Efficient Inhibition of Protein Aggregation, Disintegration of Aggregates

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Efficient Inhibition of Protein Aggregation, Disintegration of Aggregates and Lowering of Cytotoxicity by Green Tea Polyphenol-Based Self-Assembled Polymer Nanoparticle Koushik Debnath, Shashi Shekhar, Vipendra Kumar, Nihar R Jana, and Nikhil R. Jana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06853 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

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ACS Applied Materials & Interfaces

Efficient Inhibition of Protein Aggregation, Disintegration of Aggregates and Lowering of Cytotoxicity by Green Tea Polyphenol-Based Self-Assembled Polymer Nanoparticle Koushik Debnath,1 Shashi Shekhar,2 Vipendra Kumar,2 Nihar R. Jana2,* and Nikhil R. Jana1,* 1

Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata-700032, India

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

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ABSTRACT: Green tea polyphenol epigallocatechin-3-gallate (EGCG) is known for its anti-amyloidogenic property and it is observed that molecular EGCG binds with amyloid structure, redirects fibrillation kinetics, remodels mature fibril and lowers the amyloid derived toxicity. However, this unique property of EGCG is difficult to utilize because of their poor chemical stability and substandard bioavailability. Here we report a nanoparticle form of EGCG of 25 nm size (nano-EGCG) which is 10-100 times efficient than molecular EGCG in inhibiting protein aggregation, disintegrating mature protein aggregates and lowering of amyloidogenic cytotoxicity. Most attractive advantage of nano-EGCG is that it efficiently protects neuronal cells from toxic effect of extracellular amyloid beta or intracellular mutant huntingtin proteins aggregates by preventing their aggregation. We found that the better performance of nano-EGCG is due to the combined effect of increased chemical stability of EGCG against degradation, stronger binding with protein aggregates and efficient entry into the cell for interaction with aggregated protein structure. This result indicates that nanoparticle form of anti-amyloidogenic molecules can be more powerful in prevention and curing of protein aggregation derived diseases.

KEYWORDS: green tea polyphenol, EGCG, nanoparticle, amyloid, Huntington’s disease, biopolymer

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INTRODUCTION Protein misfolding and formation of β-sheet enriched amyloid fibrils or aggregates is linked to cellular toxicity and variety of amyloid diseases.1 Large variety of chemicals have been screened to identify the molecule that can bind and inhibit protein fibrillation and notable examples include peptide,2 curcumin,3 thioflavin derivative,4 metal ion chelators,5 trehalose,6 redox active small molecules7 and green tea polyphenol (epigallocatechin-3-gallate, EGCG).8-11 Among them, EGCG is unique as it is most efficient in inhibiting amyloid fibrillation and has shown to reduce the amyloidogenic cytotoxicity induced by huntingtin,12 α-synuclein8,13 and amyloid β.8,14 It is observed that EGCG directly binds with unfolded protein and inhibits formation of β-sheet structure which is an early event of amyloid formation cascade.11 In the molecular level, autooxidized EGCG reacts with free primary amine groups of proteins, forms Schiff base and induces fibril remodelling.9 However, this unique property of EGCG is difficult to utilize because of their poor chemical stability,15 substandard bioavailability16 and decreased anti-amyloidogenic performance in presence of cell membrane.17 Recent reports show that nanoscale materials have great advantages to influence amyloid fibril nucleation, disintegrating matured amyloid fibril and targeting of amyloid plaques via crossing the blood-brain barrier.18-21 For example, polymer nanoparticle,22 quantum dot,23 protein microsphere,24 gold nanoparticle,25,26 carbon nanoparticle27 and selenium nanoparticle28 have been reported to inhibit amyloid fibrillation. In addition gold nanoparticle29 and graphene oxide30 have been designed for photothermal disintegration of matured fibril. In those design, nanoparticles are appropriately functionalized for interaction with amyloid structure and commonly used functional

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molecules include peptide,23,29 amino acid,26 sialic acid,21 thioflavin,30 EGCG28 and curcumin.25 Reported advantage of nanoparticle form include increased water solubility of amyloid targeting molecule and enhanced binding affinity with amyloid structure.25,28 These findings indicate that nanoscale material based approach might be a promising option for prevention and curing of various neurodegenerative diseases. Here we have demonstrated a nanoparticle form of EGCG (nano-EGCG) with greatly enhanced performance in inhibiting amyloid fibrillation, disintegrating mature fibril and lowering of amyloidogenic cytotoxicity. Nano-EGCG is composed of 25 nm or 160 nm polyaspartic acid-based micelle incorporated with EGCG via covalent bonding or non-covalent interaction. Nano-EGCG is chemically more stable, strongly binds with amyloid structure and efficiently enters into cell to interact with amyloid structure. These facts lead to better performance in inhibiting amyloid fibrillation, disintegrating mature fibril and decreasing amylogenic cytotoxicity. Although nanoparticle form of EGCG is shown to enhance anticancer activity31 and EGCG coated selenium nanoparticle has been shown to decrease amylogenic cytotoxicity,28 present study clearly demonstrates 10-100 times better performance of nanoparticle form of EGCG and origin of such performance has been established due to increased chemical stability, stronger binding with amyloid structure and efficient cellular entry for interaction with amyloid structure.

EXPERIMENTAL SECTION Materials.

