Engineered Polymer Nanoparticles Containing Hydrophobic

Aug 17, 2012 - A β 17-20 Peptide-Guided Structuring of Polymeric Conjugates and Their pH-Triggered Dynamic Response. Sonu Kumar , Varun Bheemireddy ...
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Engineered Polymer Nanoparticles Containing Hydrophobic Dipeptide for Inhibition of Amyloid‑β Fibrillation Hadas Skaat,† Ravit Chen,†,‡ Igor Grinberg, and Shlomo Margel* Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan 52900, Israel S Supporting Information *

ABSTRACT: Protein aggregation into amyloid fibrils is implicated in the pathogenesis of many neurodegenerative diseases. Engineered nanoparticles have emerged as a potential approach to alter the kinetics of protein fibrillation process. Yet, there are only a few reports describing the use of nanoparticles for inhibition of amyloid-β 40 (Aβ40) peptide aggregation, involved in Alzheimer’s disease (AD). In the present study, we designed new uniform biocompatible aminoacid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains. The dipeptide residues were designed similarly to the hydrophobic core sequence of Aβ. Poly(N-acryloyl-L-phenylalanyl-L-phenylalanine methyl ester) (polyA-FF-ME) nanoparticles of 57 ± 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2methoxy ethanol, followed by precipitation of the obtained polymer in aqueous solution. Cell viability assay confirmed that no significant cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g., PC-12 and SH-SY5Y, was observed. A significantly slow secondary structure transition from random coil to β-sheets during Aβ40 fibril formation was observed in the presence of these nanoparticles, resulting in significant inhibition of Aβ40 fibrillation kinetics. However, the polyA-FF-ME analogous nanoparticles containing the L-alanyl-L-alanine (AA) dipeptide in the polymer side groups, polyA-AAME nanoparticles, accelerate the Aβ40 fibrillation kinetics. The polyA-FF-ME nanoparticles and the polyA-AA-ME nanoparticles may therefore contribute to a mechanistic understanding of the fibrillation process, leading to the development of therapeutic strategies against amyloid-related diseases.

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INTRODUCTION

Several studies on the aggregation process of Aβ have identified the critical peptidic sequence involved in amyloid aggregates formation.14−18 The hydrophobic core from residues 17−20 of Aβ, LVFF sequence, is crucial for the formation of βsheet structures.14−18 It was also demonstrated that this peptidic region binds to its homologous sequence in Aβ and prevents its aggregation into amyloid fibrils.19−21 Such inhibition is mainly based on recognition between the pairs of phenylalanine residues (FF) through hydrophobic interactions.14−21 This FF motif has served as a key platform for the development of peptide and peptidomimetic inhibitors of Aβ fibrillation.17 Although peptide or protein analogues with specific binding sites may be of primary importance for studies of neurodegenerative disorders, therapeutic agents have not been developed within this strategy. The main problems are the blood-brain barrier (BBB) permeability, the complexity of their synthesis, and their low in vivo stability and efficacy.22 Recent literature concerning novel biomaterials indicates increasing interest in developing nanoparticles to detect, prevent, and treat protein-misfolding diseases.23−25 Their potential influence on protein fibrillation is a function of both

The formation of amyloid aggregates is a pathological hallmark of many diseases, including Parkinson’s, Huntington’s, mad cow, prion, and Alzheimer’s disease (AD).1−3 The common feature of amyloid diseases is the transition of proteins from the normally soluble form into amyloid fibrils, organized mainly into cross β-sheets, which accumulate in the extracellular space of various tissues.4−6 The extracellular deposits that characterize AD are composed of fibrils of a small peptide with 39−43 amino acids, the amyloid-β (Aβ) peptide.7 Aβ is continuously secreted by normal cells in culture and is detected as a soluble peptide in the plasma and cerebrospinal fluid of healthy individuals.8,9 In AD patients, Aβ exists as insoluble fibrillar aggregates.10 It is generally accepted that the Aβ peptides can self-assemble to form neurological toxic aggregates with various morphologies, such as dimers, oligomers, protofibrils, and fibrils, which eventually deposit as insoluble plaques and cause the death of brain cells.11 Currently, there are no effective drugs to cure these diseases and treatment options are extremely limited.12 A coherent pharmacological approach for preventing amyloid aggregation encompasses agents able to specifically stabilize the initial conformations through interfering with the intermediate species and/or destabilizing the β-sheet conformations.13 © 2012 American Chemical Society

Received: April 8, 2012 Revised: August 17, 2012 Published: August 17, 2012 2662

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Figure 1. Synthetic route of the polyA-FF-ME nanoparticles.

