Inhibition of IAPP and IAPP(20−29) Fibrillation by Polymeric

Dec 17, 2009 - The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20−29)) was studied by means of Thioflavin T ...
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Inhibition of IAPP and IAPP(20-29) Fibrillation by Polymeric Nanoparticles C. Cabaleiro-Lago,*,† I. Lynch,† K. A. Dawson,† and S. Linse*,‡ †

Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland and ‡Biochemistry Department, Lund University, P.O. Box 124, 22100 Lund, Sweden Received August 12, 2009. Revised Manuscript Received November 7, 2009 The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20-29)) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation as a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied from 50:50 to 100:0. The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP(20-29) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed as a delayed onset of ThT fluorescence increase, plus a reduction of the amount of fibrils formed as indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP(20-29)), the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.

Introduction The formation of amyloid aggregates by association of peptides into ordered structures is a hallmark of many serious human disorders.1 Amyloid fibers can also have a functional role in bacteria, fungi, insects, invertebrates, and humans.2-4 Amyloid forming proteins do not show any striking similarity, either in sequence or in their secondary or tertiary structure, yet all aggregate into a common core structure, the amyloid fibril.5 In many cases, the exposure of a protein to nanoparticles will cause adsorption of the protein onto the particle surface. Increased local protein concentration on the nanoparticle surface and/or changes in protein conformation upon binding could promote aggregation while trapping of early intermediates (oligomers) may inhibit further aggregation.6-9 Exploring the effect of specific nanoparticles on the formation of amyloid fibrils may contribute toward a mechanistic understanding of the aggregation processes, and may open the way to designing nanoparticles that modulate the formation of toxic amyloid species. *To whom correspondence should be addressed. (S.L.) Address: Biochemistry Department, Lund University, P.O. Box 124, 22100 Lund, Sweden. Telephone/Fax: þ46-46-222 8246/4543. E-mail: Sara.Linse@biochemistry. lu.se. (C.C.-L.) Present address: Departamento de Quı´ mica Fı´ sica, Facultade de Quı´ micas, Universidade de Vigo, Lagoas-Marcosende s/n, 36310 Vigo, Spain. Telephone: þ34 986 818617. E-mail: [email protected]. (1) Serpell, L. C.; Sunde, M.; Blake, C. C. F. Cell. Mol. Life Sci. 1997, 53 (11-12), 871–887. (2) Fowler, D. M.; Koulov, A. V.; Balch, W. E.; Kelly, J. W. Trends Biochem. Sci. 2007, 32, 217–224. (3) Mostaert, A. S.; Higgins, M. J.; Fukuma, T.; Rindi, F.; Jarvis, S. P. J. Biol. Phys. 2006, 32, 393–401. (4) Gebbink, M.; Claessen, D.; Bouma, B.; Dijkhuizen, L.; Wosten, H. A. B. Nat. Rev. Microbiol. 2005, 3(4), 333–341. (5) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. F. J. Mol. Biol. 1997, 273(3), 729–739. (6) Giacomelli, C. E.; Norde, W. Biomacromolecules 2003, 4(6), 1719–1726. (7) Giacomelli, C. E.; Norde, W. Macromol. Biosci. 2005, 5(5), 401–407. (8) Rocha, S.; Thuneman, A. F.; Pereira, M. D.; Coelho, M.; Mohwald, H.; Brezesinski, G. Biophys. Chem. 2008, 137(1), 35–42. (9) Gazit, E. Curr. Med. Chem. 2002, 9(19), 1725–1735.

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Our group has recently reported intriguing effects of copolymeric nanoparticles on the fribrillation process of the amyloidogenic proteins β2-microglobulin (β2m)10 and amyloid β peptide (Aβ).11 These proteins, with very different sequence and fold structures, react to the presence of nanoparticles in a divergent way. We found that the presence of nanoparticles leads, in the case of the β2m, rich in β-sheets, to an acceleration of the fibrillation process by shortening the lag phase. On the contrary, the addition of nanoparticles slows down the formation of fibrils of Aβ, which is mostly unfolded as a monomer. Moreover, the inhibitory effect is dependent on the surface characteristics of the nanoparticles, and for both proteins more hydrophobic particles have a smaller effect than less hydrophobic ones which have increased possibility to hydrogen bond with the proteins.10-12 The likely role of the nanoparticles in β2-microglobulin fibril formation is to bind protein monomers on the surface of the particles, thereby increasing the local protein concentration and the likelihood of formation of critical nuclei that will initiate the growth into fibrils.10 The proposed role of the nanoparticles in Aβ fibril formation is to disturb the monomer-critical nuclei equilibrium by trapping the monomer or prefibrillar oligomers onto the particle surface, thereby decreasing their solution concentration. As a result, the overall rate of fibrillation is affected. Similar effects have been found when fluorinated and hydrogenated particles are used. Fluorinated particles in particular stabilize Aβ by interacting with the hydrophobic core of the peptide and inducing short-range interactions that favor an R-helix conformation.8 In a (10) Linse, S.; Cabaleiro-Lago, C.; Xue, W. F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S. E.; Dawson, K. A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104(21), 8691–8696. (11) Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Lindman, S.; Minogue, A. M.; Thulin, E.; Walsh, D. M.; Dawson, K. A.; Linse, S. J. Am. Chem. Soc. 2008, 130(46), 15437–15443. (12) Lynch, I.; Blute, I. A.; Zhmud, B.; MacArtain, P.; Tosetto, M.; Allen, L. T.; Byrne, H. J.; Farrell, G. F.; Keenan, A. K.; Gallagher, W. M.; Dawson, K. A. Chem. Mater. 2005, 17(15), 3889–3898.