L-aspartic

hydrochloride,

acid,

dimethyl

octadecylamine

sulfoxide-d6,

(ODA),

triethylamine,

(−)epigallocatechin

gallate

dopamine (EGCG),

ethylenediamine, thioflavin T (ThT) and hen egg white lysozyme (HEWL) and amyloid

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β-protein fragment 1–40 (Aβ) were purchased from Sigma-Aldrich. Phosphoric acid (88 %, H3PO4) was purchased from Merck. Mesitylene and dry dimethylformamide (DMF) were purchased from Spectrochem. Dulbecco’s modified eagle medium (DMEM) and penicillin/streptomycin were purchased from Sigma-Aldrich. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Hi Media (India). GFP antibody was purchased from Roche, Ponasterone A was obtained from Invitrogen and all reagents for SDS-PAGE were purchased from Sigma. Instrumentation. UV-visible absorption and fluorescence spectral studies were carried out using Shimadzu UV-2550 UV-visible spectrophotometer and BioTek SynergyMx microplate reader, respectively. The field emission scanning electron microscopy (FESEM) analysis was performed with a Supra 40, Carl Zeiss Pvt. Ltd. instrument. The 1

H NMR (400 MHz) spectra were recorded on a Bruker DPX-400 spectrometer using

DMSO-d6 as solvent at room temperature. FTIR spectra of the samples compressed into KBr pellets were measured on Perkin Elmer Spectrum 100 FTIR spectrometer. 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 using image-pro plus version 7.0 software. Synthesis of polysuccinimide (PSI). Polysuccinimide (PSI) was synthesized from Laspartic acid by our reported method with some modifications.32,33 Briefly, 3 g of Laspartic acid was suspended in a mixture of 10 mL mesitylene and 165 µL phosphoric acid (88 %) and heated at 150 °C for 4 h under inert atmosphere. After cooling to the room temperature, 20 mL DMF was added to dissolve all precipitate and then excess water was added to re-precipitate the PSI. The precipitate was washed with water for

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several times to remove phosphoric acid and DMF and then washed with methanol for several times. Finally, solid PSI was dried in vacuum for overnight. Synthesis of octadecylamine, ethylenediamine and dopamine functionalized polysuccinimide (H2N-PSI-dopamine). 500 mg PSI was dissolved in 20 mL dry DMF, mixed with 270 mg octadecylamine (1 mmole) and heated at 70 °C for 24 h under inert atmosphere. The solution was cooled down to room temperature and mixed with 33 µL ethylenediamine (0.5 mmole) and heated at 50 °C for 8 h under inert atmosphere. Then the solution was cooled down to room temperature and mixed with 76 mg dopamine hydrochloride (0.5 mmole) and 56 µL triethyl amine. The mixture was further heated at 70 °C under inert atmosphere for 24 h. The resultant H2N-PSI-dopamine was precipitated by adding excess methanol. The precipitate was washed with methanol for several times and dried under vacuum. Similar procedure was used for preparation of octadecylamine and dopamine functionalized polysuccinimide (PSI-dopamine) and used as control polymer. Synthesis of nano-EGCG. EGCG solution was prepared in DMF with a concentration of 10 mg/mL. In a separate vial 10 mg H2N-PSI-dopamine (or PSI-dopamine) was dissolved in 500 µL DMF and mixed with 10-100 µL of EGCG solution. This mixture was then drop wise added to 5 mL water under vigorously stirring condition and stirring was continued for 6 h. Next, DMF and free EGCG were removed via dialysis (MWCO ~ 12000-14000 Da) of the whole colloidal solution against distilled water. Resultant nanoEGCG was used as stock solution. EGCG loading in nano-EGCG has been estimated by following procedure. At first a calibration graph was prepared from the concentration dependent absorbance at 272 nm. (Supporting Information, Figure S2) Next, the

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absorbance value of a colloidal solution of nano-EGCG at 272 nm is used to determine the wt % of EGCG. In vitro EGCG release study was performed by loading one mL of colloidal nanoEGCG solution inside the dialysis tube (MWCO ~ 12000-14000 Da) and dialysis was performed against 100 mL PBS buffer solution of pH 7.4 under stirring condition. One mL of phosphate buffer solutions was collected at different time interval and absorbance of the solution was measured at 272 nm. Amyloid fibrillation study. HEWL and Aβ fibrillation kinetics were monitored by using ThT assay. Solution of HEWL (2 mg/mL, 140 µM) or Aβ (0.10 mg/mL, 25 µM) were prepared separately by dissolving them in water containing NaCl (137 mM) and KCl (2.7 mM) and by adjustment of pH at 5.0 by HCl. Next, 100 µL nano-EGCG solution was added to HEWL/Aβ solution and solution mixture was heated to 70 °C and kept under magnetically stirring condition for 24 h. Aggregation kinetics was monitored by collecting 20 µL of solution at different time interval and mixed with one mL of aqueous ThT (10 µM) solution. Next, fluorescence of ThT was measured using 440 nm excitation. Fibril disaggregation study. Matured fibrils of HEWL and Aβ were prepared from respective protein/peptide by pre-incubating their solutions under standard condition for sufficient time, as described before. Fibrils were isolated by centrifuge, followed by redispersing them in PBS buffer solution of pH 7.4. Next, 900 µL HEWL (or 100 µL for Aβ) of dispersed fibril was incubated with 100 µL nano-EGCG at 37 °C for 15 days. Disintegration of fibrils was investigated by collecting 20 µL aliquots at different time interval and subjecting them to ThT assay as described above.

