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Poly(trehalose) Nanoparticle Prevents Amyloid Aggregation and Suppress Polyglutamine Aggregation in Huntington’s Disease Model Mouse Koushik Debnath, Nibedita Pradhan, Brijesh Kumar Singh, Nihar R Jana, and Nikhil R. Jana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06510 • Publication Date (Web): 20 Jun 2017 Downloaded from http://pubs.acs.org on June 21, 2017

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ACS Applied Materials & Interfaces

Poly(trehalose) Nanoparticle Prevents Amyloid Aggregation and Suppress Polyglutamine Aggregation in Huntington’s Disease Model Mouse

Koushik Debnath,1 Nibedita Pradhan,1 Brijesh Kumar Singh,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]

1

ACS Paragon Plus Environment


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ABSTRACT: Prevention and therapeutic strategy of various neurodegenerative disease focus on inhibiting protein fibrillation, clearing of aggregated protein plaques from brain and lowering of protein aggregate-induced toxicity. We have designed poly(trehalose) nanoparticle that can inhibit amyloid/polyglutamine aggregation under extra-/intra-cellular condition, reduce such aggregation-derived cytotoxicity and prevents polyglutamine aggregation in Huntington’s disease (HD) model mouse brain. Nanoparticle has 20-30 nm hydrodynamic size, composed of 6 nm iron oxide core and zwitterionic polymer shell with covalently linked trehalose of ~5-12 wt %. Designed poly(trehalose) nanoparticle is 1,000-10,000 times efficient than molecular trehalose in inhibiting protein fibrillation under extra-cellular space, in blocking aggregation of polyglutamine containing mutant huntingtin protein in model neuronal cell and in suppressing of mutant huntingtin aggregates in HD mouse brain. We show that nanoparticle form of trehalose with zwitterionic surface charge and trehalose multivalency of ~ 80-200 (number of trehalose per nanoparticle) are crucial for efficient brain targeting, entry into neuron cell and suppressing mutant huntingtin aggregation. Present work shows that nanoscale trehalose can offer highly efficient anti-amyloidogenic performance at micromolar concentration, as compared to millimollar to molar concentration of molecular trehalose. This approach can be extended for in vivo application to combat protein aggregation-derived neurodegenerative diseases. KEYWORDS: nanoparticle, trehalose, amyloid aggregation, polyglutamine aggregation, neurodegenerative disease, Huntington’s, Alzheimer’s

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INTRODUCTION Protein aggregation is pathogenic hallmarks in various neurodegenerative and other diseases that include Alzheimer’s, Parkinson’s, Huntington’s and diabetes type II.1-8 Variety of proteins have been identified to fold abnormally consisting of crossed β–sheet structure, in forming pathological amyloid deposit/aggregate.9,10 Current research focus on diagnostic tool for early detection of fibrillation, understanding the mechanism of

protein fibrillation, origin of

cytotoxicity by protein oligomers, identification of chemicals to inhibit protein fibrillation, disintegrating protein fibrils by physical/chemical approach and lowering of amyloidogenic toxicity by chemical/biochemical processes.10 It is now well established that protein fibrillation follows nucleation-growth mechanism where small nuclei is formed through oligomerization and then elongated into fibril via protofibril formation. Thus attention has been paid to identify chemical/material that can inhibit protein fibrillation, decrease the toxicity of oligomeric protein and disintegrate/clear aggregated deposits from brain.10 Nanotechnology based approach has made notable impact on the preventive strategy of neurodegenerative diseases.11-16 These include design and engineering of nanoparticle that influence protein fibril nucleation,17-26 degrading matured protein fibrils23,24,27,28 and targeting aggregated protein plaques via crossing the blood-brain barrier.29,30 For example hydrophobic polymer nanoparticle,18 quantum dot,19,21 protein microsphere,20 carbon nanoparticle,26 gold nanoparticle23 and selenium nanoparticle24 are reported to inhibit protein fibrillation. Peptide functionalized gold nanoparticle27 and thioflavin conjugated graphene oxide28 are designed to disintegrate preformed fibrils under light. Nanoparticle based systems have been designed to cross the blood-brain barrier29-31 and target the amyloid fibril/plaque in brain.32-34 These results suggest that nanoparticle-based platform might be an exemplary approach to inhibit protein 3



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fibrillation occurring under intra- and extra-cellular environment35-39 and appropriate nanoparticle should be designed to target, disintegrate and clear the toxic protein plaque from brain. In order to enhance the interaction with protein, nanoparticle is functionalized with peptide,21,25,27 amino acid,19,22 sialic acid,15 thioflavin,28 epigallocatechin-3-gallate24 and curcumin.23 These molecules are selected as they are known to interact with amyloid structure and there is ongoing effort to identify chemicals that can efficiently inhibit protein aggregation under in vitro/in vivo conditions. Among them most promising molecules are epigallocatechin-3gallate,24 curcumin23 and trehalose3 that are effective typically above their micromolar concentration. Among them trehalose is an interesting disaccharide, which is commonly known as protecting agent for the cell,40 protein,41,42 RNA43 and nanoparticle44 against environmental stress and it is the most effective carbohydrate in hindering protein aggregation.45-51 For example, trehalose is shown to inhibit α-synuclein fibrillation,50 insulin fibrillation,47 Aβ peptide fibrillation46,49 and alleviates pollyglutamine-mediated pathology in Huntington’s disease (HD) mouse.45 However, trehalose is effective only at high concentration (typically in the millimolar to molar concentration) and it is also shown that multivalent (2-7) trehalose can enhance (by 2050 %) the amyloid inhibition and improve the performance in neutralizing amyloid derived cytotoxicity.52-54 We have recently shown that nanoparticle form of anti-amyloidogenic molecules can be more efficient than respective molecular forms.55,56 In particular, green tea polyphenol based polymer micelle shows 10-100 times better anti-amyloidogenic performance than respective molecular form and better performance of nanostructure is linked to increased chemical stability of polyphenol, multivalent binding effect with protein and high cellular uptake via endocytosis.55 Similarly, sugar-based carbon nanoparticles show 102 to 105 times better 4