Published on Web 12/17/2009

DOI: 10.1021/la902980d

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Cabaleiro-Lago et al. Scheme 1

that, upon polymerization, the CdC bond is converted to a -C-C•- radical, such that the reaction propagates.

Materials and Methods Peptide. IAPP and IAPP(20-29) were supplied by Aldrich and

similar fashion, conformational changes that favor or stop the fibrillation process have been observed for silica and Teflon particles, respectivly.7 The islet amyloid polypeptide (IAPP) is a short peptide that undergoes aggregation to form amyloid fibrils and is the amyloid protein related to diabetes type II.13 Recent studies reveal that the most toxic species are small prefibrilar oligomers14-18 as was also found for other amyloid proteins.19-22 The IAPP sequence (KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY) contains 37 residues. Two residues are positively charged (underlined) at neutral or higher pH, and 14 are hydrophobic (bold). Thus, the peptide has hydrogen bonding capacity throughout the backbone and at 23 side chains. The aggregation-prone region of the peptide (residues 20-29) has been identified through comparison of IAPP variants from different species with variable amylodogenic propensity.14,23-25 A fragment comprising residues 20-29 of IAPP is able to form amyloid fibrils on its own but lacks the toxicity of the intact protein.14 To further understand the different mechanisms that can play a role in the interaction between amyloidogenic proteins and nanoparticles and the relation to the protein structure, we have studied the fibrillation process of the islet amyloid polypeptide (IAPP) and the 20-29 fragment (IAPP(20-29)) in the absence and presence of copolymeric N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) nanoparticles.26 These particles offer the possibility of modulating the chemical composition (surface expression) by variation of the ratio of the two constituent monomers (Scheme 1) and allows us to prepare nanoparticles of controlled size where the surface properties are changed systematically along the composition series.26 The hydrogen bonding groups of the side chains are more accessible the higher the NPAM fraction due to the less bulky isopropyl group. Note (13) Kahn, S. E.; Andrikopoulos, S.; Verchere, C. B. Diabetes 1999, 48(2), 241– 253. (14) Konarkowska, B.; Aitken, J. F.; Kistler, J.; Zhang, S. P.; Cooper, G. J. S. FEBS J. 2006, 273(15), 3614–3624. (15) Westermark, P.; Engstrom, U.; Johnson, K. H.; Westermark, G. T.; Betsholtz, C. Proc. Natl. Acad. Sci. U.S.A. 1990, 87(13), 5036–5040. (16) Jayasinghe, S. A.; Langen, R. Biochim. Biophys. Acta, Biomembr. 2007, 1768(8), 2002–2009. (17) Janson, J.; Ashley, R. H.; Harrison, D.; McIntyre, S.; Butler, P. C. Diabetes 1999, 48(3), 491–498. (18) Haataja, L.; Gurlo, T.; Huang, C. J.; Butler, P. C. Endocr. Rev. 2008, 29(3), 303–316. (19) Kawahara, M.; Kuroda, Y.; Arispe, N.; Rojas, E. J. Biol. Chem. 2000, 275 (19), 14077–14083. (20) Townsend, M.; Shankar, G. M.; Mehta, T.; Walsh, D. M.; Selkoe, D. J. J. Physiol. (Oxford, U.K.) 2006, 572(2), 477–492. (21) Walsh, D. M.; Klyubin, I.; Fadeeva, J. V.; Rowan, M. J.; Selkoe, D. J. Biochem. Soc. Trans. 2002, 30, 552–557. (22) Walsh, D. M.; Klyubin, I.; Shankar, G. M.; Townsend, M.; Fadeeva, J. V.; Betts, V.; Podlisny, M. B.; Cleary, J. P.; Ashe, K. H.; Rowan, M. J.; Selkoe, D. J. Biochem. Soc. Trans. 2005, 33, 1087–1090. (23) Lim, Y. A.; Ittrier, L. M.; Lim, Y. L.; Gotz, J. FEBS Lett. 2008, 582(15), 2188–2194. (24) Green, J.; Goldsbury, C.; Min, T.; Sunderji, S.; Frey, P.; Kistler, J.; Cooper, G.; Aebi, U. J. Mol. Biol. 2003, 326(4), 1147–1156. (25) Rhoades, E.; Agarwal, J.; Gafni, A. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 2000, 1476(2), 230–238. (26) Lynch, I.; de Gregorio, P.; Dawson, K. A. J. Phys. Chem. B 2005, 109(13), 6257–6261.

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were of the highest purity. Nanoparticles. N-Isopropylacrylamide (NiPAM):N-tertbutylacrylamide (BAM) copolymer particles were synthesized in sodium dodecyl sulfate (SDS) micelles as previously described.10 Particles were produced at different comonomer ratios (100:0, 85:15, 65:35, and 50:50 NiPAM:BAM) and with a diameter of 40 nm (at 37 °C). After polymerarization, the particles were dialyzed to eliminate any trace of SDS as evidenced by proton NMR. Particles were lyophilized and stored until use. Stock solutions were prepared by dissolving particles on ice in Milli-Q water to obtain complete dispersion below the lower critical solution temperature.