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Cytotoxicity study using neuro 2a cell line. Neuro 2a cells were cultured in a tissue culture flask with DMEM medium. The cells were sub-cultured in a 24-well culture plate containing cell culture medium (0.5 mL) and incubated overnight for cell adherence to the bottom of culture plate. Ab1–40 oligomer and fibrils were prepared separately by incubating Ab1–40 monomer at pH 7.4 containing 140 mM NaCl and 2.5 mM KCl at 37 °C (1 day for oligomer and 7 days for mature fibril). Next, neuro 2a cells were incubated with aliquots (0.025 mL) of Ab1–40 oligomer/fibrils in presence of PSI/molecular EGCG/nano-EGCG(1) at 37 °C for 24 h after mixing and cell viability was determined by

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide)

assay.

Typically, each well plate containing cells was treated with a freshly prepared MTT solution and incubated for 4 h. The supernatant was then carefully removed leaving violet formazan on the plate. This formazan was then dispersed in DMF/water and its absorbance at 570 nm was measured with a microplate reader. Cell viability was correlated with the absorbance value, assuming 100 % viability for the cells without any fibrils. Western blot and Dot blot experiment for polyQ expression. 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, nano-EGCG or molecular EGCG 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

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buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonident 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.36 Blot was probed with GFP 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.

RESULTS AND DISCUSSION Synthesis strategy and characterization of nano-EGCG. Synthesis strategy of nanoEGCG is shown in Scheme 1. Amphiphilic polymer micelle is synthesized first and then loaded with EGCG. The polymer consists of polyaspartic acid backbone functionalized with octadecyl, dopamine and primary amine groups, where hydrophobic octadecyl groups induce micellar assembly and polar dopamine/amine groups expose out words. Synthesis involves transformation of aspartic acid into polyaspartimide followed by functionalization with octadecylamine, dopamine and ethylene diamine using our earlier protocol.32,33 Two different polyaspartimides have been synthesized and depending on functionalization

they

are

named

as

H2N-PSI-dopamine

(functionalized

with

octadecylamine, ethylenediamine and dopamine) and PSI-dopamine (functionalized with octadecylamine and dopamine). Presence of different functional groups has been

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characterized using FTIR spectroscopy. (Supporting Information, Figure S1) Molecular weight of the polymers has been estimated earlier by GPC and MALDI mass spectrometry which becomes in the range of 20-30 KD.32,33 In order to prepare nanoEGCG, DMF solution of mixture of H2N-PSI-dopamine/PSI-dopamine and EGCG are added drop wise into water under vigorous stirring condition. Under this condition, aspartimide groups of polymer backbone hydrolyzes in generating amphiphilic polymer micelle and encapsulate EGCG with the resultant formation of nano-EGCG. Nano-EGCG prepared from H2N-PSI-dopamine is termed as nano-EGCG(1) and from PSI-dopamine is termed as nano-EGCG(2). Loading of EGCG has been determined from the absorbance of EGCG at 272 nm and it is ~ 10 wt %. (Supporting Information, Figure S2) Characteristic property of nano-EGCG is shown in Figure 1 and Supporting Information, Figure S2. SEM image of shows an average size of 25 nm for nanoEGCG(1) and 160 nm for nano-EGCG(2). Hydrodynamic size of the nanoparticles have also been measured by dynamic light scattering study which also corroborate with SEM size. In our previous work we have demonstrated that size of polymer nanoparticle can be varied from 180 nm to 300 nm by changing the ratio of hydrophobic and hydrophilic groups.32 In addition we observed that size shape may be changed after incorporation of drug molecules inside polymer micelle.32 Here we have shown that size of the micelle can be below 50 nm after loading/binding with EGCG. Tentative number of EGCG in each nano-EGCG(1) is calculated as ~100 assuming the number of EGCG bound to each polymer and the number polymers required for 25 nm micelle. Colloidal particles show anionic surface charge in the range of –5 to –30 mV which increases with the increasing solution pH. UV-visible and fluorescence spectra show the signature of molecular EGCG

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in the colloidal solution of nano-EGCG. Nature of binding between EGCG and polymer micelle has been investigated via dialysis. Typically, colloidal solutions of nano-EGCG is kept inside dialysis tube (MWCO ~ 12000-14000 Da), dialyzed against phosphate buffer of pH 7.4 and release of EGCG has been monitored using the absorbance of EGCG at 272 nm. Results show that the release of EGCG is extremely slow for nano-EGCG(2) and almost stopped for nano-EGCG(1). (Supporting Information, Figure S3) Chemical stability of EGCG in the form of aqueous solution of nano-EGCG has been investigated in the presence of dissolved oxygen and under high temperature (70-80 °C). It has been observed that aqueous solution of EGCG develops brown color within 12 h of heating under oxygen atmosphere and produces degraded products of lower molecular weight. (Supporting Information, Figure S4-S7) In contrast EGCG in nanoEGCG(1) remains intact under such condition for 24 h and degradation of EGCG from nano-EGCG(2) occurs with slower rate. (Supporting Information, Figure S4-S7) Origin of slow leaching of EGCG from nano-EGCG and their chemical stability against oxidative degradation has been further investigated. Considering the reasonably good water solubility of EGCG (> 5 mg/mL) and weaker hydrophobic interaction with polymer, the easier leaching of EGCG from nano-EGCG has been expected. So we have assumed that slow leaching and protection of EGCG against degradation might be linked to chemical interaction between polymer and EGCG and this has been verified in the following control experiment. Aqueous solution of EGCG is heated in presence of ethylene diamine under oxygen atmosphere and chemical product has been identified. Mass spectral study shows the formation of Schiff base between oxidized EGCG and ethylene diamine. (Supporting Information, Figure S8) Thus it can be proposed that