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performance than molecular sugars in inhibiting protein aggregation and trehalose based nanoparticle appears better than glucose/sucrose/maltose-based nanoparticles.56 However, performance of nano-trehalose in inhibiting intracellular protein aggregation is observed only at high dose (0.5 mM of trehalose) and no noticeable in vivo effect is detected.56 We presumed that distorted chemical structure of trehalose during carbonization based nanoparticle synthesis, poorly defined trehalose multivalency (number of trehalose per nanoparticle) and non-ionic surface charge of nanoparticle with limited cellular endocytosis are linked to such poor in vitro/in vivo performance. Earlier we have demonstrated that zwitterionic surface chemistry of nanoparticle is ideal for efficient cellular uptake with minimum cytotoxicity57 and presumed that such surface chemistry combined with intact chemical structure of trehalose and optimum trehalose multivalency may further enhance the in vitro/in vivo performance of trehalose. Here we have synthesized zwitterionic poly(trehalose) nanoparticle with intact chemical form of trehalose. Resultant nanotrehalose offers 1,000-10,000 times better in vitro/in vivo performance than molecular trehalose. Poly(trehalose) nanoparticle has 20-30 nm hydrodynamic size that is composed of 6 nm iron oxide core and zwitterionic polymer shell with covalently linked trehalose of ~5-12 wt %. While in vitro/in vivo anti-amyloidogenic activity of molecular trehalose is observed at millimolar to molar concentration and trehalose-based carbon nanoparticle shows such effect at 0.5 mM trehalose concentration, the poly(trehalose) nanoparticle offers this anti-amyloidogenic performance at micromolar concentration. The presented poly(trehalose) nanoparticle has three unique features over molecular trehalose and our recently reported sugar-terminated nanoparticle.56 First, it strongly interacts with cell membrane via multiple numbers of cationic and anionic functional groups and induce high endocytotic uptake. Second, it has well defined number of trehalose (about 80-200 per particle) that offers 5



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strong binding with protein via multivalency and efficiently inhibit protein aggregation. Third, it efficiently blocks polyglutamine inclusions in the brain of HD mouse at micromolar trehalose concentration. EXPERIMENTAL SECTION Chemicals. Hen egg white lysozyme (HEWL) protein, amyloid β(1-40) peptide (Aβ), 3sulfopropyl methacrylate (sulfo-acrylate), N-(3-aminopropyl) methacrylamide hydrochloride (amino-acrylate),

poly(ethylene

glycol)

methacrylate

(PEG-acrylate)

and

N,N-

methylenebisacrylamide, MES (2-(N-morpholino)ethanesulfonic acid), EDC (1-ethyl-3-(3dimethylaminopropyl)carbodiimide), trehalose, rhodamine B, NHS (N-hydroxysuccinimide) and fluorescein-NHS were purchased from Sigma and used as received. Preparation of trehalose mono-carboxylic acid. About 175 mg trehalose was dissolved in 10 mL of dry dimethylformamide (DMF). In a separate vial 48 mg succinic anhydride was dissolved in one mL dry DMF and mixed with trehalose solution. Next, one mL of dry DMFtriethylamine mixture (100:7 volume ratio) was added and the reaction was continued for 12 hrs at 80 °C under nitrogen atmosphere. The product was precipitated by diethyl ether-acetone mixture (70:30 volume ratio) and dried in air. Preparation of trehalose mono-methacrylate. About 378 mg of trehalose was dissolved in 12 mL dry DMF and mixed with 836 µL dry triethylamine under Ar atmosphere. Then, 96 µL of dry crotonoyl chloride was added and the reaction was continued for 6 hrs at 55 °C under Ar atmosphere. Next, the solution was cooled to room temperature and the excess diethyl ether was added to precipitate the product and then it was dried in air. Preparation of poly(trehalose) nanoparticle. Hydrophobic γ-Fe2O3 nanoparticle was synthesized via high temperature colloid-chemical approach and then converted to polyacrylate 6


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coated water soluble nanoparticles using our previously reported method.58 In brief, hydrophobic nanoparticle and acrylate monomers are dissolved in reverse micelle and polyacrylate formation was initiated under nitrogen atmosphere in presence of persulfate. Different acrylate monomers were used in varied mole fractions as shown in Table S1. In method A three acrylates were mainly used that include sulfo-acrylate (to introduce SO3-), amino-acrylate (to introduce NH2), PEG-acrylate (to introduce PEG). The resultant polymer coated nanoparticle has ~ 100-1200 primary amine groups per nanoparticle on their surface which is used for conjugation with trehalose mono-carboxylic acid via EDC coupling. (method A) Typically, aqueous solution of trehalose mono-carboxylic acid was prepared in MES buffer of pH 5 with the concentration of 10 mg/mL. Next, 38 mg EDC and 38 mg NHS were added under stirring condition and immediately 0.5 mL aqueous solution of nanoparticle (5 mg/mL) was added and the reaction was continued for 12 hrs at room temperature. Trehalose functionalized nanoparticle was precipitated by adding ethanol and then purified by dialysis using dialysis membrane (MWCO 12,000 Dalton). In method B, acrylate coating of hydrophobic nanoparticle was performed using trehalose mono-methacrylate along with other acrylate monomers. The mole percents of trehalose-methacrylate and other acrylate were systematically varied to control the number of nanoparticle bound trehalose and overall surface charge. (see Table S1 for details) The amount of trehalose in these materials was estimated by anthrone test (see below) and the value varied from 0.5-12 wt % depending on the method and mole fraction of trehalose mono-methacrylate used. In addition the nanoparticle surface charge can be varied between -20 mV to +5 mV, depending on the acrylate monomer and their mole %. Colloidal solution of nanoparticle was prepared with the concentration of 0.5-2 mg/mL and used as stock solution.

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Preparation of rhodamine B and fluorescein conjugated poly(trehalose) nanoparticle. Rhodamine B was conjugated with poly(trehalose) nanoparticle via EDC coupling. Typically, rhodamine B (10 µmole) was mixed with 0.5 mL of poly(trehalose) nanoparticle solution (5 mg/mL) followed by addition of 4.8 mg EDC and 1.15 mg NHS. Fluorescein was conjugated with poly(trehalose) nanoparticle by the reaction between fluorescein-NHS and primary amine of poly(trehalose) nanoparticle in borate buffer of pH 8. About one mL borate buffer solution of poly(trehalose) nanoparticle was mixed with 100 µL DMF solution of fluorescein-NHS (2 mg/mL) and was allowed to react at room temperature for 2 hrs. Next, nanoparticles were precipitated by addition of excess acetone followed by high speed centrifuge. Finally, aqueous solution of nanoparticle was extensively dialyzed using dialysis membrane (MWCO 12,000) at pH 9-10 for 3 days. Purified nanoparticle solution was used for further study. Estimation of trehalose via anthrone test. Anthrone test was used for estimation of trehalose in poly(trehalose) nanoparticle.59 Furfural produced from carbohydrate (here trehalose) reacts with anthrone and forms blue-green color which was widely used for quantitative estimation of various carbohydrates.59 In the present case we have prepared anthrone solution (0.2 wt %) in 80 % conc. H2SO4. Next, 2 mL anthrone solution was mixed with 200 µL trehalose solution or poly(trehalose) nanoparticle solution and was heated in water bath for 15 min. Next, the mixture was cooled and absorption spectra were measured. A calibration graph was prepared for different known concentrations of trehalose which can be written as [trehalose] = 0.730 × absorbance (R2 value 0.99). Next, this equation was used to estimate the nanoparticle bound trehalose. Amyloid fibrillation study using model protein and peptide. Amyloid fibril formation was studied using HEWL protein and Aβ peptide. About one mL of HEWL solution (2 mg/mL, 140 µM) was prepared by dissolving in water of pH 5.0 (adjusted by HCl) and containing NaCl (137 8