ThT Fluorescence Assay with Peptide from DMSO. Proteins were dissolved in dimethyl sulfoxide (DMSO) and then diluted with 20 mM Tris/HCl, 100 mM NaCl, 0.02% NaN3, pH 7.5 to a final concentration of 12.8 and 100 μM for IAPP and IAPP(20-29), respectively. The amount of DMSO in the samples was always lower than 1%. Immediately after dilution, aliquots are distributed into a 96-well fluorescence plate in the presence of 20 μM ThT and in the absence or presence of nanoparticles at different concentrations. Fluorescence intensity was monitored using a Molecular Devices SpectraMax M2 microplate reader (Sunnyvale, CA) with excitation and emission at 435 and 485 nm, respectively. In the case of IAPP, the samples were incubated at 37 °C and shaken inside the microplate reader and points measured every 1 min. For IAPP(20-29), samples were incubated in a VorTemp 56 incubator/ shaker at 37 °C and shaken at 700 rpm and ThT fluorescence monitored in the plate reader every 5 min. Each experimental point is an average of five replicas. The data obtained were normalized to the maximum plateau of the control sample in the absence of particles. The kinetics of the fibrillation process was analyzed by fitting of a sigmoidal empirical equation to the data: Y ¼ y0 þ

ymax - y0 1 þ e -ðt -t1=2 Þk

ð1Þ

where y0 and ymax are the initial and maximum fluorescence intensity values, respectively, t1/2 is the time at half intensity, and k is the apparent first order elongation rate constant.27 The lag time, the time required to form critical nuclei that lead to the formation of fibrils, is described by lag time ¼ t1=2 -2=k

ð2Þ

ThT Fluorescence Assay after Gel Filtration. IAPP (0.5 mg) was dissolved in 1 mL of 6 M GuHCl, pH 7.5, and subjected to gel filtration in degassed buffer (20 mM Tris/HCl, 100 mM NaCl, 0.02% NaN3, pH 7.5) on a 30  1 cm Superdex 75 column. The monomer was collected at 27 μM and diluted to 13.3 μM in the same buffer and then supplemented with 22 μM ThT. Immediately after dilution, 45 μL aliquots were distributed into a 96-well fluorescence plate (black polystyrene half area plate with clear bottom and PEG coating; Corning 3881), where each well contained a total of 5 μL of buffer and/or nanoparticles in buffer to obtain final concentrations of nanoparticles of 0, 0.1, 0.3, 1, 3, 10, or 30 μg/mL in addition to 12 μM IAPP, 20 μM ThT, 20 mM Tris/HCl, 100 mM NaCl, 0.02% NaN3, pH 7.5. Parallel experiments at the same conditions were set up in the presence of seeds that had been prepared by sonication of IAPP fibrils. The plate (27) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40(20), 6036–6046.

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Figure 1. (A) ThT fluorescence signal with time for different IAPP concentrations: (9) 12.8, (0) 6.4, (b) 3.2, (O) 1.6, (2) 0.8, and (4) 0.4 μM IAPP in 20 mM TRIS, 100 mM NaCl, 0.02% NaN3, 1% DMSO, pH 7.5. (B) ThT fluorescence signal with time in the (9) absence and presence of (0) 50:50, (b) 65:35, (O) 85:15, and (2) 100:0 NiPAM:BAM copolymeric particles of 40 nm diameter. Samples contain 12.8 μM IAPP and 10 μg/mL particles (corresponding to ca. 90 pM), 1% DMSO in 20 mM TRIS, 100 mM NaCl, 0.02% NaN3, pH 7.5.

was sealed with a plastic film (Corning 3095). The experiment was initiated by placing the 96-well plate at 37 °C and shaking at 100 rpm in a plate reader (Fluostar Omega, BMG Labtech, Offenburg, Germany). The ThT fluorescence was measured through the bottom of the plate every minute (with excitation filter 440 nm and emission filter 480 nm) with continuous shaking at 100 rpm in between reads. Each experimental condition was set up as four or more replicas. Each fibrillation curve was normalized individually. Transmission Electron Microscopy. Negative staining EM was performed. Briefly, the sample was applied onto a carboncoated formvard grid, blotted with filter paper, allowed to dry for 2 min, stained with uranyl acetate 2% in water, blotted with filter paper, allowed to air-dry, and then viewed using a JEOL 2000 electron transmission microscope operated at 80 V. All reagents were supplied by Electron Microscopy Sciences (Hatfield, PA). Samples used for EM were prepared as described above from various concentrations of IAPP in the absence or presence of ThT at different incubations times. A higher peptide concentration than that for the ThT assay is required in order to obtain enough material on the TEM grid to be able to visualize the formed structures. In order to determine the extension of the reaction by this change in concentration, the ThT assay was performed simultaneously, and results are shown in the Supporting Information (Figure S1). Circular Dichroism Spectroscopy. IAPP (0.5 mg) was dissolved in 1 mL of 6 M GuHCl, pH 7.5, and subjected to gel filtration in degassed 20 mM Tris/HCl, 100 mM NaCl, 0.02% NaN3, pH 7.5 on a 30  1 cm Superdex 75 column. The monomer was collected at 27 μM, and circular dichroism spectra were recorded in a 2 mm quartz cuvette between 250 and 200 nm using a Jasco J-815 CD spectrometer, before and after the addition of 100 μg/mL 40 nm 85:15 NiPAM:BAM nanoparticles. A third spectrum was recorded of the filtrate obtained after the addition of 100 μg/mL 40 nm 85:15 NiPAM:BAM nanoparticles followed by separation by centrifugation through a 100 kDa molecular weigh cutoff filter, which retains the nanoparticles and bound peptide while free peptide goes through the filter.

