Inhibition of Protein Aggregation by Iron Oxide Nanoparticles

performed using an Olympus IX81 microscope using image-pro plus version 7.0 software. RESULTS AND DISCUSSION ..... CSIR (Grant number 02(0249)15/EMR-I...
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Inhibition of Protein Aggregation by Iron Oxide Nanoparticles Conjugated with Glutamine- and Proline-Based Osmolytes Nibedita Pradhan,† Nihar R. Jana,*,‡ and Nikhil R. Jana*,† †

Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata 700032, India Cellular and Molecular Neuroscience Laboratory, National Brain Research Centre, Manesar, Gurgaon 122051, India

‡

S Supporting Information *

ABSTRACT: Osmolytes are small organic biomolecules utilized by cells and organisms to counter unfavorable physiological conditions that challenge protein stability and function. Among them, some osmolytes are shown to prevent protein aggregation and act as chemical chaperones. We recently showed that nanoparticle forms of sugar-based osmolytes can enhance their chaperone performance typically by 1000−100 000 times. Here, we show that the nanoparticle form of amino acid-based osmolytes such as glutamine and proline can inhibit protein aggregation 1000−10 000 times better than respective molecular glutamine and proline. Specifically, we designed a glutamine/proline-conjugated nanoparticle with zwitterionic surface charge for best performance in inhibiting lysozyme aggregation in extracellular space and inhibiting mutant huntingtin protein aggregation in intracellular space. This result indicates that an efficient artificial chaperone can be made to inhibit protein aggregation using the nanoparticle form of osmolytes and other antiamyloidogenic molecules. KEYWORDS: nanoparticle, osmolyte, proline, polyglutamine, amyloid, protein aggregation

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INTRODUCTION

A variety of antiamyloidogenic chemicals and biochemicals are known which can inhibit protein aggregation and lower the toxicity of protein aggregates.9−17 Prominent examples include curcumin,9 green tea polyphenols,10 sialic acid,11 peptides,12 metal ion chelators,13 nanoparticles,14,15 sugar-based osmolytes such as trehalose,16 and amino acid-based osmolytes such as proline.17 Among them, small molecules osmolytes are unique class of materials that are known to stabilize protein under environmental stress.18−20 These include carbohydrates (e.g., glucose, sucrose, trehalose, and sorbitol), amino acids or their derivatives (e.g., glycine, proline, glutamine, ecotine, taurine, hypotaurine, betaine), and methylammonium and methyl sulfonium solutes (e.g., trimethylamine N-oxide). In particular, osmolyte proline has a profound effect in stabilizing aggregation-prone proteins and in preventing their aggregation in vitro.17 However, nearly molar concentration of proline is required for such effect, which is difficult to achieve inside cell cytoplasm or in extracellular space. Similarly, glutamine acts as an osmolyte for survival of bacteria and other animals under

Deposition of insoluble amyloid protein aggregates/plaque is the pathological hallmark of variety of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease.1,2 More than 20 different human peptides/proteins have been identified which misfold and form pathological amyloid deposits and aggregates in intracellular and extracellular space of the brain.3−5 For example, expansion of polyglutamine repeats within Huntingtin protein causes adult onset of Huntington’s disease characterized by cognitive dysfunction, movement disorder, and various psychiatric symptoms.3 Similarly, a large number of senile plaques rich in amyloid β- and α-synuclein fibrils are responsible for Alzheimer’s and Parkinson’s disease, respectively.4,5 Most of these protein aggregates are rich in cross-β sheet structure and are highly neurotoxic, which causes neuronal cell death. Current research focuses on the diagnostic tool for early detection of disease, understanding of protein aggregation mechanisms, origin of cytotoxicity by protein aggregates, and identification of chemicals and biochemicals that can inhibit protein aggregation and decrease cytotoxicity of protein aggregates.6−8 © XXXX American Chemical Society

Received: December 3, 2017 Accepted: February 23, 2018 Published: February 23, 2018 A

DOI: 10.1021/acsanm.7b00245 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials osmotic stress.21,22 It is also reported that hydrogen bonding between glutamine residues plays a key role in intermolecular association and aggregation of glutamine-rich proteins.23 Recent works show that designed functional nanoparticles can efficiently inhibit protein aggregation.24−29 In particular, nanoparticles were functionalized with multiple affinity molecules for strong interaction with aggregating protein and efficient inhibition of protein aggregation. We recently showed that the nanoparticle form of green tea polyphenol is 10−100 times efficient than its respective molecular form,30 and the nanoparticle form of sugar-based osmolytes are 103−105 times better than molecular sugars in inhibiting protein aggregation.31,32 Using these approaches, we designed a nanoparticle that can inhibit protein aggregation in intra/extracellular space and suppress protein aggregation in the mouse brain.32 Here, we demonstrate that amino acid-based osmolytes can enhance their efficiency typically up to 10 000 times, if their nanoparticle forms are appropriately designed. In particular, we designed zwitterionic nanoparticles terminated with glutamine/proline and found that they are 1000−10 000 times more effective than respective molecular glutamine/proline in inhibiting protein aggregation in intra- and extracellular space.