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mM) and KCl (2.7 mM). Protein solution was heated to 60 °C and kept under magnetically stirring condition for 24 h. HEWL aggregation kinetic was monitored using thioflavin T assay. Typically, 20 µL of protein solution was collected at different time intervals and was mixed with one mL of thioflavin T (10 µM) solution. After 5 min thioflavin T fluorescence was measured at 480 nm with 440 nm excitation. In order to study the effect of nanoparticle on the fibrillation kinetics, the nanoparticle solution was added to protein solution and aggregation kinetics was studied following the same thioflavin T titration procedure. Similarly, Aβ peptide solution (1 mM) was prepared first by dissolving the lyophilized peptide in dry DMSO and kept at -30 °C to maintain the monomeric stock solution. In order to perform thioflavin T fibrillation assay, stock solution was diluted to 25 µM with aqueous solution containing 140 µM NaCl and 2.7 µM KCl. Here, pH of the solution was maintained by adding 50 µL of MES buffer of pH 5. The peptide solution was then incubated at 37 °C for 15 days for complete fibrillation. In order to study the effect of poly(trehalose) nanoparticle on the kinetics of fibrillation, 50 µL solution of poly(trehalose) nanoparticle was mixed with amyloid solution and the progress of fibrillation was monitored by taking the aliquots time to time. Thioflavin T assay, HRTEM, circular dichroism spectra, western blot assay and dot blot assay were used to determine the fibrillation. In order to observe the structure of Aβ fibril in presence or absence of poly(trehalose), each solution was centrifuged for 30 min at 15000 x g after 15 days of fibrillation process, precipitate was re-dispersed in PBS buffer and used for 8 % SDS-PAGE gel based immunoblot analysis. Blot was probed with 6E10 antibody. For dot blot analysis amyloid beta fibril solution was allowed to filter through nitrocellulose membrane. Membrane was then probed with 6E10 antibody.

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Polyglutamine-based mutant huntingtin protein aggregation study using HD150Q cell line. HD150Q cell is a mutant mouse neuroblastoma cell line (N2A) expressing a truncated Nterminal huntingtin containing 150 glutamine residues, fused to an enhanced green fluorescent protein. Cells were cultured in DMEM media with 10 % fetal bovine serum. In order to study the effect of nanoparticle on the aggregation kinetics, the cells were cultured in a 12 well plate at 37 °C with 5 % CO2 incubation. After 24 h cells were incubated with 0.5 mL fresh media along with nanoparticles at 37 °C for 1-4 h. Next, media was removed and cells were washed with PBS for 2 times. Next, 0.5 mL fresh media along with one µM ponasterone A was added and incubated at 37 °C to induce aggregation of mutant huntingtin protein. This condition was maintained upto the cell death and protein aggregation kinetics was studied time to time. The effect of nanoparticle on mutant huntingtin aggregation was further quantified from cell lysate. Localization of poly(trehalose) nanoparticle within the cell was studied using their flourescein/rhodamine B conjugate. In particular rhodamine B conjugated poly(trehalose) nanoparticle was used for study in HD150Q cell and fluorescein conjugated poly(trehalose) nanoparticle was used for study in N2A and chinese hamster ovary (CHO) cell. Cells were cultured in a 24 well plate in modified DMEM media. Then cells were incubated with rhodamine B/fluorescein conjugated poly(trehalose) nanoparticle for 4 h in CO2 incubator at 37 °C and under 5% CO2. In the case of HD150Q cell, polyglutamine aggregation was induced by treating with one µM ponasterone A for 1 day and washed cells were then incubated with rhodamine B conjugated poly(trehalose) nanoparticle for 4 h. Next, cells were washed with phosphate buffer and used for imaging study. Blue excitation was used for mutant hunting protein imaging and green excitation was used for rhodamine B conjugated poly(trehalose) nanoparticle imaging.

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Western blot and dot blot experiment for mutant huntingtin quantification. HD150Q cells were grown in DMEM supplemented with 10 % heat-inactivated fetal bovine serum and antibiotics containing 0.4 mg/mL Zeocin and 0.4 mg/mL G418. For experimental purpose, cells were platted into 6-well tissue cultured plates and induced with ponasterone A. Next, cells were incubated with poly(trehalose) nanoparticle or molecular trehalose or control nanoparticle 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 phosphatebuffered saline (PBS), collected by centrifugation, and sonicated on ice for 30 min with Nonident P-40 lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and complete protease inhibitor cocktail). Cell lysates were 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.55 Blot was probed with GFP or ubiquitin antibody. For dot blot analysis, HD150Q cells were homogenized in homogenization buffer (50 mM Tris, pH, 7.4, 150 mM NaCl, 1 % Triton X-100, 1 mM PMSF, and complete protease inhibitor tablet), sonicated briefly and then centrifuged at 13,000 x g for 15 min. Pellet was collected and treated with 2 % SDS at room temperature for 5 min and were allowed to filtered through nitrocellulose membrane. Membranes were then probed with GFP antibody. Animal experiment. The transgenic mice for HD [strain B6CBA-Tg (HDexon1) 62Gpb/3J] was purchased from the Jackson Laboratory (USA) and maintained in the animal house facility of National Brain Research Centre. Animals had free access to a pelleted diet and water ad libitum. All animal experimentation were conducted as per the institutional guidelines for the use and care of animals and all experimental protocols were approved by the Institutional Animal Ethics 11



