Note also the higher abundance of hIAPP fibrils observedin the vesicle solutions upon pre-incubation with both PS NPs and TiO2

NPs. The inhibition of hIAPP fiber formation in case of Au(+) NPs(Fig. 3B), echoing the ThT fluorescence data in Fig. 2B [a seemingdiscrepancy is the observation in the TEM image of hIAPP fibrils in caseof Au(+) addition in buffer, Fig. 3A, which appears to contrast thenegligible ThT signal, Fig. 2A; however we noticed that significantagglomeration occurred in that sample, possibly accounting for the lackof ThT fluorescence signal].

A notable result in the TEM images in Fig. 3 is the association of theNPs with the hIAPP fibrils assembled, further corroborating the ThTfluorescence data and pointing to interactions of the NPs with hIAPPthroughout the fibril assembly process. It should be emphasized that theTEM analysis indicates that the overall hIAPP fibril morphology in thepresence of membranes was not disrupted by co-incubation with theNPs, suggesting that the particles did not interfere with the basic con-tours of membrane-associated hIAPP fibrillation.

To further illuminate the effects of the different NPs upon thestructural transformations of hIAPP occurring during fibril formationwe carried out circular dichroism (CD) spectroscopy (Fig. 4). CD

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Fig. 4. Secondary structure of hIAPP co-incubated with NPs. A. Buffer; B. DMPC:DMPG vesicle solution. hIAPP concentration was 15 μM; NP concentrations were7.24 nM (PS NPs); 1 μg/mL (TiO2 NPs); 1.5 nM (the three Au NPs). Temperature was 25C.

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Fig. 5. Effects of NPs upon hIAPP-modified bilayer fluidity. Fluorescence ani-sotropy of TMA-DPH:DMPC:DMPG (0.004:1:1)) following addition of hIAPPand NPs. Temperature was 25 °C.(For interpretation of the references to color inthis figure, the reader is referred to the web version of this article.)

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spectroscopy has been previously employed to monitor the conforma-tional transformations of amyloidogenic peptides in general, hIAPP inparticular, occurring throughout fibril formation processes [64,65]. TheCD signatures in Fig. 4, recorded approximately 10min after dissolutionof hIAPP, alone or together with the NPs, suggest that the NPs did notnoticeably alter the conformation of the peptide. This result is apparentboth in buffer in which hIAPP existed in a largely random structure(Fig. 4A), and in the DMPC:DMPG vesicle solution in which hIAPPadopted an α-helical conformation, an intermediate structure prior toits transformation into β-sheet-comprising fibrils [12,66,67]. Im-portantly, the CD traces of hIAPP recorded in the vesicle solutions in-dicate that the NPs tested, with the exception of Au(+) NPs, somewhatenhanced the extent of helical hIAPP conformations (e.g. lower minimaat around 208 nm and 222 nm, Fig. 4B).

The CD data further illuminate the NP effects upon bilayer-asso-ciated structural transformations and fibrillation of hIAPP. The pro-motion of helical content of hIAPP in case of the PS NPs and TiO2 NPs isconsistent with both the ThT fluorescence experiment (Fig. 2) and TEMdata (Fig. 3) which attest to enhanced bilayer-induced fibrillation ofhIAPP induced by these NPs. Somewhat divergent data were recordedin case of Au(+) NPs and Au(−) NPs. Notably, while according to theThT fluorescence experiment (Fig. 2), both Au(+) NPs and Au(−) NPsinhibited fibril growth in the presence of lipid vesicles, the CD analysissuggests that Au(−) NPs gave rise to somewhat more pronounced he-lical content of the peptide, while Au(+) NPs appears to have had anopposite effect. This result indicates that the two NPs might conferdifferent fibril remodeling pathways in the presence of the DMPC/DMPG vesicles. Similar CD data were recorded after much longer in-cubation (48 h, Fig. 3, SI).

3.3. Effect of the NPs upon hIAPP/membrane interactions

While Figs. 2–4 interrogated the effects of the NPs upon bilayer-associated aggregation and fibril formation of hIAPP, we further aimedto evaluate whether the NPs also affected membrane interactions ofhIAPP. Previous studies have shown that hIAPP, particularly oligomericand pre-fibrillar species of the peptide, experience significant interac-tions with lipid bilayers comprising anionic phospholipids [6,13,53].Fig. 5 presents a fluorescence anisotropy experiment designed to assessthe impact of the NPs upon bilayer properties, particularly bilayerfluidity. The anisotropy of fluorescence dyes embedded in lipid bilayersconstitutes a useful vehicle for evaluation of dynamical properties oflipid bilayers [68,69].