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temperature for 1 h, and then particles were precipitated by adding few drops of ethanol. The particles were washed with chloroform and ethanol and finally dissolved in 5.0 mL of distilled water. Functionalization of Nanoparticle with Glutamine/Proline for Preparation of Nanoglutamine/Nanoproline. Polymer coated nanoparticles were transformed to glutamine conjugated nanoparticles by covalent linking between primary amines on the nanoparticle surface and primary amine of (L)-glutamine. At first, 1.0 mL of abovementioned polymer coated nanoparticle solution was mixed with 0.2 mL of borate buffer of pH 9.0. In a separate vial, 1.0 mL of glutamine (0.01 M) was prepared in borate buffer solution of pH 9.0 and mixed with 0.5 mL of ethanolic solution of glutaraldehyde (0.01 M). After 15 min, 100 μL of this mixture was added to 0.5 mL of polymer coated nanoparticle solution. After 1 h, NaBH4 solution was added to reduce the imine bond formed by the reaction between the aldehyde and amine. After 1 h, nanoparticles were precipitated by adding acetone followed by high speed centrifugation at 12 000 rpm. Finally, particles were redissolved in 0.5 mL of fresh water, and the solution was dialyzed for 24 h against water using a dialysis membrane (MWCO 12−14 kDa) to remove unbound reagents. Proline conjugated nanoparticles were prepared from polymer coated nanoparticles by covalent linking between primary amines of nanoparticles and carboxylic acid of proline. Polymer coated, amine terminated nanoparticles were covalently conjugated with (L)-proline via EDC coupling. Typically, 0.1 mL aqueous solution of L-proline (0.01 M) was added to 0.1 mL MES buffer of pH 4.5 and then mixed with 0.1 mL of N-hydroxy-succinamide (0.05 M) and 0.1 mL of EDC (0.05M). Immediately, a mixture of 0.5 mL of polymer coated nanoparticle solution and 0.1 mL of borate buffer of pH 9 was added. The mixture was allowed to stir overnight and finally dialyzed for 1 day against water using a dialysis membrane (MWCO 12−14 kDa) to remove unbound reagents. Extent of conjugated glutamine/proline in nanoparticle solution was measured by estimating the primary amines present on the nanoparticle surface before and after the glutamine/proline conjugation.32 Primary amines were estimated using well-known fluorescamine-based titration method. First, a calibration curve was prepared using glycine as a primary amine standard. Typically, 0.9 mL of borate buffer solution of glycine (in the concentration range of 1−50 μM) was mixed with 100 μL of acetone solution of fluorescamine (0.4 M). Next, fluorescence intensity of each sample was measured at 485 nm (by exciting at 400 nm), and the calibration curve was prepared by plotting the fluorescence intensities against the glycine concentration. The obtained calibration curve was as follows: Y = 5 × 10−10X − 3 × 10−6, where X is the fluorescence intensity and Y is the primary amine concentration with the R2 value of 0.99. Next, borate buffer solution of nanoparticles was mixed with excess fluorescamine, and fluorescence intensity was then measured at 485 nm (by exciting at 400 nm). After the fluorescence intensity was put into the calibration curve, the primary amines on the nanoparticle surface were estimated. Similarly, primary amines were estimated after functionalization with glutamine/ proline, and the difference in the amount of primary amine concentration before and after functionalization was considered as glutamine/proline bound to nanoparticles. The tentative number of glutamine/proline per nanoparticle was also calculated using the method described earlier.32 Lysozyme Aggregation Study in the Presence of Nanoglutamine and Nanoproline. Protein aggregation was studied using HEWL as model protein. As lysozyme aggregation is slow at physiological condition, we used higher temperature and lower pH condition.33 Typically, 1 mL of HEWL solution (1 mg/mL, 70 μM) was prepared in 0.01 (N) acetate buffer of pH 5 and containing 140 mM NaCl and 2.7 mM KCl. HEWL solution was then magnetically stirred at 60 °C for 24 h, and aggregation kinetics was monitored using thioflavin T-based titration. Typically, 10 μL of protein solution was collected at different time intervals and mixed with 0.5 mL of phosphate buffer (pH 7.4) solution of thioflavin T (10 μM), and after 5 min, thioflavin T fluorescence was measured under 440 nm excitation. To study the effect of nanoparticles on the fibrillation

