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Sugar-Terminated Nanoparticle Chaperones are 102-105 Times Better than Molecular Sugars in Inhibiting Protein Aggregation and Lowering of Amyloidogenic Cytotoxicity Nibedita Pradhan, Shashi Shekhar, Nihar R Jana, and Nikhil R. Jana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01886 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 11, 2017

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ACS Applied Materials & Interfaces

Sugar-Terminated Nanoparticle Chaperones are 102-105 Times Better than Molecular Sugars in Inhibiting Protein Aggregation and Lowering of Amyloidogenic Cytotoxicity Nibedita Pradhan,1 Shashi Shekhar,2 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]

Keywords: sugar, osmolyte, nanoparticle, chaperone, amyloid fibril, Huntington’s disease

1 ACS Paragon Plus Environment


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Abstract Sugar-based osmolyte molecules are known to stabilize proteins under stress but usually they have poor chaperone performance in inhibiting protein aggregation. Here we show that nanoparticle form of sugar molecule can enhance their chaperone performance typically by 102 to 105 times as compared to molecular sugar. Sugar-based nanoparticles of 20-40 nm size have been

synthesized

by

simple

glucose/sucrose/maltose/trehalose.

heating These

of

acidic

nanoparticles

have

aqueous

solution

excitation

of

dependent

green/yellow/orange emission and surface chemistry identical to respective sugar molecule. Fibrillation of lysozyme/insulin/amyloid beta in extracellular space, aggregation of mutant huntingtin protein inside model neuronal cell and cytotoxic effect of fibrils are investigated in presence of these sugar nanoparticles. We found that sugar nanoparticles are 102 to 105 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity arising of fibrils. We propose that better performance of nanoparticle form is linked to their stronger binding with fibril structure and enhanced cell uptake. This result suggests that nanoparticle form of osmolyte can be an attractive option in prevention and curing of protein aggregation derived diseases.


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Introduction Peptide or protein fibrillation in extracellular and intracellular space plays significant role in the development of several human diseases, such as Alzheimer’s, Parkinson’s, Huntington’s and type II diabetes.1-4 The fibrillation typically involves aggregation of disease-linked peptide/protein via nucleation-growth step. Thus significant research is directed to identify material that can effectively repair peptide/protein misfolding, inhibits fibril nucleation and restricts the fibril growth. Variety of materials have been identified that can inhibit peptide/protein fibrillation which include protein based molecular chaperone,5,6 peptide,7 green tea polyphenol,8 curcumin,9 metal ion chelator,10 nanoparticle11-16 and chemical chaperones based on small molecule osmolytes.17-24 Among them sugar based chemical chaperones are interesting class of molecules that include trehalose,17,18 fructose,19 sucrose,20 glucose22 and sorbidol.24 They are commonly known as osmolyte that accumulate in cells at high concentration and protect proteins from environmental stress via preventing their abnormal folding.20 Such responsibility as osmolyte indicates that they may be employed as efficient chaperones and for repairing/inhibiting protein aggregation.20 However, there are two common issues for sugar osmolyte as effective molecular chaperone.20 First, sugar osmolytes are generally effective as chaperones only at high concentration --typically above millimolar concentration and such a high concentration is difficult to introduce under in vivo condition. In particular molecular osmolyte cannot accumulate in cell cytoplasm or extracellular space at such a high concentration. Second, binding interaction between peptide/protein and molecular chaperone is very weak and thus alternative approach is necessary to increase this binding and to enhance the chaperone function. Thus various attempts have been made to increase the chaperone performance of sugar-based osmolytes. Polymeric sugar-based 3 ACS Paragon Plus Environment


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chaperones have been developed with enhanced protein stabilizing property against heat.25 Similarly, polymeric sugars have been used to enhance the amyloid fibril inhibition performance26-28 and it has been shown that multivalent binding of polymeric sugar is responsible for better performance.29-32 However, performance of sugar osmolytes in inhibiting protein aggregation is low (upto 50 % reduction of fibrillation and cannot completely stop fibrillation) and needs to be enhanced further for any useful application. Alternatively nanoparticle based artificial chaperones have been designed that can influence the nucleation-growth kinetics of protein fibrillation. For example nanoparticles have been designed that can retard amyloid fibrillation,11-16 accelerate amyloid fibrillation33 and disintegrate matured protein fibrils.13,16,34-36 These study show that nanoparticle surface chemistry and chemical/bio functionality can greatly induce their chaperone performance. In particular it has been found that nanoparticle can be designed for higher binding affinity with protein aggregates via multivalent interaction.16,36 Additionally nanoparticle can enter into the cell via endocytosis which can enhance their uptake as compared to small molecules37 and can be designed for efficient crossing of blood-brain barrier.38 We have recently demonstrated that nanoparticle form of green tea polyphenol has 10-100 times enhanced anti-amyloidogenic performance as compared to respective molecular form.36 Thus nanoparticle based chaperone has great potential in targeting amyloid into the brain and at intracellular/extracellular length scale. Here we show that sugar molecule based osmolytes can be very efficient chaperone in their nanoparticle form in inhibiting protein aggregation and lowering of amyloidogenic cytotoxicity. There are four distinct advantage and significance of presented nanoparticle chaperones over reported artificial chaperones. First, these nanoparticle chaperones can be easily synthesized from sugar based food products by simple heating. Second, sugar nanoparticles inhibit protein


