Trehalose-Functionalized Gold Nanoparticle for Inhibiting Intracellular

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Trehalose Functionalized Gold Nanoparticle for Inhibiting Intracellular Protein Aggregation Suman Mandal, Koushik Debnath, Nihar R Jana, and Nikhil R. Jana Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02202 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 12, 2017

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Trehalose Functionalized Gold Nanoparticle for Inhibiting Intracellular Protein Aggregation Suman Mandal,1 Koushik Debnath,1 Nihar R. Jana2,* and Nikhil R. Jana1,* 1

Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata700032, India

2

Cellular and Molecular Neuroscience Laboratory, National Brain Research Centre, Manesar, Gurgaon-122051 (India) *Corresponding authors E-mail: [email protected] and [email protected]

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Summary: Trehalose is a well-known anti-amyloidogenic molecule that inhibits protein aggregation under the intra-/extra-cellular condition and recent work shows that nanoparticle form of trehalose can further enhance this performance. Here we have designed trehalose functionalized Au nanoparticle that can inhibit aggregation of polyglutamine-containing mutant protein inside the neuronal cell. Designed nanoparticles have 20-30 nm Au core with about 350 ± 50 trehalose molecules per particle on the surface on an average. They enter into the cell, inhibit mutant protein aggregation and enhance the cell survival against toxic protein aggregates. This work extends application potential of trehalose for understanding and treatment of different diseases involving protein aggregation. Keywords: gold nanoparticle, trehalose, amyloid aggregation, polyglutamine, Huntington’s disease


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Introduction Trehalose is a well-known disaccharide that plays a significant role in protecting cell/biomolecule/nanoparticle against environmental stress (such as high/low temperature, high osmotic pressure, high salt concentration)1-4 and it is the most effective carbohydrate in inhibiting protein aggregation under intra-/extra-cellular environment.5-8 In addition trehalose receptors are present in various bacterial lipopolysaccharides.9,10 These properties of trehalose have been utilized in multiple purposes including polymeric trehalose-based stabilization

and

delivery

amphiphile/nanoparticle-based

siRNA/pDNA,11,12

of

targeting

of

pathogen,9,10

microenvironment-assisted nanoparticle endocytosis,13

trehalose-conjugated molecular

trehalose

trehalose incorporated hydrogel-

based stabilization and delivery of biomolecule14 and nanoparticle-based trehalose delivery for cryopreservation of stem cells.15 Nanotechnology-based approach offers alternative options for the treatment of protein aggregation-derived various neurodegenerative disease.16-19 Nanoparticles are designed to inhibit protein aggregation under intra-/extra-cellular space20-24 and to degrade matured protein fibrils.25-28 We have recently shown that nanoparticle form of anti-amyloidogenic molecules can be more efficient than respective molecular forms.29,30 For example, green tea polyphenol-based polymeric nanoparticles are 10-100 times efficient than respective molecular form,29 sugar-terminated nanoparticles are 102 to 105 times superior than molecular sugars30 and trehalose based nanoparticles are designed for in vivo application.31 The above studies show that better performance of nanoparticle form is linked to increased chemical stability of respective molecular form, multivalent binding effect with protein and high cellular uptake via endocytosis.29,30 These studies also show that performance of nanoparticle form of an anti-amyloidogenic molecule depends on the nature of precursor molecule and the degree of multivalency (number of molecules present at the surface of each nanoparticle


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through which protein interacts). In particular, we found that trehalose-based nanoparticles offer the best performance among all the tested carbohydrates in inhibiting protein aggregation but it has poor performance in disintegrating matured protein fibrils as compared to green tea polyphenol-based nanoparticles. Thus further works should be directed to design trehalose-based nanoparticle that can efficiently degrade matured protein fibrils. For example, trehalose-based conjugate with plasmonic (Au/Ag) nanoparticle or graphene can be explored for photothermal degradation of mature fibrils.27,28,32 However, there is no report on trehalose functionalized Au/Ag nanoparticles, although trehalose functionalized carbon nanoparticle,30 iron oxide nanoparticle,31 colloidal silica,10 hydrogel14 and polymers11,12 are reported. Here we have designed trehalose functionalized Au nanoparticle (Au-trehalose) in order to extend the application potential of the antiamyloidogenic property of trehalose. The performance of Au-trehalose is evaluated using a neuronal cell line that expresses polyglutamine-containing mutant huntingtin protein, responsible for neurodegenerative Huntington’s disease.33 We have shown that Au-trehalose enters into cells, inhibits mutant huntingtin aggregation inside the neuronal cell and plasmonic property of Au nanoparticles can be utilized to follow the cellular interaction/uptake of Au-trehalose. This approach can be extended to prepare trehalose functionalized anisotropic Au nanoparticles, for monitoring the intracellular protein aggregation using surface-enhanced Raman spectroscopy (SERS) and for efficient photothermal degradation of matured protein fibrils under intra-/extra-cellular space.32