Conjugation of Nanoparticles to Gold Surfaces for SPR Studies. The SIA Au kit (BIAcore AB, Uppsala) was used for sensor chip preparation. Thiol-linked nanoparticles with on average less than one SH group on the surface (synthesis described in detail in Cedervall et al., 2007)28 were dissolved at 0.2 mg/mL in 20 mM sodium phosphate buffer, 100 mM NaCl, pH 7.5 on ice, and 120 μL was applied to a 10  10 mm2 gold surface for 4 h or overnight, before the surface was rinsed with H2O, dried, and assembled in a sensorchip cassette. The change in response units (28) Cedervall, T.; Lynch, I.; Lindman, S.; Bergga˚rd, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104(7), 2050–2055.

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after coupling of the nanoparticles to gold reveals the amount of immobilized nanoparticles. Densely packed layers of 70% surface coverage yields a response of 35 kRU, and the increase in response obtained in separate coupling trials ranged from 50 to 70% of these numbers, indicating efficient coupling of the particles. Surface Plasmon Resonance (SPR) Experiments. SPR studies of IAPP associating to and dissociating from nanoparticles were performed using a BIAcore 3000 instrument (BIAcore AB, Uppsala). The flow buffer contained 10 mM sodium phosphate pH 8.2 with 3 mM EDTA, 150 mM NaCl, and 0.005% Tween20 and was filtered (0.2 μm filter) and degassed for at least 30 min. Each sensorchip surface with attached particles was washed for at least 5 h at a flow rate of 50-100 μL/min and then equilibrated at 10 μL/min for at least 30 min or until the baseline was stable. IAPP was dissolved in DMSO at 1 mM and diluted to 20 μM in the running buffer just prior to the SPR experiment. It was injected for 30 min to study the association kinetics. After 30 min, buffer was flown over the sensorchip surface for 24 h at 10 μL/min to study the dissociation kinetics. Association and dissociation data were fitted using eqs 3 and 4, respectively     on on off on off RðtÞ ¼ C1 k =ðk þ k Þ 1 -exp -ðk þ k Þt ð3Þ

RðtÞ ¼ A1 expð -k 1off tÞ þ A2 expð -k 2off tÞ

ð4Þ

Results Copolymeric Nanoparticles Inhibit IAPP Fibrillation. The fibrillation process of IAPP was studied in the absence and presence of copolymeric nanoparticles of 40 nm diameter. The particles used were prepared at different comonomer ratios, 50:50, 65:35, 85:15, and 100:0 NiPAM:BAM. This variation in composition yields a systematic change in the physicochemical properties of the material, and obviously, the surface chemistry of the nanoparticles with increasing amount of BAM results in increasingly hydrophobic particles. IAPP fibrillation was studied at 37 °C under agitation (see Materials and Methods). Under these conditions, IAPP rapidly aggregates and forms amyloid fibrils. Even in the absence of agitation, the reaction is finished within 2 h (data not shown). Figure 1A shows the evolution of fibril formation measured by means of the Thioflavin T (ThT) assay. ThT binds to amyloid structures, resulting in a shift and increase of the fluorescence signal that allows us to follow the kinetics of fibril formation. Fibrillation of IAPP shows the typical DOI: 10.1021/la902980d

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Figure 2. TEM images of IAPP samples in the presence and absence of particles (85:15 NiPAM:BAM, 10 μg/mL) for two different time

points, 15 and 30 min, along the fibrillation process of 12.8 μM IAPP in 1% DMSO in 20 mM TRIS, 100 mM NaCl, 0.02% NaN3, pH 7.5.

characteristics of a nucleation and growth mechanism. The time course of fibrillation shows a lag phase during which monomer and small oligomers are in equilibrium with no ThT-binding species detectable, and an elongation phase during which the initial aggregates grow exponentially into fibrils that bind ThT and enhance its fluorescence (Figure 1A). The addition of NiPAM:BAM copolymeric nanoparticles clearly slows down the aggregation process by increasing the lag time. Figure 1B shows the fibrillation time course in the absence and presence of 40 nm copolymeric nanoparticles of different composition. In the absence of particles, the lag time of the process is 7.3 ( 0.6 min (defined as the intercept of the x-axis of the tangent through the midpoint of a sigmoidal curve fitted to the data; see Materials and Methods). The lag time in the presence of particles is increased to 10.3 ( 0.7 min in the case of 50:50 NiPAM:BAM nanoparticles, to 16.9 ( 0.45 min for 65:35 NiPAM:BAM nanoparticles, and up to around 27 min for 85:15 and 100:0 NiPAM:BAM nanoparticles. Remarkably, for the 85:15 and 100:0 nanoparticles, the fluorescence value of the plateau is significantly lower than those for the rest of the cases. Even with longer incubation times, this plateau does not increase significantly, indicating that the maximum fluorescence value observed corresponds to the final state in the fibrillation process in the presence of those nanoparticles. This low equilibrium plateau value is comparable with that of the fibrillation experiments performed in the absence of particles at reduced protein concentration (Figure 1A). According to the proposed nucleation-polymerization mechanism, as the protein concentration decreases, the lag phase increases and the plateau decreases, indicating a lower amount of fibrils formed. The effect of nanoparticles on the IAPP fibrillation is confirmed by TEM experiments. In agreement with the enhanced ThT signal (Supporting Information Figure S1) at 15 min in the absence of particles, the sample shows an extensive network of amyloid fibrils with the typical features of hundreds of nanometers long and 10 nm width. On the other hand, in the sample with nanoparticles, no mature fibrils are observed at this time point 3456 DOI: 10.1021/la902980d