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Committee of the National Brain Research Centre. (Protocol Number: NBRC/IAEC/2016/117) PCR-based genotyping protocol was adapted from the earlier studies.61 After genotyping, female HD mice were intraperitoneally injected with 100 µL of either normal saline or control nanoparticle (0.4 mg/mL) or poly(trehalose) nanoparticle (0.4 mg/mL corresponding to 50 µM trehalose). There were two sets of experiments. In one experimental protocol, HD mice were injected at their age of 42 days and each mouse received total 5 injections at 2 days interval. Mice were sacrificed at their age of 56 days. In another experiment, HD mice were given injections at their age of 60 days and each mouse was given total 10 injections at 2 days interval. Mice were sacrificed at 84 days of their age. During the experiments, mice were monitored of their body weight and toxic effect of the injected chemicals. Immunohistochemical staining. Mice were anaesthetized and transcardially perfused with 4 % paraformaldehyde in PBS and brains were carefully dissected out. Brains were then processed for cryo-sectioning using freezing microtome and coronal sections (20 µm thickness) were obtained. Sections were then subjected to antigen retrieval for 45 min at 70 °C, washed with PBS, blocked with normal goat serum, and then incubated overnight with huntingtin antibody (anti-goat at 1:100 dilutions). Biotinylated secondary antibody was used at a dilution of 1:500 and signal was enhanced using ABC reagent and developed using ImmPACT Novared Peroxidase Substrate. Stained sections were observed in a Leica DM RXA2 microscope and digital images were obtained. Quantification for cell uptake and brain uptake: HD150Q Cells were cultured in serum free DMEM media and then incubated with/without poly(trehalose) nanoparticle solution for 4 hrs and then cell dispersion of 4.5x104 /mL is used for measurements. Next, cells were washed with 12


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PBS buffer for 3 times. Next, cells were isolated by centrifuge and dried overnight under hot air oven. Finally cells were dissolved in 1mL 65% suprapure HNO3 and 5.5 mL Milli-Q water and then quantitative estimation of iron was done for each sample by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Brain section of the sacrificed mice (at 84 days of their age) were collected and dried overnight under hot air oven. Then quantitative estimation of iron was done with similar protocol described above. Instrumentation. UV-visible absorption and fluorescence spectral studies were carried out using Shimadzu UV-2550 UV-visible spectrophotometer and BioTek SynergyMx microplate reader, respectively. Emission spectra were measured using SynergyTM MX multi-mode microplate reader. Transmission electron microscopic (TEM) samples were prepared by putting a drop of sample solution on carbon coated copper grid and observed with FEI Tecnai G2 F20 microscope. Circular dichroism (CD) spectra were measured using JASCO J-815 CD spectrometer (model J815-1508). The hydrodynamic sizes and zeta potentials were measured using NanoZS (Malvern) instrument. Diﬀerential interference contrast and ﬂuorescence images of live cells were performed using Olympus IX81 microscope and Axio Plan inverted Apotom microscope (Zeiss) using image-pro plus version 7.0 software. Amount of iron (after treatment) content in cell lysate and in mice brain section were estimated by Optima 2100DV inductively coupled plasma atomic emission spectroscopy (ICP-AES, Perkin Elmer). Chemi Gel documentation (Biodigital Pvt Ltd) and DOT Blot apparatus (BIO RAD) were used for western blot and DOT blot experiment. LEICA DM 2500 Bright field microscope was used for brain section imaging. QSONICA Q700 sonicator was used for cell lysate preparation.

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RESULTS AND DISCUSSION Poly(trehalose) nanoparticle with modular surface charge and trehalose multivalency. The chemical composition and structure of poly(trehalose) nanoparticle is shown in Figure 1. Nanoparticle is composed of 6 nm iron oxide core and 10-15 nm polyacrylate shell having different chemical functional groups such as primary amine (NH2), SO3-, polyethylene glycol (PEG) and trehalose. As polyacrylate shell consists of both cationic (protonated primary amine at physiological pH) and anionic (SO3-) functional groups and each nanoparticle consists of multiple number of trehalose on their surface, they are named as zwitterionic poly(trehalose) nanoparticle. Synthetic strategy involves transformation of surfactant capped hydrophobic iron oxide nanoparticle into trehalose functionalized hydrophilic nanoparticle via polyacrylate coating.58 Different acrylate monomers are used to introduce PEG, NH2 and SO3- on the polyacrylate shell. (for details see experimental section and Supporting Information, Table S1, Figure S1 and S2) Two different approaches are employed for trehalose functionalization of nanoparticle. In method A, NH2 functionalized zwitterionic nanoparticle is prepared first and then a fraction of NH2 is used for covalent conjugation with trehalose mono-carboxylic acid via EDC coupling. This method produces zwitterionic nanoparticle with 10-12 wt % of trehalose. In method B, trehalose acrylate is used as one of the acryl monomer during the polyacrylate coating. This method can produce zwitterionic nanoparticle with tunable wt % of trehalose, typically in the range from 0.5-12 wt %. Property of zwitterionic poly(trehalose) nanoparticle is summarized in Figure 1 and Supporting Information, Table S1 and Figure S3. TEM image of as synthesized hydrophobic γ-Fe2O3 shows uniform size of 6 nm and dynamic light scattering (DLS) data of poly(trehalose) nanoparticle shows average hydrodynamic size of 20-30 nm that include 6 nm γ-Fe2O3 core and 10-15 nm 14


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polyacrylate shell. Zwitterionic surface charge of nanoparticle is established from primary amine estimation and positive or negative zeta potential value (from + 6 mV to – 20 mV) depending on solution pH. Amount of primary amine on nanoparticle surface has been estimated via fluorescamine test.58 These primary amines are protonated at neutral/acidic pH and acts as source of ammonium (NH3+) cation. Most of the synthesized poly(trehalose) nanoparticles have over all anionic surface charge at pH 7.4 (typically of -5 to -10 mV) but at pH 4.5 surface charge approaches close to zero or little positive and becomes more negative at pH 10.0 (typically from -10 mV to -20 mV). The pH dependent surface charge of nanoparticle originates from different extent of protonation of primary amines that partially balance the anionic SO3- at the nanoparticle surface. As the protonation of primary amine decreases with increasing solution pH, the surface charge of particle becomes more negative. Amount of trehalose in the poly(trehalose) nanoparticle has been estimated via anthrone test.59 Trehalose produces furfural in concentrated H2SO4 that reacts with anthrone and develop bluegreen color that is used for quantitative estimation of trehalose.59 A calibration graphs has been prepared using known concentrations of trehalose and used for estimation of trehalose in poly(trehalose) nanoparticle. We have estimated that amount of trehalose can be varied from 0.5 -12 wt % and found that 5-12 wt % gives better performance. (Supporting Information, Table S1) We have estimated the tentative number of trehalose per nanoparticle for 5-12 wt % trehalose that comes in the range of 80-200 trehalose per nanoparticle. (Supporting Information, page S-1) Biochemical activity of trehalose present in poly(trehalose) nanoparticle is tested via Concavalin A induced nanoparticle aggregation.60 (Supporting Information, Figure S3) Concavalin A is known to have 4 carbohydrate binding sites and thus poly(trehalose)