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aggregation at micromolar concentration as compared to millimolar concentration of sugar molecules. Third, developed nanoparticle chaperones are effective both at extracellular and intracellular condition. In particular efficient intracellular inhibition of protein aggregation can be exploited for cellular scale and early stage prevention of protein aggregation. Fourth, earlier works show that multivalent carbohydrate polymer can provide upto 50 % reduction of fibrillation31,32 but present work show that multivalency can enhance the anti-amyloidogenic performance upto 105 times and near complete inhibition of amyloid fibrillation.

Experimental section Materials. D-(+)-glucose ≥98% (Sigma), sucrose ≥98% (Merck), D-(+)- maltose monohydrate from potato ≥98% (Sigma) , D-(+)-trehalose dihydrate ≥ 98% (TCI), anthrone ≥97% (Sigma), lysozyme from chicken egg white (Sigma), insulin from bovine pancreas (Sigma), amyloid β protein fragment 1-40 from human ≥90% (Aβ, Sigma), thioflavin T (Sigma), ponasterone A ≥65 % (Sigma), dialysis tube (MWCO ~ 12000 Da and 2000 Da, Sigma), Dulbecco’s Modified Eagle Medium (DMEM, Sigma), methylthiazolyldiphenyl-tetrazolium bromide (MTT, Himedia) and 2´, 7´-dichlorofluorescin diacetate (Sigma), propidium iodide (PI, Sigma) were used as received. Chinese hamster ovary (CHO) cell and Neuro 2A cell were purchased from National Centre for Cell Science, Pune; Generation of HD 150Q cell line in an ecdysone inducible system are described elsewhere.39 GFP antibody was purchased from Roche and all reagents for SDSPAGE were purchased from Sigma. Preparation of sugar terminated nanoparticle. Sugar terminated nanoparticles were prepared using

our

reported

method

with

some

modifications.40

In

brief

one

gram



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glucose/sucrose/maltose/trehalose was dissolved in 3 mL water. Next, one drop of concentrated hydrochloric acid was added to the solution and the mixture was heated at ~ 90-100 °C for about 2 h. The solution color changed from colorless to deep brown. Near complete carbonization of sugar was tested by adding a drop of nanoparticle solution in 1-2 mL acetone. Unreacted sugar appeared as white precipitate. Next, reaction was quenched by lowering the temperature and by adding sodium hydroxide to adjust the solution pH to 7.4. The solution was dialyzed against distilled water for overnight using dialysis membranes (MWCO 12 KDa). A part of solution was dried to calculate the yield of nanoparticle in weight percent. The yield of nanoparticle products is about 60 %. Stock solution of particle was prepared with the concentration of 20-30 mg/mL. Determination of concentration of sugars on nanoparticle surface. Concentration of sugars on the nanoparticle surface was quantified using anthrone test.41 In this processes nanoparticle or sugar solution is mixed with anthrone solution in 80 % H2SO4 (w/v) and heated at 80 ºC for 10 min to form bluish–green furfural complex. Then the solution was cooled in ice-water. Next, absorbance of furfural complex formed was measured at 630 nm for quantification of sugars. At first, calibration graph was separately prepared for each sugar by plotting absorbance against sugar concentration. Typical calibration graphs were as follows: Y=0.007[glucose], Y=0.115[sucrose], Y=0.019[maltose], Y=0.02[trehalose] where Y=Absorbance of furfural complex and concentration of sugar is in µM. Next, sugar concentration on the surface of each nanoparticle was determined using the respective calibration graph. Fluorescence quantum yield (QY) measurement. The quantum yield of the samples was measured using fluorescein disodium salt as reference (QY = 95 % at 430 nm excitation). The formula used for QY measurement is as follows: (QY)Sm = (QY)St x [(PL area/OD)Sm/(PL area/OD)St] x η2Sm/η2St where Sm indicates the sample, St indicates the standard, η is the