although some small fibrillar segments can be found on the grid. At longer experiment times, 30 min, formation of an extensive net of fibrils can be observed in both samples, as shown in Figure 2. The Effect of IAPP Fibrillation Is Concentration and Surface Property Dependent. The composition of the nanoparticles (and hence the surface chemistry) defines the effect that they have on the fibrillation process of IAPP (Figure 1B). The inhibition is more pronounced as the amount of NiPAM monomer in the particles increases. As expected, the effect of nanoparticle addition is concentration dependent. As we increase the concentration, the inhibitory effect increases due to the larger surface area present in the solution. Depending on the intrinsic tendency to slow down fibrillation, the minimum concentration of nanoparticles needed to generate a significant inhibitory effect varies with composition. For the less active nanoparticles, 50:50 NiPAM:BAM, up to 0.03 mg/mL of particles is needed in order to observe a significant increase of the lag phase. Approximately 100 times lower concentration is needed to observe a significant effect with the 85:15 NiPAM:BAM nanoparticles (Figure 3). The addition of nanoparticles to an ongoing fibrillation process has different effects depending on the point of the addition. Figure 4 shows an experiment where 100:0 NiPAM:BAM particles were added at different time points to a sample where the fibrillation process has started. When the particles are added during the lag phase, at times 0, 2, and 5 min, no increase of fluorescence intensity is observed during the length of the experiment, indicating no formation of ThT positive fibrillar species. On the other hand, after the lag time (when the critical nuclei are formed), freezing of the process is observed at the moment of the addition of particles. The fibrillar species present at a time point of the addition of the particle do not elongate by addition of other monomers. Due to the lack of specific interaction in the case of 50:50 NiPAM:BAM nanoparticles, its addition does not cause any disturbance to the fibrillation process unless added at the start of the experiment (0 min). The TEM images in Figure 4C show the status of samples after 30 min from the beginning of the reactions when 85:15 NiPAM: Langmuir 2010, 26(5), 3453–3461

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Figure 3. Variation of ThT fluorescence intensity with time in the absence and presence of (A) 50:50 NiPAM:BAM and (B) 85:15 NiPAM: BAM nanoparticles at different concentrations: (9) 0, (0) 0.1, (b) 0.3, (O) 1, (2) 3, (4) 10, and ([) 30 μg/mL. All samples contain 12.8 μM IAPP, 1% DMSO in 20 mM TRIS, 100 mM NaCl, 0.02% NaN3, pH 7.5.

Figure 4. (A) Fibrillation kinetics of IAPP at 37 °C monitored by temporal development of ThT binding disturbed by the addition of 100:0 NiPAM:BAM particles at 30 μg/mL at different time points following the start of the reaction: (9) no added particles, (0) 0, (b) 2, (O) 5, (2) 8, (4) 10, and (1) 14 min. (B) Fibrillation kinetics of IAPP at 37 °C monitored by temporal development of ThT binding disturbed by the addition of 50:50 NiPAM:BAM particles at 30 μg/mL at different time points: (9) no added particles, (0) 0, (b) 2, (O) 5, and (2) 8 min. (C) TEM images for the fibrillation of 12.8 μM IAPP after 30 min of reaction for samples where particles were not added or where 85:15 NiPAM:BAM particles were added at 0 or 10 min to give a final concentration of 30 μg/mL. Scale bar indicates 200 nm.

BAM particles were added at 0 or 10 min or not added. It is clear from the images that adding the particles at 10 min has less of an inhibitory effect than adding them at time 0, as reflected in the small numbers of mature fibrils observed for the sample at 10 min. However, compared to the very extensive network of fibrils in the absence of nanoparticles, a significant inhibitory effect is observed in both cases. IAPP Fibrillation after Gel Filtration. Direct dissolution of IAPP in DMSO followed by dilution into the experimental buffer may yield samples with preformed nuclei which may shorten the lag phase compared to starting from pure monomer. A separate set of 96 kinetic traces were therefore recorded after gel filtration Langmuir 2010, 26(5), 3453–3461

of IAPP into the experimental buffer. When collected from the gel filtration column and used immediately, the monomeric peptide displays a distinct lag phase of about 37 ( 3 min when studied at 12 μM in the absence of nanoparticles (Figure 6). The addition of 85:15 NiPAM:BAM nanoparticles in the range 0.1-10 μg/mL retards the process by prolonging the lag phase, while a much smaller effect is seen with 50:50 NiPAM:BAM particles. At 30 μg/ mL nanoparticles, we observe no retardation (85:15) or a slight acceleration (50:50). No systematic effect on the equilibrium plateau value for fluorescence was observed; the same random variation around the mean is seen at all nanoparticle concentrations as in the absence of particles. The lag time versus nanoparticle DOI: 10.1021/la902980d

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Figure 5. (A) Variation of ThT fluorescence intensity with time in the absence (black) or presence of 85:15 NiPAM:BAM nanoparticles at (dark blue) 0.1, (light blue) 0.3, (green) 1, (yellow-green) 3, (red) 10, and (purple) 30 μg/mL. In the absence of nanoparticles, we show 16 individual traces, whereas at each nanoparticle concentration we show one representative trace out of four to improve the figure clarity. All samples contain 12 μM IAPP in 20 mM TRIS, 100 mM NaCl, 0.02% NaN3, pH 7.5. (B) Lag time versus nanoparticle concentration for (blue) 85:15 and (red) 50:50 NiPAM:BAM nanoparticles, with error bars indicating the standard deviations over four replicas.