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nanoparticle with multiple number of trehalose precipitate from solution via cross-linking between Concanavalin A and nanoparticle. Poly(trehalose) nanoparticle with zwitterionic surface charge and 5-12 wt % trehalose is most efficient in inhibiting amyloid fibrillation and disintegrating matured fibril. At first performance of poly(trehalose) nanoparticle is investigated using model amyloid peptide/protein such as hen egg white lysozyme (HEWL) protein and amyloid β(1-40) peptide (Aβ). Amyloid fibrillation study has been performed by incubating the mixture of poly(trehalose) nanoparticle and amyloid protein/peptide under standard condition and then a part of solution is used at different point of time for thioflavin T assay, TEM study and circular dichroism (CD) spectral measurement. In addition western blot/dot blot analysis is performed after complete fibrillation. Figure 2a,b and Supporting Information, Table S1 show the effect of poly(trehalose) nanoparticle surface charge on inhibiting HEWL/Aβ fibrillation. Results clearly show that aggregation of Aβ/HEWL is significantly inhibited by poly(trehalose) nanoparticle and zwitterionic or anionic surface charge provides better efficiency as compared to cationic or non-ionic surface charge. In contrast nanoparticle of similar surface chemistry but without any trehalose functionalization can hardly affect the amyloid fibrillation. Matured fibrils of Aβ peptide and HEWL protein are clearly observed under TEM and such fibril structures are clearly absent when poly(trehalose) nanoparticle is present during fibrillation. CD spectroscopic study also shows that peptide/protein do not form beta-sheet structure in presence of poly(trehalose) nanoparticle. (Figure 3b and Supporting Information, Figure S5b) Western blot and dot blot analysis shows that Aβ oligomer/aggregate formation is best suppressed by zwitterionic poly(trehalose) nanoparticle, as compared to cationic or anionic 16


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poly(trehalose) nanoparticle. (Figure 2c,d) In contrast zwitterionic nanoparticle without trehalose functionalization is inefficient in suppressing Aβ oligomer/aggregate formation. Next, we have tested zwitterionic nanoparticle with varied trehalose wt % from 0.5 to 12 and found that 5-12 wt % trehalose gives best inhibition performance. (Supporting Information, Figure S4 and Table S1) These results convincingly prove that poly(trehalose) nanoparticle with zwitterionic surface chemistry and 5-12 wt % trehalose offers best performance in inhibiting fibrillation of model peptide/protein. Although anionic poly(trehalose) nanoparticle also gives reasonably good performance, we have focused on zwitterionic surface structure for further studies as this surface structure offers high cell uptake as compared to anionic surface structure.57 Aggregation inhibition performance of zwitterionic poly(trehalose) nanoparticle has been compared with respect to molecular trehalose and it is found that nanoparticle form of trehalose can be 1,000 to 10,000 times better. (Figure 3a-d and Supporting Information, Figure S5a-d) Next, we have investigated if the zwitterionic poly(trehalose) nanoparticle can disintegrate matured fibrils made of Aβ peptide and HEWL protein. At first matured fibrils are prepared under standard conditions. Next, isolated fibrils are incubated with zwitterionic poly(trehalose) nanoparticle under physiological condition and fibril dissolution is followed via thioflavin T assay, TEM imaging and CD spectral study. Results are summarized in Figure 3e,f and Supporting Information, Figure S5e,f. Thioflavin T assay clearly show the disappearance of fibril structures as the time progress and thioflavin T signal becomes negligible at 10 days (for Aβ peptide) or 20 h (for HEWL fibrils). TEM study show that long fibrils are completely transformed into dotted like structures after treatment with zwitterionic poly(trehalose) nanoparticle. These results suggest that zwitterionic poly(trehalose) nanoparticle breaks the amyloid fibril structures into the small fragments. We have investigated the binding of 17



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poly(trehalose) nanoparticle with amyloid fibril via TEM and found that nanoparticles heavily bound with fibril but no such binding is observed for nanoparticle without trehalose functionalization. (Supporting Information, Figure S6, S7) Zwitterionic poly(trehalose) nanoparticle is > 1000 times efficient than molecular trehalose in inhibiting polyglutamine-based mutant huntingtin aggregation inside neuronal cells. Next, we have investigated the role of poly(trehalose) nanoparticle in inhibiting the polyglutamine-based mutant huntingtin aggregation inside the neuronal cell. We have used HD150Q cell that is commonly used as Huntington disease cell model. It is a mutant mouse neuroblastoma cell line (N2A) expressing a truncated N-terminal huntingtin containing 150 glutamine residues, fused to an enhanced green fluorescent protein. This cell can be cultured in DMEM media and ponasterone A can be used to induce aggregation of mutant huntingtin protein. Aggregated mutant huntingtin appears as green dots under fluorescence microscope and cell dies within 7-8 days due to toxicity of aggregated mutant huntingtin. Using this cell model we have studied the kinetics of mutant huntingtin aggregation in presence of zwitterionic poly(trehalose) nanoparticle. Typically, cultured cells are incubated with zwitterionic poly(trehalose) nanoparticle and then treated with ponasterone A to induce the mutant huntingtin aggregation. Next, mutant huntingtin aggregation kinetics is studied under fluorescence microscope, till the cell death. In addition, cell lysates have been analyzed for quantification of mutant huntingtin aggregates. (Figure 4 to 6 and Supporting Information, Figure S8-S12). At first we have confirmed that zwitterionic poly(trehalose) nanoparticle has very high cell uptake. We have conjugated zwitterionic poly(trehalose) nanoparticle with rhodamine B so that they can be seen under fluorescence microscope. We have observed that nanoparticle has high uptake in CHO, N2A and HD150Q cells within 2-6 h. (Supporting Information, Figure S8, 18