EXPERIMENTAL SECTION

Materials. (L)-glutamine, (L)-proline, thioflavin T (ThT), hen egg white lysozyme (HEWL), octadecylamine, octadecence, methyl morpholine N-oxide, Fe-stearate, Igepal CO-520, glutaraldehyde, sodium borohydride, ponasterone A > 65%, dialysis tube (MWCO 12000 Da), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma-Aldrich. All the reagents were used without further purification. Generation of HD150Q cell line was described elsewhere.33 GFP antibody was purchased from Roche, and all reagents for SDS-PAGE were purchased from Sigma-Aldrich. Synthesis of Polymer Coated γ-Fe2O3 Nanoparticle. The synthetic method of polyacrylate coated nanoparticle was followed from a reported method.32 At first, hydrophobic γ-Fe2O3 nanoparticle is synthesized and then converted into polyacrylate coated nanoparticle. In brief, octadecylamine (0.37 g), methyl morpholine N-oxide (0.16 g), and 10 mL of octadecene were taken in 250 mL three-necked round-bottomed flask and kept under magnetic stirring. The solution was purged with argon for 15−30 min and then heated at 300 °C under argon, and then octadecene solution of Fe-stearate (0.32 g dissolved in 4 mL of hot octadecene) was injected. Heating was stopped after 1 min and allowed to cool to room temperature. Resultant hydrophobic γ-Fe2O3 nanoparticles were separated from excess reagents by standard methods. Typically, particles were precipitated by acetone/ethanol and separated by centrifuge, and the precipitate was dissolved in CHCl3. This precipitation−redispersion was repeated 3−4 times, and finally, particles were dissolved in 10 mL of cyclohexane prior to polymer coating. For polyacrylate coating, hydrophobic γ-Fe2O3 nanoparticles and acrylate monomers were dissolved in Igepal-cyclohexane reverse micelle, and then polymerization was initiated under nitrogen atmosphere in the presence of persulfate. Typically, reverse micelle was prepared by adding 500 μL of Igepal to 8 mL of cyclohexane, and then 0.5 mL of cyclohexane solution of hydrophobic iron oxide nanoparticles was added. Next, 9 mg of N-(3-aminopropyl) methacrylamide hydrochloride in 100 μL of water, 12.5 mg of 3sulfopropyl methyl acrylate in 100 μL of water, 36 μL of poly(ethylene glycol) methacrylate in 100 μL water, 100 μL aqueous solution of bis[2-(methacryloyloxy) ethyl] phosphate (6 μL dissolved in 100 μL water), and 100 μL of base (tetramethyl ethylenediamine) were added. An optically clear solution was formed, and the solution was taken in a three necked flask and put under oxygen-free atmosphere by purging with nitrogen for 15 min. Finally, ammonium persulfate solution (3 mg dissolved in 100 μL water) was injected as a radical initiator to start the polymerization. The polymerization was continued at room B

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ACS Applied Nano Materials kinetics, the nanoparticle solution was mixed with HEWL solution before initiating the aggregation. Polyglutamine Aggregation Study Inside the HD150Q Cell in the Presence of Nanoglutamine. We used HD150Q cell as model for studying the role of nanoglutamine in inhibiting polyglutamine-based protein aggregation inside the cell.34 HD150Q cell is a stable and inducible mouse neuroblastoma cell line, expressing green fluorescent protein tagged mutant huntingtin protein with 150 glutamine residues. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and kept in a incubator at 37 °C with 5% CO2 environment. Penicillin and streptomycin were added to the medium. The cells were plated at a density of 100 000 cells/well on 24-well plates in 0.5 mL of medium. After 24 h, the cells were given fresh media and mixed with nanoparticle solution (or amino acids) for 2 h. Then, ponasterone A solution (final concentration 1 μM) was added to induce polyglutamine aggregation within the cells, which were incubated at 37 °C for different times. This condition was maintained until cell death. Aggregation of protein was monitored by fluorescence microscopy. For immunoblot analysis, cells were washed with chilled phosphatebuffered saline (PBS), collected by centrifugation, and then sonicated on ice for 30 min with Nonident P-40 lysis buffer (consist of 50 mM Tris buffer of pH 8.0, 150 mM NaCl, 1% NP-40, and complete protease inhibitor cocktail). Cell lysate was then centrifuged for 10 min at 15 000g at 4 °C, and the supernatant was used for SDS-PAGE followed by immunoblot analysis as previously described.32 Finally, the blot was probed with GFP antibody. For dot blot analysis, HD150Q cells were homogenized by homogenization buffer (50 mM Tris buffer of pH 7.4, 150 mm NaCl, 1% Triton X-100, 1 mM PMSF, and complete protease inhibitor tablet), sonicated briefly, and then centrifuged at 13 000g for 15 min. Pellet was collected and treated with 2% SDS at room temperature for 5 min and quantified, and an equal amount of protein was allowed to filter through nitrocellulose membrane. Membranes were then probed with GFP antibody. Instrumentation. All UV−visible spectra were measured in a Shimadzu UV-2550 UV visible spectrophotometer using a quartz cell of 1 cm path length. Fluorescence spectra were measured in a BioTek Synergy MX microplate reader. Fourier transform infrared spectroscopy on KBr pellet was performed using PerkinElmer Spectrum 100 FTIR spectrometer. DLS and ζ-potential studies were performed using NanoZS (Malvern) instrument. CD spectra were measured using a CD spectrometer (Jasco, model J-815-1508). TEM study was done using an FEI Tecnai G2 F20 microscope with a field-emission gun operating at 200 kV. Differential interference contrast and fluorescence images of live cells were performed using an Olympus IX81 microscope using image-pro plus version 7.0 software.