Figure 6. Surface plasmon resonance data for 25 μM IAPP injected over immobilized 85:15 (blue) or 50:50 (red) NIPAM:BAM nanoparticles with 40 nm diameter given as the response in RU after subtraction of the baseline value (1 RU corresponds to 1 pg/ mm2 bound protein). The peptide was in constant flow for 1800 s to study the association to the nanoparticles, followed by buffer flow for 24 h to study the dissociation of peptide (not shown).

concentration shows a biphasic behavior (Figure 5B), indicating the coexistence of two competing mechanisms, with one leading to retardation (seen at the lower nanoparticle concentrations) and one leading to acceleration (seen at higher nanoparticle concentrations) of fibrillation. Overall the observed effects of nanoparticles are smaller compared to those seen with peptide diluted from DMSO, suggesting that a significant effect of the nanoparticles is to interfere with the fibrillation process by binding small oligomers or nuclei. However, for the seeded reaction, where sonicated fibrils were added at time zero, we see no significant effect of addition of the nanoparticles on the lag time or fibrillation rate (data not shown). IAPP Binding to Nanoparticles. The binding of IAPP to 85:15 and 50:50 NIPAM:BAM copolymer nanoparticles linked to a gold surface was studied using surface plasmon resonance. The data (Figure 6) indicate that the IAPP peptide binds with high affinity to both kinds of nanoparticles. The dissociation of IAPP from the 85:15 NiPAM:BAM particles is biphasic and is well fitted by a single exponential decay plus a constant. The exponential phase corresponds to 12% of the total amplitude, and the fitted value for koff is 5.3  10-3 s-1. The remaining 88% of the bound peptide does not appear to dissociate at all over 24 h, 3458 DOI: 10.1021/la902980d

Figure 7. CD spectrum of IAPP after gel filtration (reported as mean residue ellipticity) in the absence (black) and presence (blue) of 100 μg/mL 85:15 NiPAM:BAM nanoparticles. The IAPPnanoparticle mixture was separated on a 100 kDa Mw cutoff filter, and the dashed blue line shows the CD spectrum of the filtrate.

meaning that for the majority of the bound peptide koff is around 10-6 s-1 or lower. For the 50:50 NiPAM:BAM nanoparticles, the dissociation process is too slow to show any significant dissociation over 24 h, meaning that koff is around 10-6 s-1 or lower. The fitted values of the association rate constant are 1.3  102 M-1 s-1 for IAPP to 50:50 NiPAM:BAM particles and 2.1  102 M-1 s-1 for IAPP to 85:15 NIPAM:BAM particles. From the ratio of kon and koff, we conclude that the affinity for both kinds of nanoparticles is around 108 M-1 or higher (KD e 10-8 M). Structural Effects of Binding and Monomer Depletion. The CD spectrum of 27 μM IAPP does not change upon addition of 100 μg/mL 85:15 NiPAM:BAM nanoparticles (Figure 7). This indicates that the interaction between IAPP and nanoparticles takes place without any major changes to the IAPP secondary structure. No peptide could be detected in the 100 kDa filtrate of the IAPP-nanoparticle mixture, neither by CD spectroscopy (Figure 7) nor by UV absorbance spectroscopy (data not shown), indicating that all of the peptide is tightly bound to the nanoparticles and that monomer is depleted from the solution. Aggregation of IAPP(20-29) Fragment. The fibrillation process was also studied for the case of IAPP(20-29), which has Langmuir 2010, 26(5), 3453–3461

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Figure 8. (A) Fibrillation kinetics of IAPP(20-29) at 37 °C monitored by the temporal development of ThT fluorescence for different

nanoparticles at final concentration of 10 μg/mL: (9) no particles, (0) 50:50, (b) 65:35, (O) 85:15, and (2) 100:0 NiPAM:BAM polymeric particles of 40 nm diameter. All samples contain 100 μM IAPP(20-29), 1% DMSO in 20 mM TRIS buffer, 100 mM NaCl 0.02% NaN3, pH 7.5. (B) TEM images of the fibrillation of 100 μM IAPP(20-29) after 75, 360, and 480 min of reaction for samples in the absence and presence of 85:15 NiPAM:BAM particles (10 μg/mL). Scale bar indicates 500 nm.

been described as a highly fibrillogenic segment in the IAPP sequence. Similar experiments as described above were performed, and the results show an analogous effect of the particles on the fibrillation of IAPP(20-29), even though the fibrillation process by itself shows different features. In the experimental conditions chosen, the fibrillation kinetics are much slower than in the case of the full length IAPP. In order to follow the kinetics in a more consistent way, IAPP(20-29) samples were incubated at 37 °C with shaking at 700 rpm, and the kinetics followed for approximately 700 min (10 times longer than in the case of full length IAPP). The second feature to notice (Figure 6) is that, in the absence of particles, there is a transient maximum in the fibrillation curve that later on evolves toward a stable plateau. This could indicate an initial (and fast) formation of a ThT reactive oligomeric species, or fast formation of amyloid fibrils that subsequently undergo a change in conformation, or strong bundling of the fibrils. Bundling may decrease the access of ThT to the binding sites, and/or lead to partial decrease of the fluorescence, and therefore reduce the fluorescence signal. Upon the addition of nanoparticles, the fibrillation process is again inhibited by their presence, and moreover the transient maximum is moved or even disappears in the presence of the most active nanoparticles. This could indicate that the nanoparticles actually affect the mechanism of association from monomers to fibrils and modulate the possible structural rearrangements of young fibrils. The fibrils observed by TEM do not show an apparent change in shape at the different time points, although an increase of bundling is observed over time (Figure 8). As reported before,29 the appearance of the fibrils formed by the IAPP fragment is different from those formed by the intact protein, with less defined and truncated ends.