ization behavior, structures, and properties.42−44 Yet, to the best of our knowledge, there is no report on the preparation of polymeric nanoparticles from N-acryloyl amino acid monomers. The present article describes a novel designed synthesis of uniform biocompatible amino-acid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains. The dipeptide residues were designed similarly to the hydrophobic core sequence of Aβ. Poly(N-acryloyl-L-phenylalanyl-L-phenylalanine methyl ester) (polyA-FF-ME) nanoparticles of 57 ± 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2-methoxy ethanol, followed by precipitation of the obtained polymer in aqueous solution. These nanoparticles were well-characterized including their cytotoxic effect on different human cell lines, i.e., PC-12 (pheochromocytoma cell line) and SH-SY5Y (neuroblastoma cell line). Kinetics of the Aβ40 fibrillation process in the absence and the presence of varying concentrations of the polyA-FF-ME nanoparticles, and their analogues polyA-AA-ME nanoparticles containing the L-alanyl-L-alanine (AA) dipeptide instead of FF in the polymer side chains, have been elucidated. A significantly slow direct secondary structure transition from random coil to β-sheets during Aβ40 fibril formation was observed in the presence of the polyA-FF-ME nanoparticles. This observation indicates that these nanoparticles significantly increase the lag time and hence the kinetics of Aβ40 fibrillation. However, the opposite behavior was observed in the presence of the polyA-AA-ME nanoparticles, indicating acceleration of Aβ40 fibrillation kinetics.

the surface interfacial properties, including charge, of the nanoparticle and its enormous surface area to volume ratio. Increased local protein concentration on the nanoparticle surface and/or changes in protein conformation upon binding could promote aggregation while trapping of early intermediates may inhibit further aggregation.26 Engineered biocompatible nanoparticles offer advantages as therapeutic agents for amyloid-related diseases since they allow modification of surface properties so as to control the adsorption and interaction processes. Moreover, in vitro and in vivo studies have shown that nanoparticles are capable of overcoming the difficulty of crossing the BBB and have greater in vivo stability.27,28 Various nanoparticles such as copolymer particles of Nisopropylacrylamide/N-tert-butylacrylamide, cerium oxide, quantum dots (QDs), carbon nanotubes, and titanium oxide have been reported to promote protein assembly into amyloid fibrils in vitro, by assisting the nucleation process.29,30 Only a few studies have been reported on the inhibitory effect of nanoparticles on the Aβ fibrillation process. Very recently, Cabaleiro-Lago et al. reported the inhibition of the Aβ40 fibril formation by copolymer nanoparticles of variable hydrophobicity,31 and also demonstrated the dual effect of commercial polystyrene (PS) nanoparticles with amino modification toward the Aβ40 and Aβ42 fibril formation.32 Yoo et al. have shown an inhibition effect of CdTe QDs on Aβ40 fibrillation.33 Fluorinated nanoparticles34 and sulfonated and sulfated PS nanoparticles35 have been also reported as potential candidates for the inhibition of Aβ fibril formation. Our previous work showed that the Leu-Pro-Phe-Phe-Asp (LPFFD) peptide conjugated iron oxide nanoparticles slightly inhibit the Aβ40 fibrillation.36 Poly(amino acid) nanoparticles have recently attracted great attention due to their potential nontoxicity, biocompatibility, nonimmunogenicity, and biofunctionality. These nanoparticles containing amino acid moieties are potentially useful in many different biomedical applications, such as drug or gene delivery agents, tissue engineering scaffolds, and chiral recognition.37−40 The synthesis and radical polymerization of various acryl monomers having amino acid moieties in the side chain have been reported.41 Their corresponding polymers, poly(Nacryloyl amino acids), are expected to be functional materials, and much attention has been paid to their unique polymer-

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MATERIALS AND METHODS

Materials. The following analytical-grade chemicals were purchased from commercial sources and used without further purification: L-phenylalanyl-L-phenylalanine (FF), acetyl chloride, methanol anhydrous, acryloyl chloride, triethylamine (TEA), dimethylaminopyridine (DMAP), dry dichloromethane (DCM), hydrochloric acid (1 M), hexane, magnesium sulfate, sodium bicarbonate, sodium hydrogen sulfate, calcium chloride, 2-methoxyethanol, polyvinylpyrrolidone (PVP, MW 360 000), benzoyl peroxide (BP), amyloid β protein fragment 1−40 (Aβ40), trifluoroacetic acid (TFA), 1,1,1,3,3,3hexafluoro-2-propanol (HFIP), dimethyl sulfoxide (DMSO), and Thioflavin T (ThT) from Sigma (Israel); L-alanyl-L-alanine methyl ester (AA-ME) hydrochloride salt from D-Chem (Israel); Dulbecco’s modified eagle medium (DMEM), fetal calf serum (FCS), L-glutamine, penicillin, streptomycin, and 2,3-bis-(2-methoxy-4-nitro-5-sulfophen2663