Experimental Section Materials. Chloroauric acid (HAuCl4·H2O), L-aspartic acid, dimethylamino pyridine (DMAP), lipoic acid, dicyclohexylcarbodiimide (DCC), Dulbecco’s modified eagle medium (DMEM) and penicillin/streptomycin were purchased from Sigma-Aldrich. D-(+)-trehalose


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dihydrate ≥ 98 % was purchased from TCI chemicals. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was purchased from Hi Media (India). GFP antibody was purchased from Roche; Ponasterone A was obtained from Invitrogen, and all reagents for SDS-PAGE were purchased from Sigma. Synthesis of lipoic acid conjugated trehalose. Trehalose (210 mg, 0.6 mmol), lipoic acid (150 mg, 0.72 mmol) and DMAP (10 mg, 3-10 mol %) were taken in 3 mL anhydrous DMF. The mixture was stirred well to dissolve the reagents at room temperature for 15 min. Then the medium was cooled at 0 °C in an ice bath. Now, DCC (165 mg, 0.9 mmol) was added to the reaction mixture that is previously dissolved in DMF. Next, stirring was continued at 0 °C for another 30 min followed by keeping at room temperature for next 24 h. Next, the product was precipitated with acetone and washed with dichloromethane trice to remove unreacted reagents and side products. Synthesis of trehalose functionalized Au nanoparticle (Au-trehalose). 200 µL aqueous solution of lipoic acid conjugated trehalose (15 mM) was added to 3 mL aqueous gold chloride (1 mM) solution. Next, 24 µL aqueous solution of ascorbic acid (100 mM) was added under the stirring condition for the subsequent reduction of gold salt within 10 min. Thereafter, Au-trehalose nanoparticles were isolated by centrifugation at 12000 rpm and redispersed in 3 mL fresh water. Citrate capped Au nanoparticles of average size ~20 nm were also synthesized following standard method.34 Characterisation was done by UV-Visible spectroscopy and transmission electron microscopy. Estimation of trehalose in Au-trehalose. The concentration of trehalose in Au-trehalose was quantified using anthrone test.35 In this process, 0.1-1.0 mL nanoparticle suspenstion or trehalose solution was mixed with 2 mL anthrone solution (prepared by mixing 20 mg anthrone in 10 mL 80 % H2SO4) and heated at 80 °C for 10 min to form a bluish-green


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furfural complex. The solution was then cooled using ice-water. Next, the absorbance of furfural complex formed was measured at 630 nm for quantification of trehalose. At first, a calibration graph was prepared by plotting the absorbance against varying trehalose concentration. Typical calibration graph was as follows: Y = 0.02 [trehalose], where Y is the absorbance of furfural complex and trehalose is in µM concentration. Next, the concentration of gold nanoparticles was determined using molar extinction coefficient of Au nanoparticle.36 The number of trehalose per nanoparticle was determined from the molar ratio of trehalose to Au nanoparticle which comes around 350 ± 50. (average of 3 measurements) Cellular uptake study of Au-trehalose in HD150Q cells. HD150Q cells were cultured overnight in 4 well chamber slide under 37 °C with 5 % CO2, using DMEM media with 10 % fetal bovine serum and antibiotics containing 0.4 mg/mL Zeocin and 0.4 mg/mL G418 (Geneticin). Next, cells were given fresh media and incubated with Au-trehalose nanoparticle suspension for 2 h. Cells were then washed carefully with PBS buffer for complete removal of unbound particles. After that cells were fixed with 4 % paraformaldehyde solution and the slide was prepared for taking bright field and dark field images under microscope. Polyglutamine aggregation study using HD150Q cell. We have used HD150Q cell line, which is a stable and inducible mouse neuro 2A cell line that expresses green fluorescent protein-tagged mutant N-terminal huntingtin protein with 150 glutamine residues.37 Cells were cultured in 24 well tissue cultured plates using the conditions described above. After 24 h, cells were given fresh media and mixed with Au-trehalose nanoparticle suspension (or molecular trehalose) for 2 h. Next, cells were washed with PBS, followed by the addition of fresh media. Then, ponasterone A solution (final concentration 1 µM) was added to induce the polyglutamine protein expression and aggregation within the cell and incubated at 37 °C for different days; this condition was maintained until cell death and protein aggregation was followed by fluorescence microscopy.