Discussion Ordered protein aggregates may be the molecular basis of several neurodegenerative and systemic diseases. Peptides and proteins that differ in their amino acid sequence and native fold share a common non-native folding topology: amyloid fibrils in a cross β-sheet fold.30 We may classify proteins that undergo (29) Goldsbury, C.; Goldie, K.; Pellaud, J.; Seelig, J.; Frey, P.; Muller, S. A.; Kistler, J.; Cooper, G. J. S.; Aebi, U. J. Struct. Biol. 2000, 130(2-3), 352–362. (30) Elgersma, R. C.; Posthuma, G.; Rijkers, D. T. S.; Liskamp, R. M. J. J. Pept. Sci. 2007, 13(11), 709–716.

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amyloid fibrillation as globular proteins with a stable unique conformation (e.g., β2m) or unstructured proteins (e.g., IAPP, Aβ, R-synuclein, etc.).9,31 However, the fibrils formed by all these proteins and peptides have the characteristics of an amyloid fibril, that is, beta strands perpendicular to the axis of the fibrils with hydrogen bonds forming the backbone of the fibrils along the long axis of the structure. A divergent effect of copolymeric NIPAM:BAM nanoparticles on the fibrillation of two very different proteins, β2m, a protein with β-sheet secondary structure, and Aβ, an unstructured peptide, was observed.10,11 In the case of β2m, the fibrillation is accelerated,10 most likely due to the increased local concentration on the nanoparticle surface and promotion of the formation of critical nuclei. On the contrary, the Aβ fibrillation is slowed down, due to depletion of the monomer in solution and/or trapping of monomer/oligomer in a nonamyloidogenic species.11 IAPP is a short peptide of 37 amino acids partially unstructured, and its fibrillation behavior in the presence of copolymeric nanoparticles resembles that of Aβ, although some distinct differences are observed. The fibrillation process for IAPP is slowed down by the presence of copolymeric nanoparticles in a similar fashion as seen for Aβ,11 pointing to a similar mechanism of action. As we can see in the case of fibrillation followed by means of ThT fluorescence at different peptide concentrations (Figure 1A), the equilibrium plateau is dependent on the total concentration of peptide and decreases when the amount of peptide decreases, in a similar manner as observed for Aβ.42 In the case of Aβ, the equilibrium plateau was roughly constant for all particles and particle concentrations. However, the equilibrium plateau for IAPP decreases for the highest concentration of nanoparticles The observed decrease of the fluorescence equilibrium value when nanoparticles are present could be due to the withdrawal of active monomer from solution, which will lead to an increase of the lag phase, a reduction of the elongation rate, and a lowering of the equilibrium plateau. However, a reduction in plateau value is not seen when the starting point is pure IAPP monomer from gel filtration, although depletion of monomer is clearly observed using the filtration assay. This suggests the existence of several competing and/or reinforcing effects of the nanoparticles on IAPP fibrillation, and the net effect will depend (31) de Groot, N. S.; Pallares, I.; Aviles, F. X.; Vendrell, J.; Ventura, S. BMC Struct. Biol. 2005, 5, 18–32.

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on both peptide and nanoparticle concentration and on the distribution of the peptide over different species at the point of nanoparticle addition. This is further illustrated by biphasic dependence on nanoparticle concentration when starting from monomeric IAPP, in contrast to the monotonous and stronger inhibitory effect when the starting solution includes a small amount of oligomers. Based on these findings, we can consider that the inhibitor species (nanoparticles) may act at three different points along the fibrillation pathway: (1) at the nucleation phase, by increasing the lag phase, (2) at the polymerization step, lowering the elongation rate, and (3) by peptide diversion from the polymerization pathway, reducing the end point at the equilibrium. Analysis of the stopping experiments (Figure 4) also suggest that once nanoparticles are added, adsorption of the peptide to the particles depletes the monomer from solution stopping the elongation process. The comparison between the equilibrium plateau for different IAPP concentrations (Figure 1A) and the plateau reached and the fibrillation process in the presence of 10 μg/ mL 85:15 NiPAM: BAM particles (Figure 3B) could give an estimate of the amount of peptide adsorbed on the surface of the particles. The equilibrium plateau in the presence of particles would be compatible with, roughly, 6 μM of IAPP, that is, 50% of the total amount present in solution. That would imply that 50% of the total protein is bound to the particles. If we consider the total surface area exposed by the particles, 0.8 m2/L, and the approximate surface area of the peptide at 6 μM, 22 m2/L, it is clear that several layers of peptide cover the nanoparticle surface if we assume that 50% of the protein is bound. The amyloidogenic fragment IAPP(20-29) retains the ability to interact with the copolymeric nanoparticles in a similar fashion as intact IAPP. The fibrillation process is slowed down by the nanoparticles, but in this case the particular feature of the fibrillation itself makes it more difficult to analyze the observed effect. Reaching the equilibrium plateau is delayed in all cases by the presence of particles as well as the formation of the transient ThT-positive structures observed in the absence of nanoparticles. However, it is clear at least from the TEM images that the addition of particles that interact positively with the peptide leads to a retardation of the formation of fibrils. The transient species observed in this case have also been observed in the case of IAPP in the presence of heparin sulfate, indicating a change in fibril conformation.32 Recent AFM studies reveal a change in the periodicity and packing for the fragment IAPP(20-29) showing that the morphology of the fragment fibrils may vary during growth. Younger protofibrils display a periodicity that disappears as they mature into older fibrils. This change in structure can affect the binding of ThT and therefore create the signal artifact observed.33 The tendency of IAPP molecules to interact with and associate to lipidic membranes is a clear indication of the driving force for the peptide to shield its hydrophobic regions from (32) Meng, F.; Abedini, A.; Song, B.; Raleigh, D. P. Biochemistry 2007, 46(43), 12091–12099. (33) Sedman, V. L.; Allen, S.; Chan, W. C.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Protein Pept. Lett. 2005, 12(1), 79–þ. (34) Brender, J. R.; Lee, E. L.; Cavitt, M. A.; Gafni, A.; Steel, D. G.; Ramamoorthy, A. J. Am. Chem. Soc. 2008, 130(20), 6424–6429. (35) Tycko, R. Biochemistry 2003, 42(11), 3151–3159. (36) Kapurniotu, A. Biopolymers 2001, 60(6), 438–459. (37) Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castano, E. M.; Frangione, B. Nat. Med. 1998, 4(7), 822–826. (38) Tjernberg, L. O.; Naslund, J.; Lindqvist, F.; Johansson, J.; Karlstrom, A. R.; Thyberg, J.; Terenius, L.; Nordstedt, C. J. Biol. Chem. 1996, 271(15), 8545– 8548.