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EGCG is chemically linked in nano-EGCG(1) via formation of Schiff base between oxidized EGCG and primary amine group of polymer. (shown as inset in Scheme 1) As a result, the chemical degradation of EGCG is protected and their release from nanoEGCG(1) becomes slower. As primary amines are absent in nano-EGCG(2) such type chemical reaction is less probable and less effective in protecting EGCG. Nano-EGCG is 10-100 times efficient than molecular EGCG in inhibiting amyloid fibrillation and disintegrating matured amyloid fibrils. Inhibition of amyloid fibrillation and disintegration of matured amyloid fibrils have been studied using lysozyme and Aβ peptide as model systems. Typically, fibrillation is performed in presence of nano-EGCG or molecular EGCG and fibrillation is monitored using thioflavin T (ThT) assay and SEM/TEM-based imaging of fibrils. Results are summarized in Figure 2-5 which clearly show that nano-EGCG is 10-100 times efficient than molecular EGCG in inhibiting amyloid fibrillation. For example, molecular EGCG is completely inefficient at 10 µg/mL concentration, partially efficient at 200 µg/mL concentration and completely inhibits lysozyme fibrillation at 2000 µg/mL concentration. In contrast, both the nano-EGCG with EGCG concentration of 10 µg/mL can completely inhibit lysozyme fibrillation. (Figure 2a) This result also corroborates with SEM/TEMbased imaging of fibrils (Figure 3) and confirms that the efficiency of nano-EGCG can be of 100 times better than molecular EGCG. Similar trends are also observed for Aβ peptide fibril inhibition, however, in this case a clear difference is observed between two types of nano-EGCG. (Figure 4, 5) For example, molecular EGCG is completely inefficient at 20 µg/mL concentration and induces near complete inhibition of fibrillation at 200 µg/mL concentration. In contrast,

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nano-EGCG(1) with EGCG concentration of 10 µg/mL can completely inhibit Aβ fibrillation and nano-EGCG(2) with same EGCG concentration can partially inhibit their fibrillation. (Figure 4a) This result also corroborate with TEM-based imaging of Aβ fibrils (Figure 5) and indicate that nano-EGCG(1) can be > 10 times efficient than molecular EGCG but nano-EGCG(2) is relatively less efficient. Disintegration of matured fibrils has been performed by mixing their dispersion with nano-EGCG or molecular EGCG and then extent of fibril disintegration is monitored using ThT assay and imaging of fibrils via SEM/TEM. Results show that the nano-EGCG is 10-20 times efficient than the molecular EGCG in disintegrating matured fibrils and nano-EGCG(1) offers better performance than nano-EGCG(2). (Figure 2b, 4b) For example, molecular EGCG at 20 µg/mL has insignificant effect and to attain near complete disintegration of lysozyme fibrils it requires the concentration as high as 200 µg/mL. In contrast nano-EGCG(1) with EGCG concentration of 10 µg/mL can perform such complete disintegration. However, nano-EGCG(2) with same EGCG concentration can disintegrate the fibrils only partially. (Figure 2b) Similar trend is also observed in the Aβ fibril disintegration study. Performance of nano-EGCG(1) with 10 µg/mL EGCG is better than 200 µg/mL of molecular EGCG and performance of nano-EGCG(2) with 10 µg/mL EGCG is better than 20 µg/mL of molecular EGCG but poor than 200 µg/mL of molecular EGCG. (Figure 4b) Two general conclusions can be derived from these data. First, nano-EGCG can perform 10-100 times better than molecular EGCG in inhibiting amyloid fibrillation and disintegrating matured fibrils. Second, nano-EGCG(1) performs better than nano-EGCG (2) in doing these performances.