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Scheme 1. Synthetic Strategy for Nanoglutamine and Nanoprolinea

a

Hydrophobic iron oxide nanoparticles are transformed into polyacrylate coated and primary amine terminated zwitterionic nanoparticles and then converted to nanoglutamine by glutamine conjugation or nanoproline by proline conjugation.

with primary amine, SO3−, and PEG.32 Next, primary amines on the nanoparticle surface have been used to conjugate with glutamine and proline. Glutamine is conjugated via glutaraldehyde-based coupling, and proline is conjugated via EDC coupling (Scheme 1). Hydrodynamic size of nanoglutamine is 30−50 nm and that of nanoproline is 20−40 nm. These values are larger than that of parent nanoparticles (20−30 nm) due to the partial aggregation of nanoparticles after functionalization and particle−particle cross-linking during conjugation. The surface charge of functional nanoparticles arises due to SO3− and primary amines. In particular, primary amines are protonated at acidic pH to generate cationic ammonium ion and thus ζ-potential changes depending on solution pH. Specifically, nanoproline has close to zero ζ-potential value at pH 7.4 but it changes to +12 mV at pH 4.5 and attains −4 mV at pH 10.0. Similarly, nanoglutamine has ζ-potential value of −13 mV at pH 7.4 but it changes to −6 mV at pH 4.5 and attains −18 mV at pH 10.0. We tested the colloidal stability of nanoglutamine and nanoproline at physiological conditions and at acidic pH or higher temperature that is used for lysozyme fibrillation study. We observed reasonably good colloidal stability at phosphate buffer solution of pH 7.4, acetate buffer of pH 4.5, and at a temperature range of 37−60 °C (Supporting Information, Figure S1). Thus, nanoglutamine and nanoproline have zwitterionic surface features. We showed earlier that this type of zwitterionic surface charge is preferred over cationic or anionic surface charge for efficient inhibition of protein aggregation inside cell with minimum cytotoxicity.32 Evidence of presence of glutamines in nanoglutamine and presence of proline in nanoproline was investigated using FTIR and mass spectroscopy (Figure 1 and Supporting Information, Figure S2). In addition, primary amines present on the nanoparticle surface were estimated before and after glutamine/proline functionalization via fluorescamine titration. This gives an estimate of the amount of conjugated glutamine/proline, and it was found that 1 mg/mL solution of nanoglutamine contains ∼50 μM

RESULTS AND DISCUSSION

Properties of Nanoglutamine and Nanoproline. Chemical composition of glutamine and proline functionalized nanoparticles is shown in Scheme 1, and their properties are summarized in Figure 1 and Table 1. Nanoparticles have 20−30 nm hydrodynamic size with 5−6 nm γ-Fe2O3 core and 7−12 nm polymer shell with covalently linked glutamine/proline. Iron oxide nanoparticles were selected as they are widely used in different biomedical applications, and functionalization with glutamine/proline can be adapted easily.32 The polymer shell has chemical functional groups such as polyethlylene glycol (PEG), anionic SO3− groups, and primary amines. Glutamine functionalized nanoparticles are abbreviated as nanoglutamine, and proline functionalized nanoparticles are abbreviated as nanoproline. At first, 5−6 nm hydrophobic γ-Fe2O3 nanoparticles were synthesized and then transformed to watersoluble nanoparticles via polyacrylate coating. Appropriate ratios of different acrylate monomers were used during polyacrylate coating so that the polymer shell is terminated C

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Figure 2. Effect of nanoglutamine and nanoproline in inhibiting HEWL fibrillation as observed via thioflavin T fluorescence assay. (a) Fibrillation kinetics of HEWL in the presence of (1) 1.0 mg/mL control nanoparticles without glutamine functionalization, (2) 100 mM molecular glutamine, (3) 0.2 mg/mL nanoglutamine with 10 μM glutamine, (4) 0.8 mg/mL nanoglutamine with 40 μM glutamine, and (5) 1.0 mg/mL nanoglutamine with 50 μM glutamine. (b) Fibrillation kinetics of HEWL in the presence of (1) 1.0 mg/mL control nanoparticles without proline functionalization, (2) 100 mM molecular proline, (3) 0.5 mg/mL nanoproline with 10 μM proline, (4) 1.5 mg/mL nanoproline with 30 μM proline, and (5) 3.0 mg/mL nanoproline with 60 μM proline. Each data point is an average of three independent measurements.