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water.18,34,39-41 The amyloidogenic region includes the most hydrophobic residues of the IAPP sequence.31 Thus, it is reasonable to expect that the peptide may adsorb onto the particle surface due to hydrophobic interactions. Copolymeric particles offer an apolar environment, and thus, the adsorption of the peptide onto the surface will minimize the exposure of hydrophobic regions of the peptide to water. Nevertheless, the characteristics of the particle surface and the use of a series of nanoparticles with different hydrophobicity indicate that hydrophobic interactions are not the only forces that play a role in the interaction with IAPP, as the observed trend in terms of inhibition of fibrillation is opposite: the nanoparticles with highest BAM content are the most hydrophobic, and these particles produce the lowest effect. Consequently, a second interaction may play a role. Amyloid fibrils result from the assembly of antiparallel oriented peptides in which the peptide backbone can form an ideal hydrogen bonding network over long distances along the length of the fibril.35 It is known that peptides preventing hydrogen bond formation or causing improper hydrogen bonding network formation inhibit or delay fibrillation, and have even been observed to redissolve preformed Aβ fibrils.30,36-38 The molecular backbone of the nanoparticles offers the possibility to establish hydrogen bonding between the particle and the peptide, and this ability to form hydrogen bonds is highest for the most active particle, with 100:0 NiPAM:BAM. Previous work on NIPAM: BAM copolymers, including on molecular simulations, indicated that the poly(NIPAM) packing is able to accommodate BAM up to 50% before a modification of the solid structure takes place. Second, the addition of BAM leads to decreased surface exposure of the N-H groups, even at small additions of BAM, despite the unmodified NIPAM polymer structure. Furthermore, as the content of BAM groups exceeds 50%, a noticeable decrease in the solvation free energy of the polymer chain is observed even though the overall solvent-accessible surface area steadily increases on moving from poly(NIPAM) to poly(BAM) surfaces, which can be explained by better shielding of the amide groups (N-H groups) by tert-butyl groups than by isopropyl groups.12 Likewise, in the case of the nanoparticles, as the BAM content in the particle polymer backbone is increased, the number of NH residues at the surface decreases, due to the additional steric hindrance of the bulkier BAM group (Scheme 1), which results in decreased hydrogen bonding capability and thus decreased adsorption of proteins which bind via hydrophobic interactions.12 This observation and the importance of the hydrogen bonds in amyloid fibril formation suggest that the nanoparticles with lower BAM content bind the peptide tightly by means of hydrogen bonds. This results in the impediment of monomers or early aggregates to grow into mature fibrils due to the competition for the hydrogen bonding partners of the peptide by the nanoparticles. In addition, the strong binding affinity of the peptides for the particles suggests a sort of polyion effect where the energy gain from multiple hydrogen bonds to the nanoparticle surface cannot be overcome by that of few hydrogen bonds upon the addition of a single monomer to the prefibrils. This can explain also the very long residence time of the peptides on the particle surface. To conclude, we have shown that the fibrillation process of an amyloidogenic peptide and its amyloidogenic fragment is affected (39) Jeworrek, C.; Hollmann, O.; Steitz, R.; Winter, R.; Czeslik, C. Biophys. J. 2009, 96, 1115–1123. (40) Brender, J. R.; Hartman, K.; Reid, K. R.; Kennedy, R. T.; Ramamoorthy, A. Biochemistry 2008, 47, 12680–12688. (41) Engel, M. F. M.; Yigittop, H.; Elgersma, R. C.; Rijkers, D. T. S.; Liskamp, R. M. J.; de Kruijff, B.; Hoppener, J. W. M.; Killian, J. A. J. Mol. Biol. 2006, 356, 783–789. (42) Hellstrand, E.; Boland, B.; Walsh, D. M.; Linse, S., ACS Chem. Neurosci. 2009, in press; DOI: 10.1021/cn900015v.

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by the presence of hydrophobic, uncharged nanoparticles of different composition. The association of the peptide and a linked depletion effect of peptide from solution is the most plausible cause of the inhibition observed in the fibrillation process. We also show that for (some) unstructured peptides with fast aggregation kinetics the mechanism of inhibition is similar although some differences arise regarding the mechanism of interaction of nanoparticles with the early stage amyloidogenic species. Hydrogen bonding of the peptides with nanoparticles may be a key inhibitor of fibrillation.

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Acknowledgment. Funding for this work came from EU FP6 NanoInteract (I.L., K.A.D., S.L.), IRCSET (C.C.-L.), Ministerio de Educacion y Ciencia postdoctoral grant (C.C.-L.), Swedish Research Council and the Linneus Centre Organizing Molecular Matter (S.L.), and SFI Walton (S.L.). Supporting Information Available: ThT kinetic assay for the TEM experiment corresponding to Figure 2. This material is available free of charge via the Internet at http://pubs.acs.org.

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