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Nano-EGCG is >10 times efficient than molecular EGCG in lowering the cytotoxicity of extracellular amyloid beta aggregates or intracellular mutant huntingtin proteins aggregates. Next, we have investigated the performance of nanoEGCG(1) in lowering the amyloidogenic toxicity using two cell lines such as neuro 2a and HD150Q cells. Nano-EGCG is designed using polyaspartic acid that has good biocompatibility and functionalized with dopamine for enhanced uptake by neuro 2a and HD150Q cells. These cells have high expression of dopamine receptors34 and thus it is expected that cellular interaction/uptake of nano-EGCG can be enhanced by dopamine functionalization. In order to confirm this, dopamine functionalized polymer nanoparticle is loaded with nile red and delivery of nile red into neuro 2a cell has been investigated. Results show that dopamine functionalized polymer nanoparticle can successfully deliver nile red into neuro 2a cell but polymer nanoparticle without dopamine functionalization is inefficient in nile red delivery. (Supporting Information, Figure S9) Next, neuro 2a cell is used for amyloidogenic toxicity study for matured Aβ fibril and Aβ oligomer. Aβ fibril and Aβ oligomer are synthesized by incubating the Aβ peptide solution under fibrillation conditions for 7 days and one day, respectively.35 (Figure 6 and Supporting Information, Figure S10) Next, Aβ fibril/oligomer is mixed with nano-EGCG(1) (or molecular EGCG) and incubated with neuro 2a cells for 48 h. Next, cell viability has been determined by conventional MTT assay, assuming 100 % viability in control sample. Results clearly show that both Aβ fibril and Aβ oligomer reduce the cell viability due to their well known cytotoxicity and presence of nanoEGCG(1) can completely stop this toxic effect above a certain dose. (Figure 6) In contrast, molecular EGCG can only partially inhibits such toxicity even at 16 times

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higher concentration. Control experiments show that polymer nanoparticle (without EGCG) cannot prevent oligomer toxicity but can partially prevent the toxicity of mature fibrils. This partial prevention may be due to the interaction between polymer nanoparticle and fibril that restrict their interaction with cell membrane. Nevertheless, these experiments clearly demonstrate that efficiency of nano-EGCG(1) in reducing the amyloidogenic toxicity can be > 10 times than molecular EGCG. Next, performance of nano-EGCG(1) has been investigated using HD150Q cells that produce polyglutamine-based truncated N-terminal huntingtin protein aggregates inside cell. In particular the role of nano-EGCG(1) in protecting HD150Q cells from the toxic effect of huntingtin aggregates has been investigated. The HD150Q cell is an interesting model system as they have regulated expression of green fluorescent protein (GFP) tagged mutant huntingtin proteins and the formation/progress of these protein aggregates can be observed/monitored under fluorescence microscope.36 Thus performance of nano-EGCG(1) can be directly tested under fluorescence microscope. In usual practice cells are treated with ponasterone A to induce the expression of mutant protein and aggregated huntingtins are observed inside cells as green fluorescent dots under fluorescence microscope. (Figure 7) As time progress the fluorescent dots inside cells increases in size and number and finally cells die in 6-7 days. Amylogenic performance is studied by incubating HD150Q cells with nanoEGCG(1) solution (or molecular EGCG solution as control) along with ponasterone A inducer for different time points and washed cells are then used for imaging. Results show that nano-EGCG(1) efficiently blocks the expression of mutant huntingtin and their aggregation. Fluorescence image of nano-EGCG(1) treated cells at 5th day shows very

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low number of green fluorescent dots while cells without nano-EGCG(1) treatment shows significant number of green fluorescent dots. (Figure 7) In order to understand the effect of nano-EGCG on mutant huntingtin-induced-cell death, cell viability has been studied by MTT assay. Results show that cell viability increases to 95 % for nanoEGCG(1) treated cells as compared to 35 % viability for non-treated cells. (Supporting Information, Figure S11) Similar study is also performed using molecular EGCG using varied concentration from 0.5-100 µg/mL but no significant inhibition of green fluorescent dots formation and no increase of cell viability is observed. This result confirms that nano-EGCG is > 10 times efficient than molecular EGCG in blocking the polyglutamine aggregation inside the cell and in lowering the cytotoxicity generated from those aggregates. The extracts of HD150Q cells have been used for identification of soluble polyglutamine-containing mutant huntingtin via Western blot analysis. (Figure 8) Soluble mutant huntingtin appears as multiple band (top band correspond to 150Q) because of instability of CAG repeats during cell division. Insoluble mutant huntingtin can be seen in the stacking gel. Interestingly, the extent of soluble and insoluble polyglutamine aggregates are significantly decreased in cells treated with nano-EGCG(1) with EGCG concentration of 1 µg/mL. In contrast, cell extract treated with molecular EGCG upto 20120 µg/mL shows unchanged signals of soluble and insoluble mutant huntingtin. This result suggests that nano-EGCG(1) is >100 times efficient than molecular EGCG in blocking the polyglutamine expression in cells. One interesting aspect of Western blot data is that extent of insoluble polyglutamine-containing huntingtin is significantly low in presence of nano-EGCG(1).

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In order to quantify the insoluble huntingtin we have further performed the dot blot analysis of insoluble huntingtin extracted from HD150Q cells. Representative results are summarized in Figure 9. It clearly shows that insoluble mutant huntingtin level is suppressed by nano-EGCG(1) but neither by molecular EGCG nor by polymer nanoparticle. This result indicates that nano-EGCG not only prevents polyglutamine aggregation but also probably enhance the clearance of soluble mutant huntingtin. Origin of high performance of nano-EGCG: Increased chemical stability of EGCG, multivalent binding with protein aggregates and efficient cellular entry. All the experimental data suggest that presented nano-EGCG perform 10-100 times better than molecular EGCG in inhibiting amyloid fibrillation, disintegrating mature fibrils and lowering of amylogenic cytotoxicity. Such better performance of nano-EGCG is linked to three possible reasons. First, chemical stability of EGCG is enhanced while they are in the form of nano-EGCG. We have shown that EGCG is chemically very unstable and degrades into smaller components under physiological condition. In contrast EGCG in nanoparticle form is chemically stable due to Schiff base formation between oxidized EGCG and primary amine of polymer. Thus stable EGCG can interact throughout the whole process of amyloid fibril nucleation-growth stages that continue for several days. Second, nano-EGCG has stronger binding with amyloid structure via multivalent interaction which is not possible for molecular EGCG. We have compared the binding interaction of matured amyloid fibril with nano-EGCG and molecular EGCG and found that nano-EGCG binds 10-20 times stronger than molecular EGCG. (Figure 10) The binding interaction is measured indirectly via ThT binding assay in presence of molecular EGCG/nano-EGCG. Typically, same concentration of HEWL/Aβ fibrils is incubated