Figure 1. (a) TEM images of polymer coated iron oxide nanoparticle showing the core size of 5−6 nm. (b) XRD pattern of polymer coated iron oxide nanoparticle showing the reflections corresponding to 2θ = 30.65°, 35.94°, 43.55°, 53.35°, 57.58°, and 63.22°, which are due to the (220), (311), (400), (422), (511), and (440) planes of γ-Fe2O3. (c) Hydrodynamic size distribution of polymer coated zwitterionic nanoparticles, nanoglutamine, and nanoproline at pH 7.4 as observed by DLS. (d) FTIR spectra of polymer coated zwitterionic nanoparticles, nanoglutamine, and nanoproline. Characteristic amide stretching vibration band at 1643 cm−1 is observed in nanoglutamine. Similarly, amide stretching vibration band at 1624 cm−1 is observed for nanoproline in addition to cyclic bending for proline C−H at 1400 cm−1. (e) MALDI-MS spectra of nanoglutamine, glutamine, and zwitterionic nanoparticles. The characteristic fragmented exact mass of glutamine for C13H27N3O3 (MW 273.2052) appears at 273.262. (f) Fluorescamine test of zwitterionic nanoparticles before functionalization (1) and after functionalization with glutamine (2) and proline (3), showing that a fraction of primary amines are consumed after functionalization.

protein that produces highly ordered fibrillar morphology. Typically, HEWL monomer is coincubated with nanoglutamine/nanoproline (or molecular glutamine/proline/control nanoparticle) under the fibrillation condition, and fibril formation is monitored by using thioflavin T fluorescence assay, circular dichroism spectroscopy, and TEM imaging (Figures 2−4). We tested that fluorescence of thioflavin T is not quenched by the nanoparticle. This is expected as the nanoparticle is coated with polymer that minimizes direct interaction of thioflavin T with iron oxide core (Supporting Information, Figure S3). Thioflavin T-based kinetics show that HEWL fibrillation process follows three distinct steps that include (i) nucleation, (ii) elongation/growth, and (iii) steady phase (Figure 2). The typical lag time for nucleation is ∼30 min followed by an elongation phase for 1−12 h, and then steady state continues up to 24 h. However, in the presence of

glutamine and 1 mg/mL solution of nanoproline contains ∼30 μM proline. The approximate number of glutamine/proline per nanoparticle has been calculated,32 which typically comes in the range of 150−250. Inhibition of Lysozyme Fibrillation by Nanoglutamine and Nanoproline. Effect of nanoglutamine and nanoproline on protein fibrillation was studied using HEWL as model

Table 1. Properties of Nanoglutamine and Nanoproline and Their Chaperone Performance nanoparticle nanoparticle (control) nanoglutamine nanoproline a

hydrodynamic size (nm)

ζ-potential (mV) at pH 4.5, 7.4, 10.0

20−30

−1, −0, −18

30−50 20−40

−6, −13, −18 +12, −1, −4

lysozyme aggregation inhibition performancea

huntingtin aggregation inhibition performancea

104 times 104 times

104 times not studied

Compared to molecular amino acid. D

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nanoglutamine or nanoproline, the nucleation−growth kinetics of HEWL fibrillation is inhibited in a dose-dependent manner. For example, 0.2 mg/mL of nanoglutamine with 10 μM glutamine can delay nucleation by 3.5 h, and 0.8 mg/mL of nanoglutamine with 40 μM glutamine can completely inhibit the fibrillation. Similarly, 0.5 mg/mL of nanoproline with 10 μM proline can delay nucleation by 1 h, and 3 mg/mL of nanoproline with 60 μM proline can completely inhibit the fibrillation. In contrast, control nanoparticles without any functionalization or molecular glutamine/proline (100 mM) can hardly inhibit fibrillation. Circular dichroism spectra further corroborate this observation (Figure 3). As shown in Figure 3, mature lysozyme fibrils with β-sheet structure shows a typical negative band at 218 nm and a positive band at 195 nm, and nanoglutamine or nanoproline can inhibit such β-sheet formation in a dosedependent manner. In particular, 1 mg/mL nanoglutamine with 50 μM glutamine and 3 mg/mL nanoproline with 60 μM proline can completely inhibit lysozyme fibrillation. In contrast, molecular glutamine/proline even at 100 mL concentration cannot inhibit such β-sheet formation. In addition, control nanoparticles without any glutamine/proline functionalization cannot inhibit such β-sheet formation. These studies conclude that nanoglutamine is 104 times more efficient than molecular glutamine, and nanoproline is 104 times more efficient than molecular proline in inhibiting HEWL fibrillation. TEM imaging of HEWL fibril is investigated to visualize fibril morphology. Results showed that in the presence of nanoglutamine, only amorphous HEWL aggregates are formed, but some short fibrils are observed in the presence of nanoproline (Figure 4). This result indicates that nanoglutamine is relatively more effective over nanoproline in inhibiting protein fibrillation. Nanoglutamine Inhibits Polyglutamine-Expanded Huntingtin Aggregation Inside Cell. Next, we investigated the effect of nanoglutamine in inhibiting protein aggregation