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For Western blot, cells were washed with ice-cold phosphate-buffered saline (PBS), collected by centrifugation, and sonicated on ice for 30 min with Nonidet P-40 lysis buffer (consists of 50 mM Tris buffer of pH 8.0, 150 mM NaCl, 1 % Nonidet P-40, and complete protease inhibitor cocktail). The cell lysate was then centrifuged for 10 min at 15000g at 4 °C and the supernatants were used for SDS-PAGE followed by immunoblot analysis as previously described.31 Blot was probed with GFP/ubiquitin antibody. For dot blot analysis, HD150Q cells were homogenized in 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 15000g for 15 min. Pellet was collected and treated with 2 % SDS at room temperature for 5 min, quantified and equal amounts of proteins were allowed to filter through nitrocellulose membrane. Membranes were then probed with GFP/ubiquitin antibody. For counting aggregates, fluorescence microscopic images of treated HD150Q cells were taken into account where we have calculated cells and number of green dots due to polyglutamine aggregation. Dot number % was calculated from the ratio of the total number of green dots to that of the total number of cells counted (500 cells were considered). Dot area % was calculated as the ratio of total area of green dots to that of the total area of cells (only the cells with green dots were considered). Viability assay of HD150Q cells in the presence of Au-trehalose. HD150Q cells were cultured in 12 well plates using the conditions described above. After 24 h, cells were washed with PBS, followed by the addition of fresh media. Then, ponasterone A solution (final concentration 1 µM) was added to induce the polyglutamine aggregation within the cell in presence of 200 µL of Au-trehalose nanoparticle suspension (with trehalose conc. 0.5 mg/mL) and incubated at 37 °C for different days. After 4 days, 50 µL of freshly prepared MTT solution (5 mg/mL) was mixed to each well and incubated for 4 h. The produced violet-


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colored formazan then was dissolved in 50 % aqueous DMF solution, and the absorbance was measured at 570 nm using a microplate reader. The optical density was correlated with cell viability, assuming 100 % viability for the control sample without any ponasterone A and nanoparticle. Instrumentation. Ultraviolet-visible absorption spectra were measured using a Shimadzu Model UV-2550 UV-visible spectrophotometer. Emission spectra were measured using Synergy MX multimode microplate reader. Transmission electron microscopy (TEM) samples were prepared by placing a drop of nanoparticle suspension on carbon-coated copper grid and observed with FEI Tecnai G2 F20 microscope. Mass spectra were measured using a Quadruple TOF (Q-TOF) micro-MS system using the electrospray ionization (ESI) technique. (Thermo Scientific LTQ Orbitrap XL hybrid Fourier Transform Mass Spectrometer) Proton NMR spectra were recorded using 500 MHz spectrometer in the appropriate solvent. (Bruker ultrashield 500) Fourier transform infrared (FTIR) spectroscopy was performed on PerkinElmer Spectrum 100 FTIR spectrometer after making pellets with solid KBr. Bright field and dark field images of live cells were performed using Zeiss Axio Observer A1 microscope. The fluorescence image of the cells was captured using a fluorescence microscope (Olympus, Model IX-81) and Axio Plan inverted Apotome microscope (Zeiss). QSONICA Q700 sonicator was used for cell lysate preparation. Chemi Gel documentation (Biodigital Pvt Ltd.) and dot blot apparatus (BioRad) were used for Western blot and dot blot experiments.