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with molecular EGCG/nano-EGCG followed by addition of excess ThT and then fluorescence of ThT is measured. Under this condition binding of ThT is low due to competition with molecular EGCG/nano-EGCG and lowering of ThT fluorescence is translated into % binding of molecular EGCG/nano-EGCG, assuming 100 % for binding of ThT in absence of any EGCG. (Figure 10) Results clearly show that nano-EGCG binds 10-20 times strongly than molecular EGCG. This stronger binding of nano-EGCG induces efficient inhibition of amyloid fibril nucleation/growth, disintegration of matured fibrils and decreases the direct interaction of amyloid fibril with cell membrane. Third, nano-EGCG can efficiently interact with amyloid inside cell due to their high cell uptake as compared to molecular EGCG. It is well know that nanoscale materials of 10-100 nm size has stronger interaction with cell as compared to molecules of sub-nanometer size and induces endocytotic uptake processes.37 In addition dopamine functionalizaion of nanoparticle further enhances their uptake as the studied cells have dopamine receptors. Thus intracellular polyglutamines are readily exposed with nanoEGCG and induce antiamylogenic performance. All these results indicate that our designed nano-EGCG is ideal for in vitro and in vivo application with better performance as compared to conventionally used molecular EGCG. Based on these results we propose a generalized scheme showing the mechanism of action of nano-EGCG. (Scheme 2) Nano-EGCG can interact with aggregating amyloids proteins via multivalent interaction either at extracellular space or inside cell. This interaction can lead to the near complete inhibition of fibrillation and disintegration of matured amyloid fibrils. Inhibition of fibrillation and lowering of amyloidogenic cytoxicity are the result of stronger interaction of nano-EGCG with extracellular amyloid

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protein that effectively isolates them from interacting with cell and insufficient formation of toxic aggregates. This work shows that nano-EGCG reported here has three distinct roles as compared to most of the reported anti-amyloidogenic materials. First it can inhibit amyloid aggregation either in extracellular space or inside the cell. Second, it can efficiently disintegrate mature fibrils without any external energy. Third, it can protect cells from toxic effect of amyloid aggregates. As the amyloid aggregates can be formed either inside cell (in case of Huntington’s disease) or extracellular space (in case of Alzheimer’s disease), developed material should be equally useful for all cases.38-41 In addition aggregated amyloid beta and polyglutamine are known to disrupt cell membrane and induces toxicity.41-43 Thus effective treatment requires their isolation/inactivation in the extracellular or intracellular space. We have demonstrated that nano-EGCG is equally effective at extracellular and intracellular space. Moreover, EGCG is known to cross the blood brain barrier.12 Therefore, its nano-formulation is likely to cross blood brain barrier and suitable for in vivo disease model.

CONCLUSION We have demonstrated that nanoparticle form of anti-amyloidogenic molecule can be more efficient than the respective molecular form in prevention and curing of amyloid derived diseases. In particular we have shown that anti-amyloidogenic EGCG molecule can be 10 -100 times efficient in their nanoparticle form in inhibiting amyloid fibrillation, disintegrating matured amyloid fibrils and in lowering of amyloidogenic cytoxicity. Most attractive advantage of nanoparticle form is that it can strongly bind with amyloid structure via multivalent interaction which is not possible in molecular form. Additionally

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nanoparticle form can be designed for enhanced solubility, enhanced stability and better intracellular targeting of amyloids. Research should be directed to investigate the performance of nanoparticle form of other anti-amyloidogenic molecules on prevention and curing of amyloid derived diseases.

ASSOCIATED CONTENT Supporting Information Details of characterization of nano-EGCG, control cell labeling experiments, characterization of amyloid oligomer and cytoxicity study. 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 SB/S1/IC-13/2013) We also acknowledge Department of Biotechnology (DBT), Government of India for providing financial support to National Brain Research Centre.

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Scheme 1. Strategy for synthesis of EGCG based polymer nanoparticle. Polyaspartimide is synthesized from aspartic acid and transformed into amphiphilic polymer functionalized with dopamine and primary amine (H2N-PEI-dopamine) or dopamine (PEI-dopamine). Next, EGCG is covalently conjugated with primary amines to produce nano-EGCG(1) or incorporated non-covalently to produce nano-EGCG(2). Inset shows the mode of chemical conjugation of EGCG in the nano-EGCG(1).