Figure 3. Circular dichroism spectra of HEWL, showing that β-sheet formation is inhibited in the presence of nanoglutamine and nanoproline. (a) Circular dichroism spectra of HEWL in the presence of (1) 1 mg/mL control nanoparticles without glutamine functionalization, (2) 100 mM molecular glutamine, (3) 0.2 mg/mL nanoglutamine with 10 μM glutamine, and (4) 1 mg/mL nanoglutamine with 50 μM glutamine. (b) Circular dichroism spectra of HEWL in the presence of (1) 100 mM molecular proline, (2) 1 mg/mL control nanoparticles without proline functionalization, (3) 0.5 mg/ mL nanoproline with 10 μM proline, (4) 1.5 mg/mL nanoproline with 30 μM glutamine, and (5) 3 mg/mL nanoproline with 60 μM glutamine.

Figure 4. Representative TEM images of HEWL fibrils/amorphous aggregates formed in the presence of nanoproline and nanoglutamine. Fibrils are formed without nanoparticles (control) or in the presence of molecular glutamine/proline, but very short fibrils are formed in the presence of nanoproline with 50 μM proline, and only amorphous aggregates are found in the presence of nanoglutamine with 50 μM glutamine. Red arrows indicate nanoparticles. E

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nanoglutamine along with inducer (ponasterone A) for mutant huntingtin expression for 2−3 days. Cells are imaged under fluorescence microscopy at different time points to investigate protein aggregation inside the cell. At first, we tested the ability of nanoglutamine to label and enter HD150Q cells. Cells are induced by ponasterone A for 1 day followed by incubation with rhodamine conjugated nanoglutamine for 6 h, and washed cells are used to image mutant huntingtin and nanoglutamine. Results show that nanoglutamine labels cells and partially colocalizes with mutant huntingtin aggregates (Figure 5). This result suggests the interaction between nanoglutamine and mutant huntingtin inside cells. Figure 6 and Supporting Information, Figure S4 show fluorescence images of nanoglutamine treated cells under different time points. Results show that nanoglutamine blocks the aggregation of GFP tagged mutant huntingtin inside cells. Fluorescence image of cells at 2 or 3 days clearly shows a low number of green fluorescent dots in nanoglutamine treated cells as compared to control cells that are not treated with nanoparticles. In addition, molecular glutamine with a concentration up to 10 000 times higher is much less efficient in inhibiting protein aggregation. This result suggests that aggregation of GFP tagged mutant huntingtin is blocked by nanoglutamine. Next, the aggregated soluble huntingtin is quantified by immunoblot, and aggregated insoluble huntingtin is quantified by dot blot assay (Figure 7). Typically, cell lysates are collected after 3 days of treatment of nanoglutamine and used for detection of protein by immunoblot analysis using GFP antibody. Results clearly show that the level of soluble mutant huntingtin (3 bands ranging between 75 and 100 kDa) is significantly reduced by nanoglutamine but not by control nanoparticles or 10 000 times excess of molecular glutamine. Similarly, cell homogenates are collected after three days of incubation with nanoglutamine and processed for dot blot analysis using GFP antibody. Results clearly show that insoluble pool of mutant huntingtin is dramatically suppressed by nanoglutamine but not by 10 000 times excess of molecular glutamine or control nanoparticle. All these results clearly show that nanoglutamine efficiently suppresses the aggregation of mutant protein typically at micromolar glutamine concentration, but molecular glutamine as high as 100 mM concentration cannot suppress such aggregation. Finally, we tested the viability of nanoglutamine treated HD150Q cells via MTT assay (Figure 8). Typically, cells are incubated with nanoglutamine (or molecular glutamine) followed by incubation with ponasterone A to induce mutant huntingtin protein expression for 3 or 5 days. Next, cell viability is measured via MTT assay, assuming 100% viability for samples without any sample treatment. Results clearly show that nanoglutamine treated cells have increased survival (Figure 8). In particular, cell survival at 5 days is only 40% for 50 mM glutamine treated cells, but cell survival at 5 days is 65−80% for nanoglutamine (with 5−20 μM glutamine) treated cells. We also studied nanoproline-based polyglutamine aggregation inside the cell. However, we observed that cells experience stressed conditions in the presence of nanoproline, and further studies are necessary to understand the reasons (Supporting Information, Figure S5). Nanoglutamine as Artificial Chaperone. Osmolytes are small organic solutes that accumulate inside the cell to counter cellular stresses and provide protection against cell shrinkage or