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refractive index of the solvent, PL area indicates fluorescence area and OD indicates absorbance value. QY values are 1-2 % for all nanoparticles. Study of insulin/lysozyme/amyloid beta fibrillation by nanoparticle. Lysozyme, insulin, Aβ proteins were used for this study. Lysozyme stock solution (5 mg/mL) was prepared in acetate buffer (pH 5.5) with 137 mM sodium chloride (NaCl) and 2.68 mM potassium chloride (KCl). Next, solution mixture of lysozyme and nanoparticle (or molecular sugar) was prepared. The final concentration of lysozyme was kept as one mg/mL in each set and concentration of nanoparticles (or molecular sugar) was varied. Solution pH was adjusted to 5.5 using acetate buffer along with 137 mM NaCl, 2.68 mM KCl. Then fibrillation was initiated by heating the solution at 70 ºC. The fibrillation was continued for 24 h and a part of aliquot was collected at different time point for analysis. Similarly, insulin solution (one mg/mL) was prepared in aqueous HCl solution (pH 1.6) along with 0.1 M sodium chloride (NaCl). Then mixed with different concentration of nanoparticle (or molecular sugar) keeping the final concentration of insulin at 0.2 mg/mL and NaCl was added with final concentration of 0.1M. The solution temperature was kept at 65 ºC and a part of aliquot was taken for analysis at different time points. Stock solution of Aβ (~ one mM) was prepared by dissolving 0.5 mg lyophilized powder in 122 µL anhydrous DMF. Next, nanoparticle (or molecular sugar) with varied concentration was mixed with Aβ solution with final concentration of 25 µM. The solution pH was adjusted at 5.5 with acetate buffer along with NaCl concentration of 14 mM and KCl concentration of 2.7 mM. Next, solution was incubated at 37 ºC for 8 days and a part of solution was taken for analysis at different time interval.



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Fibrillation kinetics was measured by ThT assay. Typically, 20 µL of aliquot of protein/peptide solution was collected at different time point and mixed with one mL solution of ThT (10 µM), prepared in phosphate buffer solution of pH 7.4. The emission intensity of ThT at 480 nm was measured under excitation at 440 nm. For AFM analysis, protein/peptide solution at different time points was appropriately diluted with water, drop casted on mica disk and air dried before the AFM study. For TEM analysis, protein/peptide solution was appropriately diluted with water, drop casted on carbon coated copper grid and dried in air before TEM study. For circular dichroism (CD) study, protein/peptide solution (prepared in milli-Q water) at different time points was appropriately diluted with water and used for analysis. Lysozyme oligomer cytotoxicity assay in presence of nanoparticle. Lysozyme oligomer was prepared following the fibrillation procedure described above and at the 30 min time point the protein solution was collected as oligomer.36 CHO cells (2x106/mL) were taken in 24 well plates and incubated with 0.1 mL of lysozyme oligomer (0.4 mg/mL) and nanoparticles (or sugar molecule) of different concentrations. After 24 h, cells were washed using phosphate buffer solution. Then, 50 µL of freshly prepared MTT solution (5 mg/mL) was mixed to each well and incubated for 4 h. After that the produced violet colored formazan was dissolved in 50 % aqueous DMF solution and absorbance was recorded at 570 nm using microplate reader. The optical density was correlated with cell viability, assuming 100 % viability for the control sample without any nanoparticle. Similarly, PI staining experiment was done to determine the extent of cell death. In brief washed cells were treated with 10 µg/mL PI staining solution and then cells were incubated in dark at 37 °C for 10 min. Finally, cells were washed in PBS solution and imaged under green excitation.


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Inhibition of polyglutamine aggregation inside HD150Q cell by nanoparticle. HD150Q cell is a stable and inducible mouse neuroblastoma cell line, expressing green fluorescent protein tagged mutant N-terminal huntingtin protein with 150 glutamine residues. Cells were cultured in 24 well plate 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). After 24 h cells were given fresh media and mixed with nanoparticle solution (or sugar molecule) for 10 h. Next, cells were washed with PBS followed by addition of fresh media. Then ponasterone A solution (final concentration 1 µM) was added to induce the polyglutamine aggregation within cell and incubated at 37 °C for different days and this condition was maintained until cell death and protein aggregation was analyzed by fluorescence microscopy. Western blot and dot blot analysis of mutant huntingtin expression. HD150Q cells were grown in DMEM supplemented with 10 % heat-inactivated fetal bovine serum and antibiotics. For experimental purpose, cells were platted into 6-well tissue cultured plates and induced with ponasterone A, incubated with molecular sugars or sugar nanoparticles for different time periods and then observed under a fluorescence microscope or processed for immunoblot analysis. For immunoblot analysis, cells were washed with ice cold phosphate-buffered saline (PBS), collected by centrifugation, and sonicated on ice for 30 min with Nonident P-40 lysis buffer (consists of 50 mM Tris buffer of pH 8.0, 150 mM NaCl, 1% Nonident P-40, and complete protease inhibitor cocktail). Cell lysate was then centrifuged for 10 min at 15 000 x g at 4 ºC and the supernatants were used for SDS-PAGE followed by immunoblot analysis as previously described.48 Blot was probed with GFP 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