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was then dried over MgSO4, filtered, and evaporated to produce white solid. Pure white crystals of N-acryloyl-AA-ME (A-AA-ME) were obtained by recrystallization of the white solid twice from DCM/ hexane (yield 44%). 1 H NMR (CDCl3, 300 MHz): δ 6.6 (broad doublet, J3 = 6.8 Hz, NH near the double bond, 1H), 6.3 (dd, J2 = 0.6 Hz, J3 = 16.8 Hz, 1H, CH2CH), 6.28 (broad, NH, 1H), 6.16−6.05 (dd, J3cis = 10.5 Hz, J3trans = 16.8 Hz, 1H, CH2CH), 5.69 (dd, J2 = 0.6 Hz, J3 = 10.5 Hz, 1H, CH2CH), 4.59 (double-quartet, J3 = 6.8 H, J3 = 7.2 Hz, 1H, C(O)NHCH), 4.54 (double-quartet, J3 = 6.8 H, J3 = 7.2 Hz, 1H, C(O)NHCH), 3.76 (s, 3H, OCH3), 1.43 (d, J3 = 6.8 3H, CHCH3), 1.41 (d, J3 = 7.2, 3H, CHCH3). 13C NMR (CDCl3, 200 MHz): δ 173.4 (C(O)NH, 1C), 172.0 (C(O)OCH3, 1C), 165.4 (C(O)CHCH, 1C), 130.6, 127.6 (2C, double bond), 52.9 (1C, OCH3), 49.0, 48.5 (2C, C(O)NHCHCH3), 18.8, 18.5 (2C, CHCH3). TOF MS ES+: 229 (MH+, 12%), 251 (MNa+, 100%). HRMS MALDI: 229.1215 (MH+). Elemental analysis (calculated): C, 52.62; N, 12.27; H, 7.07. Found: C, 51.96; N, 12.30; H, 7.02. Synthesis of PolyA-AA-ME Nanoparticles. The polyA-AA-ME was prepared by a similar procedure to that described above for the polyA-FF-ME, substituting the monomer A-FF-ME for the A-AA-ME. The formed soluble polyA-FF-ME was precipitated as uniform nanoparticles by adding 100 μL of its solution to 0.5 mL distilled water. The formed polyA-AA-ME nanoparticles were then washed by extensive dialysis against water. Dried polyA-AA-ME nanoparticles were then obtained by lyophilization. 1 H NMR (D2O, 300 MHz): δ 4.6−4.2 (broad, C(O)NHCH, 2H), 3.7−3.6 (broad, 3H, OCH3), 2.3−2.1 (broad, 1H, CHCH2), 2.1−1.8 (broad, 2H, CHCH2), 1.5−1.3 (broad 6H, CHCH3). Cytotoxicity Assay. XTT assay was performed to determine the cytotoxicity of the polyA-FF-ME nanoparticles on PC-12 and SHSY5Y cell lines.47 Cells were seeded in a 96-well plate at a density of 1 × 104 cells/well in 100 μL culture medium and grown in a humidified 5% CO2 atmosphere at 37 οC. After 18 h at 37 °C, different volumes of polyA-FF-ME nanoparticles dispersed in water were added to the cells, giving final concentrations of 0.02 and 0.2 mg/mL per well. After incubation for 24 or 48 h at 37 °C, 50 μL XTT solution was added to each well according to the kit manufacturer’s instructions. Absorbance was read at 480 nm, and the absorbance of corresponding concentrations of the nanoparticles was subtracted from the reading. Cell viability was determined as a percentage of the negative control (cultured cells in medium without nanoparticles). Preparation of the Aβ40 Fibrils in the Absence and Presence of the PolyA-FF-ME Nanoparticles or PolyA-AA-ME Nanoparticles. Lyophilized Aβ40, synthesized by Sigma Israel, was stored at −20 °C immediately upon arrival. To obtain a homogeneous solution free of aggregates, a variant of Zagorski’s protocol48 was followed, as discussed in our previous publication.36 Briefly, 0.5 mg of the Aβ40 was first dissolved with TFA, followed by TFA evaporation with N2. This process was then repeated two more times. To remove TFA thoroughly, HFIP was added and then evaporated with N2. This HFIP treatment was also repeated another two times. The dry aliquot was completely resuspended in a solution containing 0.5 M DMSO and 0.1 M PBS (pH 7.4) to a final volume of 2.88 mL, in order to reach a final Aβ40 concentration of 40 μM. Before use, all solutions were filtered through 0.20 μm pore size filters. For initiating the Aβ40 fibrillation process, samples of the Aβ40 solutions (40 μM) were incubated in 1.5 mL eppendorf tubes in a bath heater at 37 °C with gentle shaking. To monitor the appearance and growth of fibrils, aliquots from the tubes were taken at different times and added to a black 96-well plate, and the ThT fluorescence (20 μM ThT added to each well) was measured at 485 nm with excitation at 435 nm in a plate reader.49 A similar process to that described above was also performed in the presence of different concentrations of the polyA-FF-ME nanoparticles or polyA-AA-ME nanoparticles. Briefly, different volumes, 2.6−26 μL, (10−100% (w/wAβ40)) of an aqueous dispersion of nanoparticles (3.4 mg/mL) were added to 0.5 mL of 40 μM Aβ40 PBS solution, as described above. The formation of the fibrils was then initiated by