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S9, S10) Next, blocking of mutant huntingtin aggregation by zwitterionic poly(trehalose) nanoparticle is investigated via fluorescence imaging of HD150Q cells for 5 days. (Figure 4,5) The green fluorescent dots, appear as signature of mutant huntingtin protein aggregates, becomes insignificant in number and size for zwitterionic poly(trehalose) nanoparticle treated cells along with the increased cell survival. (Figure 4,5 and Supporting Information, Figure S12) Comparative investigation shows that zwitterionic poly(trehalose) nanoparticle completely inhibit mutant huntingtin aggregation at 5µM trehalose concentration but molecular trehalose is inefficient even at 5 mM concentration. Blocking of mutant huntingtin aggregation by zwitterionic poly(trehalose) nanoparticle is also confirmed by suppression of soluble huntingtin aggregates in immuno blot analysis and suppression of insoluble huntingtin aggregates in dot blot analysis. (Figure 6) Control experiments show that zwitterionic nanoparticle without any trehalose functionalization is completely inefficient in blocking the mutant protein aggregation. These results clearly show that zwitterionic poly(trehalose) nanoparticle blocks the aggregation of mutant huntingtin at micromolar trehalose concentration meaning that they are >1000 times efficient than molecular trehalose. Co-localization study shows that zwitterionic poly(trehalose) nanoparticle partially localize with aggregated polyglutamine which further

suggests that

poly(trehalose) nanoparticle binds with aggregated huntingtin inside the cell. (Supporting Information, Figure S11) All these results clearly suggest that zwitterionic poly(trehalose) nanoparticle binds with mutant huntingtin inside the cell, inhibits their aggregation and thus increases the cell survival. Zwitterionic poly(trehalose) nanoparticle suppress polyglutamine-containing mutant huntingtin aggregation in HD model mouse. The effect of zwitterionic poly(trehalose) nanoparticle on the aggregation of mutant huntingtin is further tested in an animal model of HD. 19



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The HD transgenic mouse (also called R6/2 line) was produced by introducing 5’-end of human HD gene having promoter sequence and Exon1 carrying 120 CAG repeats.61 This R6/2 transgenic mouse model shows progressive increase in mutant huntingtin aggregates in their brain (visible from as early as from 40-50 days of their age in immunohistochemical staining) along with rapidly progressive neurological phenotype. Average life span of the mice varies from 110-120 days. We have observed that treatment with both zwitterionic poly(trehalose) nanoparticle and control zwitterionic nanoparticle (without any trehalose) for 20 days had no lethal effect in these mice. However, body weight has been significantly reduced upon treatment of both nanoparticles. Immunohistochemical staining has been performed using different parts of HD mouse brain. (Figure 7) Results show that both nanoparticles reduce the number of mutant huntingtin aggregates in comparison with saline control in different regions of the HD brain (Figure 8 and Supporting Information, Figure S13). When compared between two nanoparticles the zwitterionic poly(trehalose) nanoparticle is found to be more effective in reducing aggregation as compared to control nanoparticle without any trehalose functionalization. Shortterm early treatment also had similar suppressive effect on mutant huntingtin aggregation. In order to confirm that nanoparticle enters into brain we have estimated iron content in brain section via inductively coupled plasma atomic emission spectroscopy (ICP-AES). We have observed that iron content is increased by 3.5 fold in nanoparticle treated brain, suggesting that both nanoparticle cross the blood-brain barrier, target brain and influence the polyglutaminemediated pathology. (Supporting Information, Figure S14) Advantage of nano-trehalose over molecular trehalose: Endocytotic cell uptake, multivalent binding with aggregating protein and crossing of blood-brain-barrier. Developed poly(trehalose) nanoparticle has three unique features that offers 1,000-10,000 times 20


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better in vitro and in vivo performance over molecular trehalose. First, nanoparticle has zwitterionic surface charge with multiple numbers of cationic and anionic functional groups that offers interaction with cell membrane via multiple interacting points and to induce high cellular uptake of nanoparticle. We have shown in earlier work that zwitterionic surface charge offers modular interaction of nanoparticle with anionic cell membrane and induces endocytosis without membrane damage (that occurs for cationic nanoparticle).57 In present case zwitterionic surface charge induces high cell uptake, followed by interaction with intracellular mutant huntingtin. Clearly this feature is absent in molecular trehalose of nonionic structure with aliphatic alcohol functional groups. Second, multiple numbers (about 80-200) of trehalose are attached to each nanoparticle that offers multivalent binding interaction with protein. Earlier works show that trehalose-based polymers can enhance amyloid fibril inhibition performance by 50 % due to trehalose multivalency of 2-7.52-54 In the present case higher multivalency of 80-200 in combination with zwitterionic surface charge offers enhanced interaction with aggregating protein. Third, nanoparticle form and zwitterionic surface charge offers enhanced brain targeting. Earlier in vivo work mainly use protein/polymer-based nanoparticle for treatment of various neurodegenerative disease.33,34 These nanoparticles are 50-200 nm size with low surface charge and some of them are terminated with polyethylene glycol. 33,34 In contrast use of zwitterionic nanoparticle in brain delivery is limited. Thus smaller size (20-30 nm) of our nanoparticle and zwitterionic surface charge are associated with better in vivo performance. Based on our results we propose a tentative mechanism of action of poly(trehalose) nanoparticle that involves 5 consecutive steps such as circulation in blood, crossing of the blood brain barrier, entry into neuronal cell of brain, binding with mutant huntingtin and blocking the protein aggregation. (Scheme 1) 21



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Although trehalose is reported to have anti-amyloidogenic property, the effective in vitro/in vivo performance is observed only at millimolar or higher concentration.45-51 Here we show that nanoparticle form of trehalose can work at micromolar concentration. Thus enhanced antiamyloidogenic performance of trehalose based nanoparticle made it possible for testing the rescue of disease pathogenesis in animal models of neurodegenerative disorders. However, presented nanoparticle composition has non-biodegradable iron oxide component and need to be replaced with completely biodegradable and biocompatible nanoparticle. Future work should focus on preparing polymer-based biocompatible and biodegradable nano-trehalose with zwitterionic surface property.

CONCLUSION In conclusion we report trehalose functionalized zwitterionic nanoparticle that can inhibit amyloid/polyglutamine

aggregation

under

extra-/intra-cellular

condition

and

supress

polyglutamine aggregation in HD model mouse brain. The nanoparticle has zwitterionic shell that offers high endocytotic cell uptake without cytotoxicity, defined number of trehalose (about 80-200 per particle) that offers efficient inhibition of protein aggregation via multivalent binding and successfully blocks mutant huntingtin aggregates in HD mouse brain. Compared to molecular trehalose that works beyond millimolar concentration, the designed nano-trehalose works at micromolar trehalose concentration, that can be practically achieved under in vivo condition. Present work shows that nanoparticle form of anti-amyloidogenic molecule can be more efficient and approach can be extended to other anti-amyloidogenic molecules to combat amyloid-derived neurodegenerative diseases.