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inhibitor tablet), sonicated briefly and then centrifuged at 13,000 x g for 15 min. Pellet was collected and treated with 2 % SDS at room temperature for 5 min, quantified and equal amounts of protein were allowed to filtered through nitrocellulose membrane. Membranes were then probed with GFP antibody. ROS generation study inside neuronal cell. Amyloid oligomer mediated ROS generation and influence of sugar nanoparticle in lowering of ROS generation has been studied inside HD150Q cell. HD150Q was cultured in 24 well plate under 37 °C with 5 % CO2 using DMEM media with 10 % fetal bovine serum. After 24 h cells were taken in fresh media and mixed with sugar nanoparticle (or sugar molecule) solution. Next, cells were treated with 100 µL lysozyme oligomer solution (1 mg/mL) for 30 min. Next, cells were washed with PBS buffer (pH 7.4) and fresh media were added. Then 5 µL DCF solution (10 mM) was added and incubated for 30 min under 37 °C. Then cells were washed with PBS and fresh DMEM media was added. Then the cells were imaged under fluorescence microscope with blue excitation. Instrumentation. Emission spectra were measured using SynergyTM MX multi-mode microplate reader. Transmission electron microscopic (TEM) samples were prepared by putting a drop of nanoparticle solution on carbon coated copper grid and observed with FEI Tecnai G2 F20 microscope. Fourier transform infrared (FTIR) spectroscopy was performed on Perkin Elmer Spectrum 100 FTIR spectrometer after making pellet with solid KBr. UV-Visible absorption spectra were measured using Shimadzu UV-2550 UV-Visible spectrophotometer. The fluorescence image of the cells was captured using (Olympus (IX-81) fluorescence microscope. Dynamic light scattering (DLS) and Zeta potential measurements were performed using a NanoZS (Malvern) instrument. Diluted sample solution was deposited on mica disk and then AFM was measured using VEECO DICP II autoprobe (model AP 0100) instrument. Circular


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dichroism (CD) spectra were measured using JASCO J-815 CD spectrometer (model J-8151508)

Results and Discussion Sugar-terminated nanoparticle from molecular sugar. Sugar terminated nanoparticles are synthesized via carbonization of molecular form of sugar/carbohydrates. (Figure 1) We have selected four different sugar molecules such as glucose, sucrose, maltose and trehalose for following reasons. First, these sugars are well known and routinely used as food and biochemicals. Second, molecular form of these sugars is known to stabilize proteins under stressed condition.25,42 Third, these sugar molecules are known to weakly interact with amyloid aggregates and some reports show that increase of multivalency can enhance this binding.20,29,31 Fourth, carbonization of these sugars is well known and nanoparticle of 10-100 nm size can be prepared.40,43,44 Synthesis of sugar nanoparticle involves heating of acidified aqueous solution of molecular sugar at 90-100 °C.40 Colorless aqueous solution gradually turns into brown within 12 h during the carbonization and nanoparticles formation. Unreacted sugars are then removed by dialysis. Resultant nanoparticles are named as SNPglucose, SNPsucrose, SNPmaltose and SNPtrehalose depending on the sugar from which they are derived. All the nanoparticles are highly water soluble and stock solution of nanoparticle can be prepared with the concentration of 10-100 mg/mL. Sugar nanoparticles are characterized by elemental analysis, UV-visible and fluorescence spectroscopy, TEM, AFM, DLS and FTIR spectroscopy. Elemental analysis shows 46 wt % as carbon, 5-8 wt % hydrogen and rest is mainly oxygen. UV-visible spectroscopy shows broad bands in the 350-450 nm range and an absorption maxima in 290 nm which is due to conjugated



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n-π* transition (from C=O bonds) and π-π* transition (from C=C bonds), respectively. (Figure 2) Solutions of all these nanoparticles show greenish-yellow emission under hand held UV lamp and emission maxima shifts from 500 nm to 600 nm as the excitation wavelength shifts from 300 nm to 500 nm. (Figure 2) DLS study shows that colloidal solutions of the particles have hydrodynamic size in the range of 20-40 nm. (Figure 2) TEM and AFM have been used to characterize the morphology and diameter of nanoparticle. (Figure 3) TEM image shows 2-5 nm size spherical dots. The overall size of nanoparticles are imaged in AFM which show that particles have platelet like shape with 20-40 nm diameter and 1-2 nm height. Thus combined DLS, TEM and AFM data indicate that particles have plate-like shape with 2-5 nm condensed core along with 8-16 nm soft shell. In order to explore the functional groups around nanoparticle, FTIR study has been performed. (Supporting Information, Figure S1) Vibration stretching bands of hydroxyl groups at 3100-3500 cm-1, CH stretching bands at 3000 cm-1 and C=O stretching bands at 1720-1640 cm-1 are observed for sugar nanoparticle, suggesting the similarity of functional groups as of sugar molecules. (Supporting Information, Figure S1) As nanoparticles are formed via condensation-polymerization-dehydration of molecules, it is expected that their surface can be terminated with respective sugar molecules.43,44 We have tested the presence of sugar molecule on their surface and quantified them using anthrone test.41 In anthrone test sugar molecule reacts with anthrone in concentrated H2SO4 under heating and produces colored furfural complex. Similar type of colored complex is also formed when sugar nanoparticles are reacted with anthrone, suggesting that nanoparticles are actually terminated with respective sugars. (Supporting Information, Figure S2) We have also quantified the amount of sugars on the surface of each nanoparticle and the values are in the range of 8-10 wt % of nanoparticles.