Results and Discussion Design and synthesis of trehalose functionalized Au nanoparticle (Au-trehalose). First, we have transformed trehalose to a thiolated trehalose and then Au nanoparticle is synthesized using the thiolated trehalose as the stabilizer. The synthetic approach of Au-


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trehalose is shown in Scheme 1. DCC coupling is used for esterification between the primary alcoholic group of trehalose and carboxylic group of lipoic acid. About one equivalent of trehalose is reacted with the lipoic acid in order to ensure only one primary alcohol of trehalose is reacted. Formation of thiolated trehalose is confirmed via mass spectroscopy and NMR. (Supporting Information, Figure S1, S2) Synthesis of Au-trehalose involves reduction of gold salt in presence of thiolated trehalose. Typically an aqueous solution of gold chloride is mixed with thiolated trehalose and then ascorbic acid is added for reduction of gold salt. Under this condition, the gold nanoparticles are capped with thiolated trehalose via thiol groups. Au core is thus stabilized and protected from the aqueous environment by hydrophobic lipoic acid moiety and strongly hydrophilic trehalose component decorates the gold nanoparticle surface leading to good colloidal stability. Thereafter, Au-trehalose nanoparticles are isolated by centrifugation and redispersed in the fresh water. In contrast, ascorbic acid reduction of gold salt in absence of thiolated trehalose or ascorbic acid reduction of gold salt in presence of trehalose produce colloidally unstable Au nanoparticles. (Supporting Information, Figure S3) Figure 1 and Supporting Information, Figure S4 summarizes the property of Au-trehalose. The colloidal suspension of Au-trehalose appears pink colour due to plasmonic property of Au nanoparticle with the absorption band at 530 nm. TEM image shows the size of Au nanoparticle is about 20-30 nm. The trehalose functional groups on the nanoparticle surface are characterized by FTIR. (Figure 1) Vibrational stretching bands of hydroxyl groups at 3100−3500 cm−1 and CH stretching bands at 3000 cm−1 are observed for Au-trehalose. (Figure 1d) Solution phase proton NMR spectroscopy of colloidal solution of Au-trehalose gives two distinct set of chemical shifts. (Supporting Information, Figure S4) CH protons of lipoic acid moiety arise at lower chemical shift region (2-3 ppm), whereas the chemical shift of hydroxyl and CH protons of trehalose group arise at relatively downfield region (δ 3-5 ppm). This NMR


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spectral data of Au-trehalose well correlate with the NMR spectra of free trehalose-lipoic acid ligand. Further, the presence of trehalose on nanoparticle surface was verified and quantified using the anthrone test, which is specific only for carbohydrate detection.35 In the anthrone test, the trehalose molecule reacts with anthrone in concentrated H2SO4 under the heating condition and produce a bluish-green colored furfural complex. A similar type of colored complex is also formed when Au-trehalose is reacted with anthrone, suggesting the presence of trehalose molecule (Figure 1c). We have also quantified the trehalose concentration in colloidal suspension of Au-trehalose and estimated the average number of trehalose per nanoparticle as 350 ± 50. (see detailed procedure for estimation in Supporting Information). However, this result can’t clearly indicate whether the trivial form of trehalose ligand is intact. To determine the trehalose structure we replaced the trehalose-lipoic acid ligand from Au-trehalose surface by ligand exchange using 2, 3-dimarcapto propanol in an aqueous medium. The exchanged trehalose-lipoic acid ligands (that remains in the aqueous medium) are then isolated by removing Au nanoparticles via centrifugation and then characterized by mass spectrometry. (Supporting Information, Figure S5) Mass spectrometry clearly represents the trivial trehalose-lipoic acid ligand’s presence. Thus it can be said that Au-trehalose is stabilized by chemically intact trehalose-lipoic acid ligand. The Au-trehalose nanoparticle suspension has reasonably good colloidal stability in phosphate buffer solution (pH 7.4). In addition, extensive experiments are also done to understand the colloidal stability of Au-trehalose in different pH conditions. (Supporting Information, Figure S6). Phosphate buffer solution of pH 7.4, acetate buffer solution of pH 4.5 and bicarbonate buffer solution of pH 10 are used for these tests. Digital images show that in all conditions colloidal stability of Au-trehalose appears very good without any trace of precipitation.