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ASSOCIATED CONTENT Supporting Information Details of estimation of number of trehalose per nanoparticle, details of experimental conditions of making different types of poly(trehalose) nanoparticles and their properties, characterization of trehalose-monocarboxylic acid and trehalose-monomethacrylate, characterization of poly(trehalose) nanoparticle and various control labeling and imaging experiments. This material is available free of charge via the Internet at http://pubs.acs.org. Authors Contribution Nikhil RJ and Nihar RJ designed the experiments; NP prepared poly(trehalose) nanoparticle via method A and performed intra-/extra-cellular protein aggregation study; KD prepared poly(trehalose) nanoparticle via method B and performed intra-/extra-cellular protein aggregation study; Nihar RJ, KD and BKS performed animal experiments. Nikhil RJ and Nihar RJ analyzed the data and prepared the manuscript. All authors discussed the results. 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. Reference 23



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(56) Pradhan, N.; Shekhar, S.; Jana, N. R.; Jana, N. R. Sugar-Terminated Nanoparticle Chaperones Are 102−105 Times Better Than Molecular Sugars in Inhibiting Protein Aggregation and Reducing Amyloidogenic Cytotoxicity. ACS Appl. Mater. Interfaces 2017, 9, 10554−10566. (57) Chakraborty, A.; Jana, N. R. Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle. J. Phys. Chem. Lett. 2015, 6, 3688−3697. (58) Saha, A.; Basiruddin, S. K.; Maity; A. R.; Jana, N. R. Synthesis of Nanobioconjugates with a Controlled Average Number of Biomolecules between 1 and 100 per Nanoparticle and Observation of Multivalency Dependent Interaction with Proteins and Cells. Langmuir 2013, 29, 13917−13924. (59) Trevelyan, W. E.; Forrest, R. S.; Harrison, J. S. Determination of Yeast Carbohydrates with the Anthrone Reagent. Nature 1952, 170, 626–627. (60) Ambrosino, R.; Barone, G.; Castronuovo, G.; Ceccarini, C.; Cultrera, O.; Elia, V. Protein-Ligand Interaction. A Calorimetric Study of the Interaction of Oligosaccharides and Hen Ovalbumin Glycopeptides with Concanavalin A. Biochemistry, 1987, 26, 3971–3975. (61) Mangiarini, L.; Sathasivam, K.; Seller, M.; Cozens, B.; Harper, A.; Hetherington, C.; Lawton, M.; Trottier, Y.; Lehrach, H.; Davies, S.W.; Bates, G. P.; Exon 1 of the HD Gene with an Expanded CAG Repeat is Sufficient to Cause a Progressive Neurological Phenotype in Transgenic Mice. Cell, 1996, 87, 493–506.

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a)

HO

Method - B

Method - A

OH

HO

HO O

HO

O

NH2+

OH

SO3-

O

O

OH

O

γ-Fe2O3

NH2

~ 6 nm

SO3-

Polyacrylate coating with other acrylates HO

Polyacrylate coating

HO

NH3+

SO3NH3+

OH HO O

HO

O

OH

c)

O O

O

OH

OH O

SO3-

NH2

SO3-

b)

EDC coupling

NH2 SO3NH3+

d) 0.8

Correlation coefficient

30 25 Number %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20 15

0.6 0.4 0.2 0.0 0.1

10

10

103

10 5

Time (µs)

5 0 0

20

20 nm

40 60 80 Size (nm)

100

Figure 1. a) Synthetic strategy of poly(trehalose) nanoparticle. In method A, hydrophobic iron oxide nanoparticle is transformed into polyacrylate coated nanoparticle and then covalently conjugated with trehalose monocarboxylic acid, using primary amines on the particle surface. In method B, hydrophobic nanoparticle is transformed into polyacrylate coated nanoparticle using 32


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trehalose acrylate as one of the monomer. Method A produces poly(trehalose) nanoparticle with 10-12 wt % of trehalose and in method B trehalose can be varied from 0.5-12 wt %. Polyethylene glycol, amine and SO3- are introduced in the polyacrylate shells using other acrylayes. (see experimental section for details). b) TEM image of hydrophobic γ-Fe2O3 nanoparticle used to prepare poly(trehalose) nanoparticle. c) Digital image of colloidal solution of poly(trehalose) nanoparticle with one mg/mL particle concentration. d) Hydrodynamic size of colloidal solution of poly(trehalose) nanoparticle at pH 7.4 as observed in dynamic light scattering study. Inset shows the corresponding autocorrelation function curve.

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b)

100000

1 2 3 4 5 6

80000 60000 40000 20000 0

2000 1 2 3 4 5 6

1750 1500 1250 1000 750 500 250

0

c)

Fluorescence Intensity (a.u.)

a) Fluorescence Intensity (a.u.)

5

1

10 15 Time (days)

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0

25

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d) 1

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1

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6

100 Insoluble Aß1-40 fibrils

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20 0 6

Dot

Figure 2. Effect of poly(trehalose) nanoparticle surface charge on inhibiting HEWL/Aβ fibrillation showing that zwitterionic or anionic surface charge offers better performance as compared to cationic surface charge. (a) HEWL fibrillation study via thoflavin T fluorescence assay. b) Aβ fibrillation study via thoflavin T fluorescence assay and c) Suppression of Aβ oligomer formation by poly(trehalose) nanoparticle, as observed via Western blot analysis. d) Suppression of insoluble Aβ aggregate formation by poly(trehalose) nanoparticle, as observed via Dot Blot analysis. Poly(trehalose) nanoparticle with 5 wt % tehalose is used for all 34


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experiments with nanoparticle and trehalose concentrations maintaining at 0.4 mg/mL and 10 mM, respectively. [1 - without nanoparticle, 2 - zwitterionic nanoparticle without any trehalose functionalization, 3 - anionic poly(trehalose) nanoparticle, 4 - non-ionic poly(trehalose) nanoparticle, 5 - zwitterionic poly(trehalose) nanoparticle, 6 – cationic poly(trehalose) nanoparticle]

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b) 20 1 4 2

3000 2500 2000 1500

3

1 2 3

15

Ellipticity

Fluorescence Intensity (a.u.)

a)

10 5 0 -5

1000

-10

500 0

4

8

12

200

16

Incubation time (days)

c)

d)

220

240

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Wavelength (nm)

f)

e) Fluorescence intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.0 0.8

1 2

0.6 0.4 0.2 0.0

0

2

4

6

8

10

12

14

Time (days)

Figure 3. Zwitterionic poly(trehalose) nanoparticle inhibits Aβ aggregation 1000 times better than molecular trehalose and able to disintegrate mature Aβ fibrils. a) Aβ aggregation study via 36