Figure 5. Colocalization study of nanoglutamine and GFP tagged mutant huntingtin inside HD150Q cells. Cells are induced by ponasterone A for 1 day and incubated with rhodamine conjugated nanoglutamine for 6 h, and washed cells are then used for imaging. Mutant huntingtin is imaged under blue excitation, and nanoglutamine is imaged under green excitation. Mutant huntingtin aggregate appears as green fluorescent dots and partially colocalized (yellow color) with nanoglutamine, suggesting the interaction between them. Scale bar represents 50 μm.

inside the cell. We used HD150Q cell as a model that expresses GFP tagged polyglutamine expanded huntingtin protein.34 Cells emit green fluorescence as the mutant huntingtin is expressed, and with time they form cytoplasmic and nuclear aggregates that appear as green fluorescent dots under fluorescence microscopy. Typically, cells are treated with F

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Figure 6. Nanoglutamine blocks the aggregation of GFP tagged mutant huntingtin in HD150Q cells. Typically, cells were first treated with 50 mM glutamine or nanoglutamine with 5 μM glutamine followed by incubation with ponasterone A to induce protein aggregation. In control experiments, cells are incubated with ponasterone A only. Next, cells are imaged under fluorescence microscopy after 2 (a) or 3 (b) days. Absence of green fluorescent dots in nanoglutamine treated cells indicates that formation of protein aggregates is blocked. (white arrows indicate green dots of protein aggregates). Scale bar represents 50 μm.

100−10 000 times as compared to molecular sugars. Moreover, the trehalose-based nanoparticle was designed for suppression of mutant huntingtin aggregation in cell and mouse models, typically with performance 1000−10 000 times better than that of molecular trehalose. Here, we demonstrated that antiamyloidogenic performance of amino acid-based osmolytes can also be enhanced using nanoform, typically by 1000−10 000, as compared to molecular glutamine and proline. We anticipate two specific reasons for such enhanced chaperone activity. First, nanoglutamine and nanoproline bind to growing fibrils much stronger than do molecular glutamine and proline (Supporting Information, Figure S6). This is mainly due to multiple numbers (150−250) of glutamine/ proline present on nanoparticle surface that offers multivalent binding interaction with protein. Second, nanoparticles are designed with zwitterionic surface charge for high cell uptake via endocytosis that offers easier accessibility inside cells and for interaction with intracellular protein. The combined effect of these two features provides nanoglutamine as a chemical chaperone at micromolar concentration. These results suggest that future research should be directed to develop nanoscale drugs with following criteria: First, molecular forms of antiamyloidogenic materials should be transformed to nanoparticle form to enhance their performance in inhibiting protein aggregation. Although many antiamyloidogenic molecules are reported, they have limited in vivo application potential either due to poor chemical stability or low water solubility. Nanoparticle form of these molecules can be designed for enhanced bioavailability. Second, nanodrug formulation should consider appropriate surface engineering for enhanced binding to aggregating protein, and this would enhance their potential for inhibiting protein aggregation. Third, nanodrug should be designed to induce autophagy as this formulation can be used to clear protein aggregates from cell.46 This approach can be used for treatment of protein aggregation induced toxicity. Fourth, nanodrugs should be composed of biocompatible and biodegradable materials so that they can be used for in vivo application.