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Au-trehalose inhibits mutant huntingtin aggregation inside HD150Q cells. We have studied the performance of Au-trehalose in inhibiting the polyglutamine-based mutant huntingtin aggregation inside the HD150Q cell, expressing a truncated N-terminal huntingtin containing 150 glutamine residues, fused to an enhanced green fluorescent protein.37 This is a commonly used model cell line for Huntington’s disease. In presence of ponasterone A inducer the aggregation of mutant huntingtin occurs inside the cell and the aggregation appears as green dots under fluorescence microscope. Due to the toxicity of protein aggregates the cell dies within 7-8 days. Here we have incubated cells with Au-trehalose nanoparticle suspension and then treated with ponasterone A to induce the mutant huntingtin aggregation. Next, mutant huntingtin aggregation is studied under the fluorescence microscope and cell lysate is used to analyse the level of soluble mutant huntingtin. First, we have investigated that Au-trehalose has high uptake in the HD150Q cell. When cells are incubated with Au-trehalose for one hour and washed cells are imaged under dark field microscope, we found the strong yellow scattering of Au nanoparticle, suggesting that cells are labeled with Au-trehalose. (Figure 2) In addition plasmon band of Au nanoparticle is detected in Au-trehalose nanoparticle suspension treated HD150Q cells. (Supporting Information, Figure S7) Next, aggregation of mutant huntingtin in presence/absence of Au-trehalose (or molecular trehalose) is investigated under the fluorescence microscope. Typically, cells are incubated with Au-trehalose nanoparticle suspension along with ponasterone A inducer for 3 days and then observed under fluorescence microscope. (Figure 3) We found that Au-trehalose treated cells show less number of green fluorescent dots as compared to control sets that use molecular trehalose or no Au-trehalose/molecular trehalose. In addition, when we have used citrate-capped ~ 20 nm Au nanoparticle suspension as control (~1 nM gold content, same as of Au-trehalose), no significant change in the number of green fluorescent dots are observed.


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(Figure 3b and Supporting Information, Figure S8, S9) Results clearly show that Au-trehalose suppresses mutant huntingtin aggregation at one µM trehalose concentration but molecular trehalose is inefficient even at one mM concentration. We have further counted the number of aggregated huntingtin dots and area coverage of these dots in each cell for a large number of cells (about 500 cells with one/two dots in each cell). Results show that dot number is 10 % less and dot area is 4 % less for Au-trehalose with 1 µM trehalose, as compared to 1 mM molecular trehalose. (Figure 3b) This result clearly shows that Au-trehalose is more efficient than molecular trehalose in inhibiting mutant huntingtin aggregation inside the cell. Next, the level of soluble and insoluble mutant huntingtin is quantified by dot blot and Western blot analysis. Typically, cells are treated with ponasterone A along with Autrehalose or molecular trehalose for 3 days and cell lysates are then processed for immuno blot/dot blot analysis using ubiquitin and GFP antibody. (Figure 4a,b) Both antibodies are used to detect insoluble polyglutamine protein and they should give similar results. In addition, band intensities corresponding to soluble huntingtin protein (tNhtt) in immune blot and insoluble huntingtin protein in dot blot (detected by GFP and ubiquitin antibodies) are quantified using NIH image analysis software. (Figure 4c) It is observed that insoluble mutant huntingtin bands are about 40 % less intense for Au-trehalose (with one µM trehalose) as compared to 5 mM molecular trehalose. Results clearly show that Au-trehalose suppresses huntingtin aggregation in HD150Q cells. We have also observed that Au-trehalose is able to increase the survival of HD150Q cells against toxic protein aggregates. Au-trehalose labeled HD150Q cells are incubated with ponasterone A to induce mutant huntingtin protein aggregation and cell viability is measured after 4 days via MTT assay. It is observed that Au-trehalose treated cells have 90 % viability as compared to 60 % viability of control sample that is not treated with Au-trehalose. (Supporting Information, Figure S10)


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Conclusion We have synthesized trehalose functionalized Au nanoparticle and shown that they can inhibit aggregation of polyglutamine-containing mutant protein inside the neuronal cell. Designed nanoparticles have 20-30 nm Au core with about 350 ± 50 trehalose molecules per particle on the surface on an average. Advantage of nanoparticle form is that it offers enhanced cell uptake via endocytosis and multiple number of trehalose present at the nanoparticle surface offers multivalent interaction with aggregating protein. Thus Autrehalose efficiently inhibits mutant protein aggregation inside cell and enhances the cell survival against toxic protein aggregates. These nanoparticles can be used for monitoring of intracellular protein aggregation using surface-enhanced Raman spectroscopy (SERS) and for efficient photothermal degradation of matured protein fibrils under intra-/extra-cellular space.