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thoflavin T fluorescence assay in absence of any nanoparticle (1), in presence of zwitterionic nanoparticle without any trehalose functionalization (2), in presence of zwitterionic poly(trehalose) nanoparticle with 5 µM trehalose (3) and in presence of 5 mM molecular trehalose (4). b) Circular dichroism spectra of Aβ fibril without any nanoparticle (1), in presence of zwitterionic nanoparticle without any trehalose functionalization (2) and in presence of zwitterionic poly(trehalose) nanoparticle with 5 µM trehalose. c) TEM image of Aβ fibrils formed in absence of any nanoparticle. d) TEM image of Aβ fibrils formed in presence of zwitterionic poly(trehalose) nanoparticle. e) Thioflavin T fluorescence assay of Aβ fibril disintegration in presence of zwitterionic nanoparticle (1) and in presence of zwitterionic poly(trehalose) nanoparticle (2). f) TEM image of disintegrated Aβ fibrils. Poly(trehalose) nanoparticle with 12 wt % trehalose is used for all the experiments, keeping the nanoparticle concentration at 0.02 mg/mL.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Day 1

control 1 Day 2

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control 1

Day 3

control 1

control 2

Day 3

control 2

Day 1

control 2

Day 1

control 3

Day 2

control 3

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control 3

Day 1

nanotrehalose (5 µM)

Day 2


Day 3


Day 1

trehalose (5 mM)

Day 2

trehalose (5 mM)

Day 2

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Day 3

trehalose (5 mM)

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Figure 4. Zwitterionic poly(trehalose) nanoparticle blocks the aggregation of GFP tagged mutant huntingtin in HD150Q cells, 1000 times better than molecular trehalose. Typically, cells are incubated with zwitterionic poly(trehalose) nanoparticle having 5 µM trehalose along with ponasterone A inducer for different time points and washed cells are then used for imaging. Absence of green fluorescent dots in nanoparticle treated cells indicates blocking of protein aggregates. In control 1 no ponasterone A inducer is used, in control 2 no nanoparticle is used and in control 3 zwitterionic nanoparticle is used without any trehalose functionalization. Results show that zwitterionic poly(trehalose) nanoparticle completely inhibit mutant huntingtin aggregation at 5 µM trehalose concentration but molecular trehalose is inefficient event at 5 mM concentration. Poly(trehalose) nanoparticle with 5 wt % trehalose is used for all the experiments, keeping the nanoparticle concentration at 0.2 mg/mL. Blue emitting nuclear probe is used to label cell nucleus. Scale bar represents 100 µm.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Day 1

control 1

Day 3

10 µM

Day 1

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control 1

10 µM


Day 3

10 µM


10 µM

Figure 5. High magnification fluorescence image of single nucleus of HD150Q cell, showing that

mutant huntingtin forms in cell nucleus and with time they aggregate into larger size. In contrast, such aggregation is inhibited by zwitterionic poly(trehalose) nanoparticle treated cell nucleus. Blue color indicates DAPI stained nucleus and green color indicates mutant huntingtin protein aggregates. Scale bar represents 10 µm.

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a)

1

2

3

4

5

b) Relative level of mutant huntingtin-GFP

mutant huntingtin-GFP (soluble)

75kDa 100kDa

100

30kDa

GFP

80 60 40 20 0 1

2

Actin

c)

1

2

3

4

d)

5

3 Lane

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100 Mutant huntingtin-GFP (Insoluble aggregates)

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80 60 40 20

80 60 40 20 0

0 1

2

3 DOT

4

5

5

Figure 6. Suppression of mutant huntingtin aggregation in HD150Q cells by zwitterionic poly(trehalose) nanoparticle. a) Immunoblot analysis data of soluble huntingtin aggregates using GFP antibody. b) Quantification of band intensities of soluble huntingtin (tNhtt) shown in (a) using NIH image analysis software. Data are normalized against beta-actin. c) Dot blot analysis data of insoluble huntingtin aggregates using GFP antibody. d) Dot blot analysis using ubiquitin antibody. Cells are either untreated (lane 1) or treated with ponasterone A (lanes 2-5) along with nanoparticle or molecular trehalose for 3 days and cell lysates are then processed for immuno blot/dot blot analysis using GFP or ubiquitin antibody. Cells are used at day 3 and all values are 41



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Page 42 of 45

mean ± SD of 3 different experiments. [1 - Ponerteron A un-induced (control 1), 2 - without nanoparticle/trehalose (control 2), 3 - molecular trehalose (5 mM), 4 - zwitterionic nanoparticle without any trehalose functionalization, 5 - zwitterionic poly(trehalose) nanoparticle with 5 µM trehalose] All other conditions are same as of Figure 5.

Figure 7. Representative picture of HD mouse brain and Nissl-stained images of brain section (coronal) showing different areas of the brain used for assessing huntingtin immunostaining and quantifying huntingtin-positive nuclear aggregates.Scale bar for 1, 2 and 3 is 20 µm.

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1

1

1

cerebral cortex

striatum

hippocampus

2

cerebral cortex

striatum

2

2

hippocampus

3

3

3

cerebral cortex

striatum

hippocampus

Figure 8. Representative immunohistochemical staining results of different parts of HD mouse

brain, showing the suppression of huntingtin aggregates upon treatment of zwitterionic nanoparticles. Female HD mice were intraperitoneally injected with either normal saline (1) or zwitterionic nanoparticle without trehalose functionalization (2) or zwitterionic poly(trehalose) nanoparticle with 5 wt % trehalose (3) at their age of 60 days. Mice were sacrificed at their age of 84 days after receiving 10 injections at 2 days interval, brains were collected and processed

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for cryo-sectioning followed by immunohistochemical staining using huntingtin antibody. White arrows indicate huntingting aggregates. Scale bar represents 20 µm. Scheme 1. Mechanism of action of poly(trehalose) nanoparticle in HD mouse. Nanoparticles flow through blood, cross the blood brain barrier, enter into neuronal cell in brain, bind with polyglutamine containing mutant huntingtin and block their aggregation.

untreated

treated Blood-Brain-Barrier +

-

+

+

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+

+

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-

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+

+

-

+

m-HTT aggregates

NH3 + SO3 -

low m-HTT aggregation

SO3 NH2

5 µM

trehalose

5 µM

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TOC

HD150Q cell nucleus treated

10 µM

Huntington’s mice brain section

untreated

untreated

treated

5 µM

5 µM

10 µM

polyglutamine aggregate

cell membrane blood-brain-barrier Toxic NH2+ SO3-

anionic

NH2

cationic zwitter-ionic

SO3-

poly(trehalose) nanoparticle trehalose

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SO3NH3+

Poly(trehalose) - ACS Publications - American Chemical Society

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