yl)-2H-tetrazolium-5-carboxanilide (XTT) cell viability assay kit from Biological-Industries (Israel). Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga, High Wycombe, UK). The cell lines PC-12 and SH-SY5Y were purchased from ATCC (USA). The culture medium used in this study was composed of 90% DMEM, 10% FCS, 2 mM L-glutamine, 50 units/mL penicillin, and 50 μg/mL streptomycin. Synthesis of A-FF-ME. The monomer A-FF-ME was synthesized by esterification of FF followed by its reaction with acryloyl chloride, according to a procedure reported previously,45,46 as shown in Figure 1 (steps I−II). Briefly, FF (1 g, 3.2 mmol) was dissolved in a flask containing anhydrous methanol (50 mL). The flask was immersed in an ice bath and acetyl chloride (5 mL) was then added dropwise. The reaction mixture was stirred for 2 h at 0 °C and then stirred at room temperature overnight. The obtained FF methyl ester (FF-ME) hydrochloride salt was then isolated by removal under reduced pressure of methyl acetate and methanol. Pure white brilliant crystals of FF-ME hydrochloride salt were obtained in quantitative yield (98%). The obtained FF-ME hydrochloride salt (1.08 g, 3.0 mmol) was dissolved in dried DCM (45 mL). TEA (890 μL, 6.4 mmol) and DMAP (40 mg, 0.32 mmol) were then added to the solution. The reaction mixture was immersed in an ice bath. Acryloyl chloride (410 μL, 5 mmol) dissolved in dry DCM (5 mL) was then added dropwise to the reaction mixture. The solution was stirred at room temperature overnight and then diluted with further amount of DCM (40 mL). The DCM solution was then washed with HCl (1 M) and brine. The organic phase was then dried over MgSO4, filtered, and evaporated to produce a white solid. Pure white crystals of N-acryloyl-FF-ME (A-FFME) were obtained by recrystallization of the white solid twice from DCM/hexane (yield 44%). 1 H NMR (CDCl3, 200 MHz): δ 7.26−7.17 (m, 8H, aromatic hydrogen), 6.95−7.00 (m, aromatic hydrogen, 2H), 6.3 (dd, J2 = 1.6 Hz, J3 = 16.8 Hz, 1H, CH2CH), 6.14 (m, NH, 2H), 6.1 (dd, J3cis = 10.0 Hz J3trans = 16.8 Hz, 1H, CH2CH), 5.65−5.70 (dd, J2 = 1.6 Hz, J3 = 10.0 Hz, 1H, CH2CH), 4.75 (m, 2H, C(O)NHCH), 3.68 (s, 3H, OCH3), 2.93−3.13 (m, 4H, PhCH2). 13C NMR (CDCl3, 200 MHz): δ 171.1 (C(O)NH, 1C), 170.1 (C(O)OCH3, 1C), 165.1 (C(O)CHCH, 1C), 130.2, 135.4 (2C, aromatic quaternary carbon), 130.2, 129.3, 129.1, 128.67, 128.60, 127.29 (aromatic carbon, 10C), 127.14, 127.08 (2C, double bond), 54.0, 53.2 (2C, C(O)NHCH), 52.2 (1C, OCH3), 38.1, 37.8 (2C, PhCH2). TOF MS ES+: 381 (MH+, 100%), 403 (MNa+, 50%). HRMS MALDI: 403.1628 (MNa+). Elemental analysis (calculated): C, 69.46; N, 7.36; H, 6.36; O, 16.82. Found: C, 68.22; N, 7.21; H, 6.47; O, 16.45. Synthesis of PolyA-FF-ME Nanoparticles. In a typical experiment, 30 mg of the vinylic monomer A-FF-ME, 10 mg PVP, and 2 mg of BP were added to 1 mL of nitrogen-bubbled 2-methoxy ethanol. The mixture was shaken at room temperature to dissolve the solids, giving concentrations of 3%, 1%, and 0.2% (w/v) A-FF-ME, PVP, and BP, respectively. The mixture was then shaken at 75 οC for 18 h (Figure 1, step III). The formed soluble polyA-FF-ME was precipitated as uniform nanoparticles by adding 100 μL of its solution to 1 mL distilled water (Figure 1, step IV). The formed polyA-FF-ME nanoparticles were then washed by extensive dialysis against water. Dried polyA-FF-ME nanoparticles were then obtained by lyophilization. 1 H NMR (CDCl3, 200 MHz): δ 7.3−6.8 (broad, aromatic hydrogens and amide hydrogens, 12H), 4.8−4.3 (broad, C(O)NHCH, 2H), 3.7−3.3 (broad, 3H, OCH3), 3.2−2.7 (broad, 4H, PhCH2), 2.4− 2.1 (broad, 1H, CHCH2), 2.1−1.8 (broad, 2H, CHCH2). Synthesis of A-AA-ME. The monomer A-AA-ME was synthesized by the reaction of commercial AA-ME hydrochloride salt with acryloyl chloride in cold water, according to a procedure that was reported previously.42 Briefly, the AA-ME hydrochloride salt (1 g, 4.74 mmol) was dissolved in water (10 mL). NaHCO3 (880 mg, 9.49 mmol) was then added to the solution. The reaction mixture was immersed in an ice bath. Acryloyl chloride (470 μL, 5.7 mmol) was then added dropwise to the solution. The chilled solution was vigorously stirred for 30 min. The solution was acidified with NaHSO4 (1 M) to pH 3 and was then washed three times with ethyl acetate. The organic phase 2664

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Figure 2. HRSEM image (A) and size histogram (B) of the polyA-FF-ME nanoparticles.