ACKNOWLEDGEMENT The authors acknowledge DST Nano Mission and CSIR, government of India for financial assistance. (Grant numbers: SR/NM/NB-1009/2016 and 02(0249)/15/EMR-II) We also acknowledge Department of Biotechnology (DBT), Government of India for providing financial support to National Brain Research Centre. S.M. acknowledges CSIR, India for providing research fellowship. ASSOCIATED CONTENT Supporting Information. Detailed procedures for calculating the number of trehalose per nanoparticle, characterization of thiolated trehalose and Au-trehalose, the effect of experimental conditions on the preparation of Au-trehalose, colloidal stability data of Autrehalose nanoparticle suspension, cytotoxicity data using Au-trehalose and control experiments using citrate-capped Au nanoparticle. This material is available free of charge via the Internet at http://pubs.acs.org. 13 ACS Paragon Plus Environment

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Notes The authors declare no competing financial interests. References (1) Li, H.-W.; Zang, B.-S.; Deng, X.-W.; Wang, X.-P. Overexpression of the Trehalose-6Phosphate Synthase Gene OsTPS1 Enhances Abiotic Stress Tolerance in Rice. Planta 2011, 234, 1007–1018. (2) James, S.; McManus, J. J. Thermal and Solution Stability of Lysozyme in the Presence of Sucrose, Glucose, and Trehalose. J. Phys. Chem. B 2012, 116, 10182−10188. (3) Lee, J.; Lin, E.-W.; Lau, U. Y.; Hedrick, J. L.; Bat, E.; Maynard, H. D. Trehalose Glycopolymers as Excipients for Protein Stabilization. Biomacromolecules 2013, 14, 2561−2569. (4) Alkilany, A. M.; Abulateefeh, S. R.; Mills, K. K.; Yaseen, A. I. B.; Hamaly, M. A.; Alkhatib, H. S.; Aiedeh, K. M.; Stone, J. W. Colloidal Stability of Citrate and Mercaptoacetic Acid Capped Gold Nanoparticles upon Lyophilization: Effect of Capping Ligand Attachment and Type of Cryoprotectants. Langmuir 2014, 30, 13799−13808. (5) Tanaka, M.; Machida, Y.; Niu, S.; Ikeda, T.; Jana, N. R.; Doi, H.; Kurosawa, M.; Nekooki, M; Nukina, Trehalose Alleviates Pollyglutamine-Mediated Pathology in a Mouse Model of Huntington Disease. Nat. Medecine 2004, 10, 148–154. (6) Vilasi, S.; Iannuzzi, C.; Portaccio, M.; Irace, G.; Sirangelo, I. Effect of Trehalose on W7FW14F Apomyoglobin and Insulin Fibrillization: New Insight into Inhibition Activity. Biochemistry 2008, 47, 1789–1796. (7) Qi, W.; Zhang, A.; Good, T. A.; Fernandez, E. J. Two Disaccharides and Trimethylamine N-Oxide Affect Aβ Aggregation Differently, but All Attenuate Oligomer-Induced Membrane Permeability. Biochemistry 2009, 48, 8908–8919.