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Inhibition of insulin/lysozyme/amyloid beta fibrillation by nanoparticle. Three different proteins/peptides such as human insulin, lysozyme, amyloid beta 40(Aβ) have been used for aggregation inhibition study by sugar nanoparticles. Among them lysozyme is a known amyloid protein model,45 insulin fibrillation is responsible for diabetes type II46 and Aβ peptides fibrillation is linked to neurodegenerative (e.g. Alzheimer’s) diseases.3 In a typical procedure solution of human insulin/lysozyme/Aβ is incubated with sugar nanoparticle under fibrillation condition and then formation of fibrils has been investigated via thioflavin T(ThT) assay, TEM/AFM-based imaging of fibrils and circular dichroism (CD) based analysis of protein secondary conformation. Experimental results are summarized in Figure 4-8, Table 1 and Supporting Information, Figure S3-S5. ThT is known to emit blue fluorescence after binding with protein beta-sheet structure and appearance of emission of protein/peptide solution indicates formation of fibril structure. Formation of fibril structure can be directly imaged under TEM or AFM. In the present cases insulin fibrils are best imaged under AFM using their solution at different stage of aggregation. Similarly, formation of lysozyme/Aβ fibrils is investigated under AFM/TEM using their solution at different stage of aggregation. CD spectra are widely used to find out the secondary conformational changes of proteins and to obtain information of beta sheet structure. For example appearance of negative peak at 218 nm indicates formation of beta sheet structure, negative peak at 193 nm indicated formation of random coil and positive peak at 220 nm indicates alpha helical conformation. (Figure 4 and 6) Three distinct conclusions can be made from the results obtained on nanoparticle based inhibition of protein/peptide aggregation. First, all the nanoparticles are able to inhibit protein/peptide aggregation in a dose dependent manner. At low dose nanoparticle can inhibit fibrillation only partially but at high dose all the nanoparticle inhibit nucleation and fibrillation.



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Second, fibrillation of insulin, lysozyme and Aβ can be completely inhibited by all the four nanoparticles. Complete inhibition of fibrillation can be observed from insignificant emission signal in ThT assay, absence of fibril structure in TEM/AFM images and absence of respective signal of secondary structure in CD spectra. Third, sugar nanoparticles are 103-105 times efficient than respective molecular sugar. For example, sugar nanoparticles are 103 to 104 times efficient than molecular sugars in inhibiting insulin fibrillation (Figure 4, 5), more than 104 times efficient than molecular sugars in inhibiting lysozyme fibrillation (Figure 6, 7), and 104 to 105 times efficient than molecular sugars in inhibiting Aβ fibrillation. (Figure 8 and Supporting Information, Figure S5) It is also observed that inhibition performance of trehalose nanoparticle (SNPtrehalose) appears 10-100 times better than other three nanoparticles. For example, Aβ fibrillation can be stopped by SNPtrehalose with corresponding trehalose concentration of 0.5x10-5 M, but other nanoparticle can stop fibrillation with corresponding sugar concentration higher than 0.5x10-4 M and molecular trehalose or other sugar cannot stop fibrillation even at 500 mM concentration. (Figure 8 and Supporting Information, Figure S5) Lowering of amyloidogenic cytotoxicity by nanoparticle. Next, we have tested the role of sugar terminated nanoparticle in inhibiting amyloidogenic cytotoxicity. Amyloid oligomers are toxic to cell as they induce membrane damage and generate ROS inside cell.47 We have investigated the amyloidogenic cytotoxicity using two different system. In the first case HD150Q cell has been used as model that produces cytotoxic mutant huntingtin protein intra-cellularly. In

the second case extracellular cytotoxicity of lysozyme oligomer has been studied using CHO cell. HD150Q cell is a unique model for Huntington’s disease that produces mutant huntingtin protein inside the cell and generate toxicity via intracellular aggregation.39 In our present model HD150Q cells produce green fluorescent protein tagged mutant huntingtin, when exposed with