O

O OH

HO

H3PO4, mesitylene

N

150 ºC, 5 hrs

NH2 O

O

i. octadecylamine, 80 °C ii. ethylenediamine, 80 °C iii. dopamine, triethylamine, 80 °C

n

i. octadecylamine, 80 °C ii. dopamine, triethylamine, 80 °C O

O

O

O

NH NH NH O O O n-x-y-z z x O NH HN HN

N

NH O

HN O

N O

O

O

O

n-x-y HN

polysuccinimide backbone

y

x HN

16

16

NH2

ethylenediamine OH

(PEI-dopamine) EGCG, water

dopamine

octadecylamine

(H2N-PEI-dopamine) HO

EGCG OH

EGCG, water

OH O OH O O HO

N

NH

OH

O

OH OH

nano-EGCG(2)

nano-EGCG(1)

27


OH

y


a)

nano-EGCG(1)

nano-EGCG(1)

b)

200 nm nano-EGCG(1)

e)

nano-EGCG(2)

30 20 10

0

0

50

100

150

200

Size (nm)

250

300

pH 7.4

pH 10

f) 5

-10 -15 -20 -25 -30 -35

0

pH 4.5

2 µm

100 nm nano-EGCG(1) nano-EGCG(2)

800

-5

Zeta potential

Number %

40

nano-EGCG(2)

100 nm

Absorbance

d) 50

c)

-40

4

600

3

400 2

200

1

nano-EGCG(1) nano-EGCG(2)

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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0 200

300

400

0 500

Wavelength (nm)

Figure 1. a,b) SEM image of nano-EGCG(1) at two different magnifications. c) SEM image of nano-EGCG(2) with magnified view of single particle at inset. d) Hydrodynamic size of nano-EGCG(1) and nano-EGCG(2) as observed from dynamic light scattering study. e) Surface charge of nano-EGCG(1) and nano-EGCG(2) at three different solution pH and f) UV-visible and fluorescence spectra of colloidal solution of nano-EGCG(1) and nano-EGCG(2) where the emission is coming due to EGCG.

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a)

ThT fluorescence intensity (a.u)

100000 (i) (ii) (iii) (iv) (v) (vi)

80000 60000 40000 20000 0 0

5

10

15

20

25

Time (hour)

30000 ThT 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


b)

20000 (i) (ii) (iii) (iv)

10000

0 0

2

4

6

8

10

12

Time (day)

Figure 2. a) ThT based lysozyme fibrillation study at pH 4.5 showing that nanoparticle form of EGCG is > 100 times efficient than molecular EGCG in inhibiting amyloid fibrillation. i) control without EGCG, ii) molecular EGCG (10 µg/mL), iii) molecular EGCG (200 µg/mL), iv) molecular EGCG (2000 µg/mL), v) nano-EGCG(2) (10 µg/mL EGCG) and vi) nano-EGCG(1) (10 µg/mL EGCG). b) ThT based lysozyme fibril disintegration study at pH 7.4 showing that nanoparticle form of EGCG is >10 times efficient than molecular EGCG and nano-EGCG(1) is more efficient than nano-EGCG(2) in disintegrating amyloid fibrils. i) molecular EGCG (20 µg/mL), ii) nano-EGCG(2) (10 µg/mL EGCG), iii) molecular EGCG(200 µg/mL) and iv) nano-EGCG(1) (10 µg/mL EGCG).

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i) a)

c)

b)

1 µm ii) c)

1 µm

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10 µm

0.5 µm b)

c)

0.5 µm

1 µm

Figure 3. i) SEM image of lysozyme fibrils formed in absence of any EGCG (a), in presence of molecular EGCG (200 µg/mL) (b) and nano-EGCG(1) (with 10 µg/mL EGCG) (c). ii) TEM image of lysozyme fibrils formed in absence of any EGCG (a) and disintegrated fibrils formed in presence of nano-EGCG(2) (with 10 µg/mL EGCG)(b) and nanoEGCG(1) (with 10 µg/mL EGCG)(c).

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a)

1.0 (i) (ii) (iii) (iv) (v)

0.8 0.6 0.4 0.2 0.0

0

2

4

6

8 10 12 14 16 18 20 Time (day)

ThT fluorescence intensity (normalized)

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


ThT fluorescence intensity (normalized)

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b)

(i) (ii) (iii) (iv)

1.0 0.8 0.6 0.4 0.2 0.0

0

2

4

6

8

10 12 14 16

Time (day)

Figure 4. a) ThT based Aβ1–40 fibrillation study at pH 4.5 showing that nanoparticle form of EGCG is 10-20 times efficient than molecular EGCG in inhibiting amyloid fibrillation and nano-EGCG(1) is more efficient than nano-EGCG(2). i) control without EGCG, ii) molecular EGCG (20 µg/mL), iii) nano-EGCG(2) (10 µg/mL EGCG), iv) nano-EGCG(1) (10 µg/mL EGCG) and v) molecular EGCG (200 µg/mL). b) ThT based Aβ1–40 fibril disintegration study at pH 7.4 showing that nanoparticle form of EGCG is >10 times efficient than molecular EGCG and nano-EGCG(1) is more efficient than nano-EGCG(2) in disintegrating amyloid fibrils. i) molecular EGCG (20 µg/mL), ii) nano-EGCG(2) (10 µg/mL EGCG), iii) molecular EGCG (200 µg/mL) and iv) nano-EGCG(1) (10 µg/mL EGCG).