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(8) Liu, F.-F.; Ji, L.; Dong, X.-Y.; Sun, Y. Molecular Insight into the Inhibition Effect of Trehalose on the Nucleation and Elongation of Amyloid-Peptide Oligomers. J. Phys. Chem. B 2009, 113, 11320–11329. (9) Lavado, J. R.; Sestito, S. E.; Cighetti, R.; Moncayo, E. M. A.; Oblak, A. Lainšček, D.; Blanco, J. L. J.; Fernández, J. M. G.; Mellet, C. O.; Jerala, R.; Calabrese, V.; Peri, F. Trehalose- and Glucose-Derived Glycoamphiphiles: Small-Molecule and Nanoparticle TollLike Receptor 4 (TLR4) Modulators. J. Med. Chem. 2014, 57, 9105−9123. (10) Zhou, J.; Jayawardana, K. W.; Kong, N; Ren, Y.; Hao, N.; Yan, M.; Ramström, O. Trehalose-Conjugated, Photofunctionalized Mesoporous Silica Nanoparticles for Efficient Delivery of Isoniazid into Mycobacteria. ACS Biomater. Sci. Eng. 2015, 1, 1250−1255. (11) Sizovs, A.; Xue, L.; Tolstyka, Z. P.; Ingle, N. P.; Wu, Y.; Cortez, M.; Reineke, T. M. Poly(trehalose): Sugar-Coated Nanocomplexes Promote Stabilization and Effective PolyplexMediated siRNA Delivery. J. Am. Chem. Soc. 2013, 135, 15417−15424. (12) Tolstyka, Z. P.; Phillips, H.; Cortez, M.; Wu, Y.; Ingle, N.; Bell, J. B.; Hackett, P. B.; Reineke, T. M. Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery. ACS Biomater. Sci. Eng. 2016, 2, 43−55. (13) Siddhanta, S.; Zheng, C.; Narayana, C.; Barman, I. An Impediment to Random Walk: Trehalose Microenvironment Drives Preferential Endocytic Uptake of Plasmonic Nanoparticles. Chem. Sci. 2016, 7, 3730–3736. (14) O’Shea, T. M.; Webber, M. J.; Aimetti, A. A.; Langer, R. Covalent Incorporation of Trehalose within Hydrogels for Enhanced Long-Term Functional Stability and Controlled Release of Biomacromolecules. Adv. Healthcare Mater. 2015, 4, 1802–1812. (15) Rao, W.; Huang, H.; Wang, H.; Zhao, S.; Dumbleton, J.; Zhao, G.; He, X.; NanoparticleMediated Intracellular Delivery Enables Cryopreservation of Human Adipose-Derived Stem


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Scheme 1. a) Chemical transformation approach from trehalose to thiolated trehalose. b) Synthesis strategy of trehalose functionalized colloidal gold nanoparticle (Au-trehalose). Details of synthetic conditions are described in experimental section.


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Figure 1. a) Optical property of colloidal suspension of Au-trehalose. b) TEM image of Autrehalose. c) Anthrone test result for Au-trehalose with the evidence of 625 nm absorption band corresponding to furfural complex. i) Au-trehalose, ii) trehalose (control) and iii) Autrehalose under conc. H2SO4 (Anthrone test condition). d) FTIR spectra of Au-trehalose showing the vibrational fingerprint of trehalose.


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Figure 2. Evidence of Au-trehalose interaction/uptake in the HD150Q cell via dark field microscopy. Cells are cultured overnight, incubated with Au-trehalose (or control without any Au-trehalose) nanoparticle suspension for 2 h and washed cells are used for bright field (BF) and dark field (DF) imaging. The yellow colour of Au-trehalose nanoparticle suspension treated cell appears due to scattering from Au nanoparticle.


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Figure 3. a) Suppression of protein aggregation inside HD150Q cell by Au-trehalose. Cells are incubated with Au-trehalose nanoparticle suspension along with ponasterone A inducer for 3 days and washed cells are then used for imaging. Less number of green fluorescent dots in Au-trehalose treated cells indicates suppression of mutant huntingtin aggregation. In 22 ACS Paragon Plus Environment

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control 1 no ponasterone A inducer is used and in control 2 neither Au-trehalose nor molecular trehalose is used. Results show that Au-trehalose significantly suppresses mutant huntingtin aggregation at 1 µM trehalose concentration but molecular trehalose is hardly efficient even at 1 mM concentration. b) Quantitative estimation of the % of protein aggregates present in 500 counted cells and the area % covered by the aggregates inside cells. Results show that both the number of aggregate and their area are significantly lower for Autrehalose nanoparticle suspension treated cells as compared to trehalose or citrate-capped Au nanoparticles.


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Figure 4. Au-trehalose suppresses polyglutamine-containing mutant huntingtin aggregation in HD150Q cells as observed from dot blot and Western blot analysis. a) Dot blot analysis of insoluble huntingtin aggregates using GFP and ubiquitin antibodies. b) Immunoblot analysis data of soluble huntingtin using GFP antibody. c) Quantification of band intensities of soluble and insoluble huntingtin (tNhtt), shown in (a,b) using NIH image analysis software. All values are mean ± SD of 3 different experiments. Cells are either untreated or treated with ponasterone A along with Au-trehalose nanoparticle suspension or molecular trehalose for 3 days and cell lysates are then processed for immunoblot/dot blot analysis.


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Trehalose-Functionalized Gold Nanoparticle for Inhibiting Intracellular

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