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quickly raising the temperature of the aqueous mixture from room temperature to 37 °C at different time intervals. Characterization. 1H and 13C NMR spectra were obtained with a Bruker AC 200 or 300 MHz spectrometer. Chloroform-d chemical shifts are expressed in ppm downfield from tetramethylsilane used as internal standard. High-resolution mass spectra were obtained on an AutoFlex III Tof/Tof (Bruker, Germany) in MALDI mode. Elemental analysis was performed using an elemental analyzer, model FlashEA1112 (Thermo Electron Corporation). Fourier transform infrared (FTIR) analysis was performed with a ALPHA FTIR spectrophotometer equipped with attenuated total reflection (ATR) model A220/D-01, Bruker. Low-resolution transmission electron microscopy (TEM) pictures were obtained with a FEI TECNAI C2 BIOTWIN electron microscope with 120 kV accelerating voltage. Samples for TEM were prepared by placing a drop of diluted sample on a 400-mesh carbon-coated copper grid. High-resolution scanning electron microscopy (HRSEM) images were obtained with a FEI Magellan 400 L electron microscope. Samples for HRSEM were prepared by spreading a drop of diluted sample on a glass surface and then drying it at room temperature. The dried sample was coated with gold in a vacuum for 4 min before viewing under HRSEM. The average size and size distribution of the dry nanoparticles were determined by measuring the diameter of more than 100 particles with the image analysis software AnalySIS Auto (Soft Imaging System GmbH, Germany). Hydrodynamic diameter and size distribution of the nanoparticles dispersed in an aqueous phase were measured using a particle analyzer, model NANOPHOX (Sympatec GmbH, Germany). The thermal behavior of the nanoparticles was measured by ThermoGravimetric Analysis (TGA) and differential scanning calorimetric (DSC), model STAR-1 System, Mettler Toledo. The analysis was performed with approximately 10 mg of dried samples in a dynamic nitrogen atmosphere (20 mL/min) at heating rate of 10 °C/ min. Fluorescence intensity and absorbance at room temperature were measured using a multiplate reader Synergy 4, using Gen 5 software. Electrokinetic properties (ζ-potential) as a function of pH were determined by Zetasizer (Zetasizer 2000, Malvern Instruments, UK). Circular dichroism (CD) spectra were measured on a Jasco J710 spectropolarimeter at 22 °C over the wavelength range 180−260 nm with 0.2 nm resolution. All measurements in solution were recorded in a 0.1 cm path length cell. Each spectrum is an average of four measurements. The spectra were corrected by subtracting the buffer solution or nanoparticles’ dispersion baseline. All samples of Aβ40 solutions were diluted with phosphate buffer (PB) solution to a final concentration of 10 μM. Aβ40 solutions sampled at long incubation times (120 and 336 h) when the fibrils were already formed were centrifuged prior to scanning.

RESULTS AND DISCUSSION Designed Synthesis of the polyA-FF-ME Nanoparticles. Recent progress in elucidating the structural properties of Aβ fibrils has enabled the design of inhibitors of Aβ fibril formation.14−21 In the present study, we designed new uniform biocompatible amino-acid-based polymer nanoparticles for inhibition of Aβ40 fibrillation process. The FF residues, the hydrophobic core of residues 19−20 of the Aβ protein, are well-known as crucial sequences for the formation of β-sheet structures that trigger the fibrillation process.14−18 It has been suggested that the Aβ assembly is partially driven by hydrophobic interactions through recognition between the FF pairs.14−18 Short peptides homologous to the hydrophobic core of Aβ, for example, LPFFD, were designed to bind specifically to full-length Aβ and used as inhibitors of its fibrillation process via the FF recognition motif.19−21 The rational of our design is to engineer polymer nanoparticles containing extremely high concentrations of the FF motif. This motif in the nanoparticles therefore should act as a recognition motif within Aβ and interferes in its fibril formation. Figure 1 describes the synthetic route of the monomer A-FF-ME, followed by its polymerization and precipitation to form the polyA-FF-ME nanoparticles. Characterization of the polyA-FF-ME Nanoparticles. Figure 2 presents the dry (A) and hydrodynamic (B) diameters and size distributions of the polyA-FF-ME nanoparticles as measured by HRSEM images and light scattering, respectively. The dry diameter is 57 ± 6 nm, while the hydrodynamic diameter is 200 ± 27 nm. The difference in the diameter measured by HRSEM and light scattering is due to the fact that the second method also takes into account the surface-adsorbed solvent (water) molecules. FTIR spectra of the monomer A-FF-ME (A) and the polyAFF-ME nanoparticles (B) are shown in Figure 3. The IR spectrum of the monomer A-FF-ME (Figure 3A) indicates stretching vibrations at 1757 and 1206 cm−1 corresponding to the methyl ester (ME) groups, and stretching vibrations at 1672, 1647, and 1552 cm−1 corresponding to the amide groups. The absorbance peak at 1624 cm−1 corresponds to the stretching vibration of CC bonds. The absorbance peaks from 2980 to 3070 and from 1456 to 1496 cm−1 are characteristic of the C−H stretching (aromatic and aliphatic) and CH2 groups, and peaks at 700 and 3269 cm−1 correspond to the stretching of C−C and N−H bonds, respectively. The IR 2665