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ponasterone A inducer and aggregation of mutant huntingtin can be followed under fluorescence microscope as green dots.39 Typically, HD150Q cells are incubated with sugar nanoparticle and then exposed with ponasterone A inducer to express the mutant huntingtin. During the incubation stage nanoparticles enter into cell which can be confirmed by fluorescence microscopy. (Figure 9) Exposure with ponasterone A inducer leads to the formation of mutant huntingtin in the form of green fluorescent dot and as time progress these green dots increase in number and size --leading to cell death in 7 days. (Figure 10 and Supporting Information, Figure S6-S9) Most interestingly, if cells are previously exposed with sugar nanoparticle, the formation of green dots is suppressed/delayed by 2-3 days. However, none of the nanoparticles can completely inhibit mutant huntingtin aggregation even at highest tested concentration. Control experiment with molecular sugars are performed for comparison, however, experiments with high molar concentration of sugar (> 100 mM) is difficult due to rapid bacterial contamination and unusual morphology development of cells. These study show that nanoparticle forms are about 102 to 104 times better than respective molecular sugars. Next, we have estimated the mutant huntingtin level in HD150Q cells which are treated with sugar nanoparticle. (Figure 11, 12) Typically, cells are treated with ponasterone A along with sugar nanoparticle (or molecular sugar) and cell lysates are then processed for immunoblot analysis using GFP antibody. Results show that expression of mutant huntingtin protein and insoluble pool of mutant huntingtin is suppressed by sugar nanoparticle but not by molecular sugars. (Figure 11, 12) This result also suggests that sugar nanoparticle effectively inhibits mutant huntingtin expression and aggregation but molecular sugars hardly affect this. In second experiment, HD150Q cells are incubated with sugar nanoparticle (or molecular sugar) followed by incubation with ponasterone A for mutant huntingtin protein expression for 4



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days. Next, cell viability is measured using MTT assay. Results show that mutant huntingtin induced toxicity of HD150Q cells can be significantly suppressed by sugar nanoparticle. (Figure 13a) For example, cell viability can be enhanced to > 60 % in presence of nanoparticle corresponding to 0.03-0.30 mM sugar, as compared to 40-50 % viability in presence of 50-100 mM of molecular sugar. Interestingly, SNPtrehalose appears more efficient than other nanoparticles. In third experiment, oligomeric forms of lysozyme is mixed with sugar nanoparticle (or molecular sugar) and incubated with live CHO cell. Next, cytotoxicity has been studied using conventional MTT assay and PI staining and compared with molecular form of sugar. Results show

that sugar nanoparticles are more efficient than respective molecular sugar in lowering of amyloidogenic toxicity. (Figure 13b) For example, only 40 % cells are viable for 0.4 mg/mL lysozyme oligomers and cell viability can be enhanced to > 60 % in presence of nanoparticle corresponding to 1-2 mM sugar. In contrast cell viability ranges between 40-50 % in presence of 100 mM of molecular sugar. This result suggests that sugar nanoparticle possibly interact with lysozyme oligomers and inhibit their interaction with cell membrane. PI staining experiment shows that lysozyme oligomer treated cells exclude PI suggesting cells remain alive. In contrast molecular sugar treated cells are labeled with PI due to membrane damage. We have further studied the lysozyme oligomers induced ROS generation and found that sugar nanoparticle can inhibit the ROS generation. (Figure 13c) This result suggests that sugar nanoparticle decreases the cytotoxicity by lowering the ROS generation. Sugar terminated nanoparticle as efficient artificial chaperone. We anticipate three unique reasons for sugar terminated nanoparticles as better chaperone than molecular sugars. First, these nanoparticles are platelike in nature with hydrophobic core made of graphitic carbons and hydrophilic soft shell with polar sugar molecules.40,43 In contrast sugar molecules are highly 16 ACS Paragon Plus Environment

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hydrophilic in nature due to polar hydroxyl and other groups. Thus protein can interact with hydrophobic core as well as hydrophilic shell of nanoparticle and leads to enhanced binding with proteins and protein aggregates. Second, binding of these nanoparticles with protein/protein aggregate is multivalent in nature. It is expected that nanoparticle of 20-40 nm size has few tens to few hundred sugar molecule around their surface. Thus each nanoparticle can bind with protein or protein aggregate via multiple sugar molecules (i.e. multivalent binding). In contrast interaction of molecular sugar with protein is weaker due to predominate monovalent (single point) binding. Such enhanced binding of sugar nanoparticle has been verified experimentally. Binding interaction of nanoparticle with lysozyme fibril is studied in presence of molecular sugar and it is found that nanoparticle can interact with fibril even in presence of 104-107 times of excess sugar molecule. (Supporting Information, Figure S10) Third, nanoparticle can enter into cell more easily via endocytosis, which is not possible for molecular sugar. It is known that cell can interact with nanoparticles much better than small molecule, as nanoparticle has multiple interacting sites that can initiate endocytotic uptake processes.37 Thus enhanced cellular entry of nanoparticle offers their increased interaction with intracellular protein. Enhanced chaperone performance of presented nanoparticle extends the possibility for practical application. It is known that sugar molecules act as chaperone at > millimolar concentration but such high concentration is difficult to achieve under physiological condition. In contrast sugar terminated nanoparticle can act as chaperone at micromolar concentration and this concentration can be easily achieved under physiological condition. Considering the low toxicity of sugar molecule and sugar based polymer/nanoparticle, presented approach can be extended for in vivo application.