swelling. These solutes include amino acids, polyols, sugars, and methylamines.18 Among them, some osmolytes are shown to prevent aggregation and act as chemical chaperones (or osmoprotectants). For example, yeast accumulates trehalose during heat shock to protect proteins against thermal denaturation/aggregation;35 deep sea organisms accumulate high concentrations of trimethylamine-N-oxide osmolyte to protect them against the destabilizing effect of urea,36 and glutamine acts as an osmolyte for survival of bacterial and other animals under osmotic stress.21,22 Experimental data convincingly prove that aggregation of mutant huntingtin protein is prevented by using osmolyte proline37 or osmolyte trehalose;16 insulin fibrillation is prevented by osmolytes proline,20 and lysozyme aggregation is prevented by glucose.18 Molecular mechanism involves macromolecular crowding effect induced by high concentration of osmolytes, forcing the protein to adopt minimum solvent exposure via entropic/enthalpic reasons.18,38−41 However, high volume fraction of osmolytes, typically above millimolar concentration, is required for such effect, which is difficult to achieve under in vitro/in vivo conditions. Recent works show that a nanotechnology-based approach can be a unique alternative for the treatment of proteinaggregation-derived diseases and colloidal nanoparticles can efficiently inhibit protein aggregation. Nanoparticle that inhibit protein aggregation include hydrophobic polymer nanoparticles,42 quantum dots,25 protein microspheres,43 carbon nanoparticles44 and gold nanoparticles.27 In addition functional gold nanoparticles24 and graphene oxide26 have been used to disintegrate preformed fibrils under light. Considering the fact that nanoparticle-based systems can be designed with multivalent binding property, with enhanced cellular uptake and to cross the blood−brain barrier; they can be used for efficient targeting of protein fibrils/plaques inside cell and brain.45 We recently showed that performance of osmoprotectants can be enhanced by using their nanoparticle form. In particular, we showed that nanoparticle form of glucose, sucrose, maltose, and trehalose can inhibit fibrillation of insulin/lysozyme/ amyloid peptide 1000−100 000 times more effectively than respective molecular sugars. In addition, the nanoparticle form can decrease the cytotoxicity of amyloid fibrils typically by G

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Figure 8. Enhanced viability of HD150Q cells in the presence of nanoglutamine. Cells are untreated (1) or treated with ponasterone A inducer (2−5) along with 0.2 mg/mL nanoglutamine with 5 μM glutamine (3), 0.8 mg/mL nanoglutamine with 20 μM glutamine (4), 50 mM molecular glutamine (5), or without any nanoglutamine/ glutamine (2). Cells are first incubated with nanoglutamine (or molecular glutamine) followed by incubation with 1 μM ponasterone A to induce mutant huntingtin protein expression for 3 or 5 days. Next, cell viability is measured via MTT assay, assuming 100% viability for samples without any nanoparticle treatment.

aggregation, and future study should be directed for more detailed understanding of the origin of such performance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.7b00245. Mass spectral characterization of nanoproline, additional data on inhibition of intracellular protein aggregation by nanoglutamine, and evidence of stronger interaction of nanoglutamine/nanoproline with protein fibrils (PDF)

Figure 7. Nanoglutamine blocks the aggregation of soluble mutant huntingtin inside the HD150Q cell. (a) Cells are untreated (lane 1) or treated with ponasterone A inducer (lanes 2−6) along with 50 mM molecular glutamine (lane 3), 0.4 mg/mL control nanoparticles without glutamine conjugation (lane 4), 0.2 mg/mL nanoglutamine with 5 μM glutamine (lane 5), or 0.4 mg/mL nanoglutamine with 10 μM glutamine (lane 6). After 3 days, cell lysates are used for detection of protein by immunoblot analysis using GFP antibody. Results show that the level of soluble mutant huntingtin (100 kDa band) is significantly reduced by nanoglutamine but not by control nanoparticles or molecular glutamine. (b) Nanoglutamine reduces the level of insoluble mutant huntingtin inside the HD150Q cell. After 3 days of treatment with nanoglutamine, cell homogenates are processed for dot blot analysis using GFP antibody. Other conditions are same as described above. (c) Quantification of relative band intensity of insoluble mutant huntingtin in dot blot assay using NIH image analysis software (error bar indicates average of three measurements). Results indicate that insoluble pool of mutant huntingtin is significantly decreased by nanoglutamine but not by molecular glutamine or control nanoparticle.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Nibedita Pradhan: 0000-0002-0303-5351 Nihar R. Jana: 0000-0002-6549-4211 Nikhil R. Jana: 0000-0002-4595-6917 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge DST Nano Mission (Grant SR/NM/ NB/1009/2016) and CSIR (Grant 02(0249)15/EMR-II) Government of India for financial assistance. We also acknowledge the Department of Biotechnology (DBT), Government of India for providing financial support to National Brain Research Centre.

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CONCLUSION In conclusion, we demonstrated that nanoparticle forms of glutamine and proline can be 1000−10 000 times more efficient than respective molecular glutamine and proline in inhibiting protein aggregation. We designed nanoglutamine and nanoproline with optimized surface properties for best performance in inhibiting lysozyme aggregation under extracellular space and inhibiting mutant huntingtin protein aggregation under intracellular space. In particular, we demonstrated that nanoglutamine can block the aggregation of polyglutamine expanded mutant huntingtin protein intracellularly in HD model cell line. This result indicates that nanoparticle form of osmolyte molecules can be more efficient in inhibiting protein

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REFERENCES

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