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Figure 3. FTIR spectra of the monomer A-FF-ME (A) and the polyAFF-ME nanoparticles (B).

spectrum of the polyA-FF-ME nanoparticles (Figure 3B) is similar to that of the monomer except for the typical stretching vibration of CC bonds that is not present in the polyA-FFME nanoparticles. The chemical structures of the monomer AFF-ME and its corresponding polymer were also confirmed by 1 H NMR analysis (in CDCl3), as shown in Figure 4. The 1H NMR spectrum of the monomer (Figure 4A) reveals the typical peaks of the acrylic protons at 6.3−6.1 ppm and 5.70−5.65 ppm, the aromatic protons at 7.2 and 7.0 ppm, the amide protons at 6.1 ppm, the proton attached to chiral carbon atom at 4.75 ppm, the methylene protons at 3.1 ppm, and the ME protons at 3.68 ppm. The 1H NMR spectrum of the polyA-FFME nanoparticles (Figure 4B) reveals the complete absence of the acrylic protons at 5.5−6.5 ppm and the presence of broad peaks of the aromatic and amide protons at 7.3−6.8 ppm, the proton attached to chiral carbon atom at 4.8−4.3 ppm, the ME protons at 3.7−3.3 ppm, the methylene protons at 3.2−2.7 ppm, and the aliphatic chain protons of the polymer backbone at 2.4−1.8 ppm. The thermal behavior of the polyA-FF-ME nanoparticles is shown in Figure 5. The TGA curve exhibits a steep slope between 280 and 450 °C, indicating a 68% weight loss due to the polymer decomposition, leaving residual carbon. The Tm of the polyA-FF-ME nanoparticles measured by DSC is ca. 165 °C (data not shown). To learn about the stability of the polyA-FF-ME nanoparticles in aqueous medium, ζ-potential measurements can be employed as an indirect indicator of their surface charge. Figure 6 illustrates the ζ-potential curve of the polyA-FF-ME nanoparticles as a function of pH. The titration curve of the polyA-FF-ME nanoparticles, obtained by adding HCl to an aqueous dispersion of the polyA-FF-ME nanoparticles, shows an increase of the ζ-potential from −13 to 2 mV while changing the pH from 11 to 2. This change in the surface potential can be attributed to the surface ME groups of the polyA-FF-ME nanoparticles. In both basic and acidic environments, the ME groups undergo partial hydrolysis. In a basic environment, the surface charge potential is negative due to hydrolysis of ME groups to CO−O−, whereas in an acidic environment, the surface charge potential is slightly positive due to the protonation of the carboxyl groups.50 The measured isoelectric point of the polyA-FF-ME nanoparticles is at pH 4.2 as shown in Figure 6.

Figure 4. 1H NMR spectra (in CDCl3) of the monomer A-FF-ME (A) and the polyA-FF-ME nanoparticles (B).

Figure 5. TGA thermogram of the polyA-FF-ME nanoparticles.

In Vitro Cytotoxicity Study of polyA-FF-ME Nanoparticles. The cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g., PC-12 and SHSY5Y, was examined using XTT cell viability assay, as shown in 2666

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Figure 8. Kinetics of the Aβ40 fibrils formation in PBS at 37 °C in the absence (A) and presence of 10 (B), 20 (C), and 100 (D) % (w/ wAβ40) of the polyA-FF-ME nanoparticles. The kinetics of the Aβ40 fibril formation were measured using ThT binding assay, as described in the Materials and Methods. The reported values are the averages of five measurements of each triplicate tested sample. The error bars indicate the standard deviation.

Figure 6. ζ-potential of the polyA-FF-ME nanoparticles as a function of pH. The reported values are the average of five measurements. The error bars indicate the standard deviation.

Figure 7. This figure shows the cell viability % in the presence of different concentrations of the nanoparticles after incubation

that the stability against agglomeration of these nanoparticles was found to be dependent at the ionic strength of the PBS, e.g., in 0.1 M PBS, they are stable, while in 0.5 M PBS, they aggregate. Due to this instability issue, the kinetics was accomplished in the presence of 0.1 M PBS as a continuous phase. In the absence of the nanoparticles (Figure 8A), the main growth of the Aβ40 fibrils, corresponding to the end of the lag time, occurred approximately 60 h after initiating the fibrillation process, and was completed after approximately 120 h. On the other hand, the kinetics of the Aβ40 fibril growth in the presence of increasing concentrations of the polyA-FF-ME nanoparticles was significantly delayed (Figure 8B−D). For example, Figure 8 exhibits that the presence of 10 (B), 20 (C), and 100 (D) % (w/wAβ40) of the polyA-FF-ME nanoparticles inhibits the initiation of the fibril formation to be only after 85, 99, and 233 h, respectively. These observations indicate that the polyA-FF-ME nanoparticles substantially increase the fibrillation lag time and thereby inhibit the kinetics of the nucleation and oligomer formation prior to the formation of the Aβ40 fibrils. This inhibitory effect might be explained by the presence of the pairs of FF residues of these nanoparticles, which are known for their high affinity to the corresponding residues of Aβ40 prefibril aggregates, e.g., monomers and oligomers, through hydrophobic interactions. This high binding affinity disturbs the monomer-critical nuclei equilibrium by trapping the monomers and/or blocking the growing oligomer ends on the surface of the nanoparticles, thereby decreasing their solution concentration and interfering with their elongation to form fibrils. However, the ability to distinguish whether these nanoparticles adsorbed the monomers, or oligomers, or both, remains to be investigated. Our results imply that the nucleation process of Aβ40 is strongly disturbed by the presence of the polyA-FF-ME nanoparticles assuming a tight interaction between the nanoparticles and Aβ40 prefibril intermediates. A similar effect was reported for the fibrillation process of Aβ and islet amyloid polypeptide (IAPP) proteins in the presence of copolymeric Nisopropylacrylamide/N-tert-butylacrylamide nanoparticles with different ratios between these monomers having variable hydrophobicity.25,26,31 In these studies, the retardation of the