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Presented work demonstrate second clear example that nanoparticle form of antiamiloidogenic molecule can be more efficient than respective molecular form. We have recently demonstrated that nanoparticle form of green tea polyphenol has 10-100 times enhanced antiamyloidogenic performance as compared to respective molecular form.36 Although polyphenol and sugars are different class of molecules with different chemical properties, both classes are known to have anti-amyloidogenic property. We have shown that nanoparticle form of green tea polyphenol can enhance the anti-amyloidogenic performance upto 100 times but nanoparticle form of sugar can enhance this performance upto 105 times. Despite the fact that sugars are not considered as conventional drug and their anti-amyloidogenic performance is poorer than green tea polyphenol, this result is significant. From chemical point of view green tea polyphenol has aromatic rings and prone to oxidation-reduction reaction36 but sugars do not have aromatic rings and they are comparatively less prone to redox reaction. This difference in chemical property may be linked to the differential anti-amyloidogenic property -- both in molecular and nanoparticle form.

Conclusion We have shown that nanoparticle form of sugar molecule can be much better chaperone than respective molecular sugar. In particular we found that 20-40 nm size sugar terminated nanoparticle can inhibit fibrillation of lysozyme/insulin/amyloid beta in extracellular space and inhibit aggregation of mutant hunting protein inside model neuron cell. In addition cytotoxic effect of fibrils is decreased in presence of these nanoparticles. In general sugar terminated nanoparticles are 102-105 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity of fibrils. We conclude from our study that better


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performance of nanoparticle form is linked to their stronger binding with fibril structure and enhanced cell uptake. This work clearly demonstrates that sugar terminated nanoparticles inhibit protein aggregation at micromolar concentration as compared to millimolar concentration requirement for sugar molecules. Similar nanoparticle may be made from poorly performing osmolyte molecules for testing of protein aggregation studies and to make them practically more useful. ASSOCIATED CONTENT Supporting Information Details of characterization of sugar nanoparticle, additional data of extracellular and intracellular protein fibrillation and binding data of sugar terminated nanoparticle with protein fibril. This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interests. Acknowledgement. The authors acknowledge Department of Science and Technology (DST), Government of India for financial assistance. (Grant number SR/NM/NS-1143/2016) We also acknowledge Department of Biotechnology (DBT), Government of India for providing financial support to National Brain Research Centre.

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Table 1. Property of four sugar terminated nanoparticles and their performance in inhibiting protein aggregation and lowering of cytotoxicity.

Sugar-based Size from nanoparticle DLS/AFM, (SNP) thickness from AFM SNPglucose SNPsucrose SNPmaltose SNPtrehalose

20-40 nm, 1-2 nm 20-40 nm, 1-2 nm 20-40 nm, 1-2 nm 20-40 nm, 1-2 nm

C (wt %), H (wt %) sugar (wt %), fluorescence QY (%) 45, 5, 8-10, 2 46, 6, 8-10, 4 44, 5, 8-10, 2 43, 6, 8-10, 2

Aggregation inhibition by SNP, compared to molecular sugar (lysozyme, insulin, Aβ) 104 times, 103 times, 104 times 104 times, 103 times, 104 times 104 times, 103 times, 104 times 104 times, 104 times, 105 times

Cytotoxicity reduction by SNP, compared to molecular sugar (CHO, neuro 2A) 103 times, 104 times 103 times, 103 times 102 times, 102 times 104 times, 104 times


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Figure 1. a) Schematic representation of preparation of sugar terminated nanoparticle from molecular sugar. Surface of these nanoparticles are terminated with respective molecular sugar and depending on sugar they are named as SNPglucose, SNPsucrose, SNPmaltose, SNPtrehalose. b) Colloidal solution of sugar terminated nanoparticle under day light (upper panel) and UV light (bottom panel).


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Figure 3. TEM image (left panels) and AFM image along with height profile (right panels) of SNPglucose (a), SNPsucrose (b), SNPmaltose (c) and SNPtrehalose (d). TEM image shows 2-5 nm size. AFM images of selected particles (i, ii, iii, iv) indicate that they have platelet like shape with 2040 nm diameter and 1-2 nm height.