The fluorescence anisotropy experiment in Fig. 5 utilizedDMPC:DMPG vesicles which further comprised 1-(4-Trimethylam-moniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) at a mole ratioof 0.004:1:1 (TMA-DPH:DMPC:DMPG). In these vesicles, the DPHfluorescent dye is localized inside the bilayer and close to the lipidheadgroup region, and its fluorescence anisotropy can be employed tomonitor lipid mobility close to the dye [70,71]. Fig. 5 shows that hIAPPinduced higher fluorescence anisotropy (Fig. 5, grey bars), consistentwith stiffening of the bilayer headgroup area ascribed to formation ofaggregate “carpeting” at the bilayer interface [72–74]. Fig. 5 furtherpoints to significant effects of co-addition of NPs to hIAPP prior to in-cubation with the TMA-DPH:DMPC:DMPG vesicles. While PS NPs in-duced bilayer rigidity similar to hIAPP, likely reflecting the strong at-tachment of the positively-charged NPs onto the anionic headgroups atthe bilayer interface, all other NPs did not noticeably alter the fluor-escence anisotropy of the DPH dye (Fig. 5, red bars).

Remarkably, however, Fig. 5 reveals that when hIAPP and each ofthe NPs were added together to the TMA-DPH:DMPC:DMPG vesicles,experimentally-significant synergistic effects were apparent (Fig. 5,orange bars). Specifically, higher fluorescence anisotropy values wereinduced compared to the effects of the individual constituents (hIAPPor NPs alone). Importantly, the synergistic, enhanced bilayer rigiditywas apparent in case of all NPs examined, suggesting that the effect is

intrinsic to NPs regardless of the NP composition or NP surface prop-erties. The synergistically-enhanced bilayer interactions induced by theNP-hIAPP mixtures are consistent with the spectroscopic and micro-scopic data presented in Figs. 2–4, suggesting that promotion of fi-brillation pathways of hIAPP upon addition of the NPs are linked withbilayer interactions of the NPs.

The experimental data presented in Figs. 2–5 point to several factorspertaining to NP properties, likely exhibiting important roles in mod-ulating hIAPP/membrane interactions. Notably, none of the NPs ex-amined seemed to affect the secondary structure transformations ofhIAPP (Fig. 4). NPs' surface charge is an important determinant for thebilayer/NP/hIAPP interaction. Indeed, the synergistic bilayer interac-tion observed for NP/hIAPP mixtures (Fig. 5) was diminished when theexperiment was carried out in 0.5 M NaCl buffer solution (Fig. 4, SI).hIAPP is positively charged hence the interactions between these po-sitive NPs and hIAPP are likely not electrostatic in nature. This sy-nergistic effect can be explained by hIAPP adsorption by NPs and mi-gration of the peptide coated NPs to the bilayer due to electrostaticattraction between hIAPP and the lipid bilayer. In comparison, NP/hIAPP interactions and resulting modulation of hIAPP fibrillation byNPs appear not to be directly correlated to the electric charge of theNPs. PS, for example, gave rise to acceleration of hIAPP fibril formation,while Au(+) showed mild inhibition even though both NPs exhibitsubstantial positive surface charges.

Au(−), PS and TiO2 showed similar effects upon hIAPP aggregationin buffer and also in the vesicle solution, despite the fact that in-troduction of lipid vesicles likely affected NP/hIAPP fibrillation path-ways. Specifically, in both solutions PS and TiO2 appeared to acceleratefibrillation while Au(−) retarded fibril formation. This observationsuggests that affinity of these three NP species to hIAPP is more pre-dominant compared to their membrane interactions. In the case of Au(+) NPs and Au (± ) NPs, attraction of the positive moieties upon theAu NP surface and the negative headgroups at the membrane surface“shielded” the bilayer, inhibiting both hIAPP/bilayer interactions andbilayer-induced fibrillation, consistent with previous findings [16,75].Altogether, our data point to NP/hIAPP interactions, NP/amyloidpeptide interactions in general, as a central factor affecting membraneinteraction of the peptides. This realization might have significant im-plications concerning possible toxicity induced by varied NP families,related to amyloidogenic proteins and peptides.

4. Conclusions

This work focuses on the rarely-studied interplay between nano-particles (NPs), amyloidogenic peptides, and membrane bilayers; inparticular, we investigated the impact of several NPs upon membrane-associated fibrillation and membrane interactions of the human Isletamyloid polypeptide (hIAPP). This study reveals that nanoparticlesexhibiting varying compositions and surface properties modulate fi-brillation pathways of hIAPP and contribute to enhanced membraneinteractions of the peptide. Specifically, spectroscopy and microscopyexperiments indicated that polymeric (polystyrene) NPs, TiO2 NPs,zwitterionic Au NPs and negatively-charged Au NPs accelerated tran-sient helical conformation of hIAPP and enhanced peptide fibrillation inthe presence of DMPC:DMPG vesicles. Positively-charged Au NPs, onthe other hand, appeared to reduce fibrillation of hIAPP, possiblythrough competing interactions with the negative vesicles. An im-portant observation was the synergistic effect of all NPs examined uponbilayer interactions of hIAPP; this result might point to higher toxicityinduced by NPs in conjunction with hIAPP in particular, amyloid pep-tides in general. This work highlights the significance of studying therelationship and interactions between nanoparticles and amyloid pep-tides in membrane environments, reflecting the actual physiologicalenvironments.

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Conflict of interest

The authors declare no conflict of interest.

Transparency document

The http://dx.doi.org/10.1016/j.bbamem.2018.03.029 associatedthis article can be found, in online version.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbamem.2018.03.029.

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