Figure 7. Viability of PC-12 and SH-SY5Y cells exposed to increasing concentrations of polyA-FF-ME nanoparticles after 24 or 48 h at 37 °C, as determined by the XTT assay described in the Materials and Methods. The reported values are the average of measurements performed on at least eight wells for each tested sample of two independent experiments, and expressed as a percentage of the negative control (cultured cells in medium without nanoparticles). The error bars indicate the standard error of the mean.

for 24 or 48 h. After 24 h of exposure of the PC-12 and SHSY5Y cells to the nanoparticles, no significant toxicity was observed for nanoparticle concentration of 0.2 mg/mL (concentration used for our fibrillation studies) or even ten times higher (ca. 2.0 mg/mL). Minor toxicity was observed after 48 h of exposure to nanoparticle concentration of 0.2 mg/ mL for PC-12 only, and to the higher concentration of 2.0 mg/ mL for both cell lines, with approximately 80% cell viability. Effect of polyA-FF-ME Nanoparticles on the Kinetics of Aβ40 Fibrillation Process. Kinetics of the Aβ40 fibril formation in PBS at 37 °C, monitored by the temporal development of ThT binding,49 in the absence (A) and presence (B−D) of increasing concentrations of the polyA-FFME nanoparticles are shown in Figure 8. It should be noted 2667

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fibrillation process was also explained by the depletion of free monomers and oligomers (prefibrils) from solution due to their adsorption on the nanoparticle surface. This observation led to an increase in the lag time of the fibrillation process but did not prevent the fibril formation. It should be noted that, until recently, it was assumed that Aβ had to be assembled into amyloid fibrils to exert its cytotoxic effect.14,16,21 However, recent studies have revealed that the soluble oligomers are significantly more toxic to neuronal cells than the monomers and the formed fibrils.17,20,21,25,35 Therefore, there is great interest in developing nanoparticles that are able to disturb the monomer-critical nuclei equilibrium by trapping active monomers and prefibril intermediates on the nanoparticle surface, thereby delaying further growth, thereby reducing their toxicity in solution.31,33−35 Our assumption that the observed inhibitory effect in the presence of the polyA-FF-ME nanoparticles is due to specific hydrophobic interactions between the nanoparticles and the Aβ prefibril aggregates, via the FF recognition motif, was examined by replacing the polyA-FF-ME nanoparticles by polyA-AA-ME nanoparticles. The chemical structures of the monomer A-AAME and its corresponding polymeric nanoparticles were confirmed by 1H NMR analysis, as shown in the Supporting Information, Figure S1. The dry and hydrodynamic diameters of the polyA-AA-ME nanoparticles are 52 ± 8 and 214 ± 23 nm, respectively, which are similar to those obtained for the polyA-FF-ME nanoparticles, as shown in the Supporting Information, Figure S2. Figure 9 presents typical sigmoidal

nanoparticles. Therefore, the increased local concentration of the Aβ40 prefibrils adsorbed on the polyA-AA-ME nanoparticle surface promoted their growth into fibrils. It is important to note that the promoting effect achieved in the presence of the polyA-AA-ME nanoparticles may also be considered a desired process. The polyA-AA-ME nanoparticles decrease the fibrillation lag time, thereby shorting the half-life time of the Aβ40 prefibrils, thereby decreasing their toxicity in solution. The secondary conformational change of Aβ40 from α-helix and/or random coil to β-sheet is probably the key step in the process to form oligomeric aggregates.11 Hence, CD measurments were performed to determine the secondary structure changes during the Aβ40 fibril formation in the absence and the presence of the polyA-FF-ME nanoparticles. Figure 10 presents

Figure 10. CD spectra of the Aβ40 in the absence (A) and the presence (B) of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles dispersed in PB solution at different time periods of the fibrillation process.

Figure 9. Kinetics of the Aβ40 fibril formation in PBS at 37 °C in the absence (A) and presence of 10 (B), 20 (C), and 100 (D) % (w/ wAβ40) of the polyA-AA-ME nanoparticles. The kinetics of the Aβ40 fibril formation was measured using ThT binding assay, as described in the Materials and Methods. The reported values are the average of five measurements of each triplicate tested sample. The error bars indicate the standard deviation.