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Figure 5. Representative AFM image of insulin aggregates after exposed under fibrillation condition in presence of 100 mM molecular glucose/sucrose/maltose/trehalose or 0.1/1.0 mM SNPglucose/SNPsucrose/SNPmaltose/SNPtrehalose. Results show that molecular sugars at 100 mM are partially efficient in inhibiting insulin fibrillation but nanoparticle forms induce near complete inhibition of insulin fibrillation at 0.1/1.0 mM concentration, meaning that nanoparticles are > 103 times efficient than molecular sugars.



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Figure 7. Representative TEM image of lysozyme fibrils/aggregates formed after exposed under fibrillation condition in presence of 100 mM molecular glucose/sucrose/maltose/trehalose or 0.01 mM SNPglucose/SNPsucrose/SNPmaltose/SNPtrehalose. Results show that molecular forms at 100 mM are partially efficient in inhibiting lysozyme fibrillation but nanoparticle form can induce near complete inhibition of lysozyme fibrillation at 0.01 mM concentration, meaning that nanoparticles are > 104 times efficient than molecular sugars.



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Figure 8. ThT based study of amyloid beta fibrillation kinetics and representative AFM image of amyloid fibrils/aggregates formed after exposed under fibrillation condition in presence of 500 mM

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Figure 9. Evidence of high cell uptake of sugar nanoparticles. HD150Q cells without any ponasterone A treatment is incubated with colloidal solution of SNPtrehalose and washed cells are imaged under bright field (BF) and fluorescence (F) mode with blue excitation.



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Figure 10. a) Bright field (BF) and fluorescence (F) image of HD150Q cells at different days after exposed with ponasterone A inducer. Green fluorescent protein tagged mutant huntingtin aggregates are observed as green fluorescent dots (shown as red arrows) at 3 to 4 days that leads to cell death (shown as blue arrows). b) Bright field (BF) and fluorescence (F) image of HD150Q cells at different days after exposed with ponasterone A inducer and SNPglucose corresponding to 0.1 mM glucose. Formation of mutant huntingtin aggregates (seen as green fluorescent dot, shown as red arrows) and cell death (shown as blue arrows) are delayed by 2 to 3 days.


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Figure 11. Suppression of mutant huntingtin level in HD150Q cells upon treatment with sugar nanoparticle. a) Cells are either untreated (lane 1) or treated with ponasterone A (lanes 2-10) along with different molecular sugars of 50 mM concentration (lane 2-no sugar, lane 3-glucose, lane 4-sucrose, lane 5-maltose, lane 6-trehalose) and sugar nanoparticle corresponding to 500 µM sugar (lane 7-SNPglucose, lane 8-SNPsucrose, lane 9-SNPmaltose, lane 10-SNPtrehalose) for 3 days and cell lysates are then processed for immunoblot analysis using GFP antibody. b) Quantification of band intensities of soluble truncated mutant N-terminal huntingtin (tNhtt) shown above using NIH image analysis software. Data were normalized against beta-actin.



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Figure 12. Treatment of HD150Q cells with sugar nanoparticle reduces the insoluble mutant huntingtin level. a) Cells are left untreated (dot 1) or induced with ponasterone A (dots 2-10) along with molecular sugars of 50 mM concentration (dot 2-no sugar, dot 3-glucose, dot 4sucrose, dot 5-maltose, dot 6-trehalose) and sugar nanoparticle corresponding to 0.5 mM sugar (dot 7-SNPglucose, dot 8-SNPsucrose, dot 9-SNPmaltose, dot 10-SNPtrehalose) for 3 days and cell homogenates are then processed for dot blot analysis using GFP antibody. b) Quantification of insoluble truncated mutant N-terminal huntingtin is shown above using NIH image analysis software. Results show that insoluble pool of mutant huntingtin is decreased by sugar nanoparticle but not by molecular sugars.


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Figure 13. a) HD150Q cell viability in presence of molecular sugar or sugar nanoparticle. Cells are incubated with sugar nanoparticle (or molecular sugar) followed by incubation with ponasterone A to induce mutant huntingtin protein expression for 4 days. Cell viability is then measured using MTT assay. b) Lysozyme oligomer induced toxicity of CHO cells and enhanced cell viability in presence of sugar nanoparticle. Cells are incubated with sugar nanoparticle (or molecular sugar) and lysozyme fibril for 24 h. Cell viability is then measured using MTT assay c) Evidence of ROS generation by lysozyme oligomers (LO) inside cells and lowering of this ROS generation in presence of sugar nanoparticles. HD150Q cell without any ponasterone A treatment is exposed with LO and SNPtrehalose followed by incubation with ROS staining dye and washed cells are imaged under bright field (BF) or fluorescence (F) mode. Green emission indicates ROS formation.



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Sugar-Terminated Nanoparticle Chaperones Are ... - ACS Publications

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