CD spectra of the Aβ40 in the absence (A) and the presence (B) of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles at different time periods during the fibrillation process. In the absence of the nanoparticles (Figure 10A) before the initiation of the fibrillation process, the CD spectrum of the freshly prepared Aβ40 aqueous solution at pH 7.4 shows one strong minimum at 198 nm indicative of a typical spectrum of a native protein dominated by random coil conformation, as described in the literature.36,51,52 By heating the Aβ40 solution to 37 °C for 120 h, the CD spectrum of the Aβ40 displays a loss of intensity of the 198 nm peak accompanied by a maximum ellipticity at 200 nm and a broad minimum ellipticity at 217 nm, which is typical of the presence of extensive β-sheet structures. Almost the same CD spectra of Aβ40 were observed in the presence of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles. It is rather interesting to note that the CD

curves showing the kinetics of the Aβ40 fibrils formation in PBS at 37 °C in the absence (A) and the presence of 10 (B), 20 (C), and 100 (D) % (w/wAβ40) of the polyA-AA-ME nanoparticles. It is rather interesting to note that the observed behavior is the opposite of the behavior obtained in the presence of similar concentrations of the polyA-FF-ME nanoparticles. The kinetics of the Aβ40 fibrils growth in the presence of 10, 20, and 100% (w/wAβ40) were accelerated, and initiated after 53, 38, and 18 h, respectively (see Figure 9). This promoting effect probably results from the absence of the FF interfering moieties in these 2668

dx.doi.org/10.1021/bm3011177 | Biomacromolecules 2012, 13, 2662−2670

Biomacromolecules

Article

Figure 11. TEM images of the Aβ40 in the absence of the polyA-FF-ME nanoparticles, 120 h after initiation of the fibrillation process (A), and in the presence of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles dispersed in PBS, 200 h (B), 233 h (C), and 330 h (D) after initiation of the fibrillation process.

absence of the nanoparticles. These observations indicate the trapping of amorphous Aβ40 aggregates by their adsorption on the surface of the polyA-FF-ME nanoparticles through specific interactions, leading to their partial depletion from solution which interferes with their elongation into massive fibrils.

spectrum of the freshly prepared Aβ40 aqueous solution in the presence of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles (Figure 10B) exhibits one strong minimum at 198 nm and a weak maximum at 224 nm, which are stable for more than 233 h of heating. Only beyond that time does the transformation to β-sheet structure start to occur. To further confirm the achieved inhibitory effect on the Aβ40 fibril formation in the presence of the polyA-FF-ME nanoparticles, TEM images were obtained to detect morphological changes in Aβ40 solution, as shown in Figure 11. In the case of control Aβ40 solution, in the absence of nanoparticles (Figure 11A), massive networks of entangled fibrils with microsized lengths were observed after 120 h of the fibrillation process. On the other hand, when Aβ40 solution was incubated in the presence of 100% (w/wAβ40) of the polyA-FF-ME nanoparticles, only free nanoparticles in the background were observed even after 200 h of the fibrillation process (Figure 11B), indicating that the formation of the fibrils had not yet occurred. After 233 h (Figure 11C), absorption of amorphous Aβ40 aggregates on the surface of the polyA-FF-ME nanoparticles was initiated. The internal dark and bright areas around the nanoparticles (as indicated by white arrows) illustrate the adsorbed dense and less dense multiple layers of the amorphous prefibril aggregates. It is further interesting to note that after 330 h (Figure 11D) the formed fibrillar networks were much less massive than those observed in the

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CONCLUSIONS The present manuscript describes the design, synthesis, and characterization of novel polyA-FF-ME nanoparticles, by polymerization of the synthesized monomer A-FF-ME, followed by precipitation of the soluble polymer in aqueous solution. These studies demonstrate the significant inhibition of the Aβ40 fibrillation process in the presence of these nanoparticles. This inhibition is probably due to the intermolecular attractive hydrophobic interactions between the pairs of FF residues of the nanoparticles with the corresponding residues of the Aβ40 prefibrillar aggregates, which disrupt the self-assembly of Aβ40 into fibrils. This observation was confirmed by the promoting effect achieved in the presence of the polyA-AA-ME nanoparticles. The present studies were performed using pure Aβ40 without competition from other proteins for binding to the nanoparticle surface. These conditions are quite unlike those in any realistic clinical situation. Still, our studies intend to provide a new mechanistic insight into amyloidogenic peptide interaction with 2669

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nanoparticles. Further work would be necessary to assess the suitable mechanistic interference of the polyA-FF-ME nanoparticles and the polyA-AA-ME nanoparticles with the Aβ40 self-assembly. In future work, we wish to extend this study to other amyloidogenic proteins under competitive conditions.

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ASSOCIATED CONTENT

* Supporting Information S

1

H NMR spectra of the monomer A-AA-ME and the polyAAA-ME nanoparticles. TEM image and size histogram of the polyA-AA-ME nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 972-3-5318861. Fax: 972-3-6355208. Present Address ‡

Currently on sabbatical from Israel Institute for Biological Research, Ness-Ziona 74100, Israel. Author Contributions †

These authors contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS These studies were partially supported by a Minerva Grant (Microscale & Nanoscale Particles and Films). REFERENCES

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