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(i)

(ii)

500 nm

500 nm

1 µm

(iv)

(v)

(vi)

500 nm

200 nm

200 nm

(iii)

Figure 5. TEM imaging based Aβ1–40 fibrillation and Aβ1–40 fibril disintegration study. (i) amyloid fibrils formed in absence of any EGCG, (ii) amyloid fibrils formed in presence of molecular EGCG (200 µg/mL), (iii) amyloid fibrils formed in presence of nanoEGCG(1)(with 10 µg/mL EGCG), iv) disintegrated Aβ fibrils in presence of molecular EGCG (200 µg/mL), v) disintegrated Aβ fibrils formed by nano-EGCG(2)(with 10 µg/mL EGCG) and vi) disintegrated Aβ fibrils formed by nano-EGCG (1)(with 10 µg/mL EGCG).

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100 Cell viability (%)

a)

80 60 40 20 0

Aβ1-40 fibril : PSI : molecular EGCG : nano EGCG(1) :

b)

-

+ -

+ -

+

+ -

+ + -

+ + -

+ +

100 Cell viability (%)

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


80 60 40 20 0

Aβ1-40 oligomer : PSI : molecular EGCG : nano EGCG(1) :

-

+ -

+ -

+

+ -

+ + -

+ + -

+ +

Figure 6. Effect of nano-EGCG(1) on neurotoxicity of matured Aβ1–40 fibril (a) and oligomeric Aβ1–40 (b) towards the neuro 2a cell lines. The first four columns relate to cell viability of polymer(PSI), free EGCG and nano-EGCG(1), column 5 relate to cell viability in presence of Aβ-fibrils/oligomers, and last three columns relate to cell viability of Aβ-fibrils/oligomers in presence of PSI, molecular EGCG and nano-EGCG(1). Fibrils were incubated with neuro 2a cells for 48 h and cytotoxicity was estimated by MTT assay. The final concentration of Aβ1–40 fibrils in terms of monomer was 1.25 µM, final concentration of EGCG in the form of nano-EGCG(1) was 1 µg/mL and final concentration of molecular EGCG was 20 µg/mL.

33



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 control

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nano-EGCG (1)

day 1

Figure 7 20 µm

20 µm day 5

control

day 5

nano-EGCG(1)

20 µm

20 µm

Figure 7. Nano-EGCG(1) reduces the aggregation of GFP tagged mutant huntingtin in HD150Q cells. Typically, cells are incubated with nano-EGCG solution (or molecular EGCG solution for control) along with ponasterone A for different time points and washed cells are then used for imaging. Low number of green fluorescent dots in nanoEGCG treated cells indicates the blocking of mutant huntingtin protein aggregation. Some of the aggregated dots are shown as red arrow.

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a)

Lane Lane Lane Lane Lane Lane 1 2 5 6 3 4

100 kDa 75 kDa

30 kDa

GFP

actin

b) Relative level of mutant huntingtin-GFP

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


mutant mutant huntingtin-GFP huntingtin-GFP (soluble) (insoluble)

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7 6 5 4 3 2 1 0 1

2

3

4

5

6

Lane

Figure 8. Suppression of mutant polyglutamine containing mutant huntingtin level upon treatment of nano-EGCG(1) in HD 150Q cells. a) Cells are either untreated (lane 1) or treated with Ponasterone A (lanes 2-6) along with nano-EGCG(1), molecular EGCG for 3 days, cell lysates were made and then processed for immunoblot analysis using GFP antibody. Lane 1: cells without ponasterone A treatment (control), Lane 2: cells with Ponasterone A only (control), Lane 3: cells treated with nano-EGCG(1) having 1 µg/mL EGCG concentration, Lane 4: molecular EGCG having 20 µg/mL concentration, Lane 5: molecular EGCG having 60 µg/mL concentration and Lane 6: molecular EGCG having 120 µg/mL concentration. b) Quantification of the band intensities of soluble truncated mutant N-terminal huntingtin (tNhtt) using NIH image analysis software. Multiple bands appear because of instability of CAG repeats. Data were normalized against beta-actin.

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a) 2

1

3

5

4

6

b)

120

insoluble aggregates expression

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

100 80 60 40 20 0

1

2

3

Dot

4

5

6

Figure 9. (a,b) Treatment of nano-EGCG(1) to HD150Q cells reduces the level of insoluble mutant huntingtin. Cells are left untreated (lane 1) or induced with Ponasterone A (lanes 2-6) along with nano-EGCG(1), molecular EGCG and polymer(PSI). Dot 1: cells without Ponasterone A treatment (control), Dot 2: cells without any EGCG (control), Dot 3: cells treated with PSI, Dot 4: cells treated with nano-EGCG(1) having 1 µg/mL EGCG concentration, Dot 5: nano-EGCG(1) having 5 µg/mL EGCG concentration, Dot 6: Free EGCG of 10 µg/mL concentration. Results show that insoluble pool of mutant huntingtin is suppressed by nano-EGCG(1) but not by PSI or 10 times excess of molecular EGCG. Values are mean ± SD of 3 different experiments.

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a) 100

molecular EGCG nano-EGCG(1)

Binding (%)

80 60 40 20 0 0

20 EGCG (µg/mL)

b) 100

Binding (%)

200

molecular EGCG nano-EGCG(1)

80 60 40 20 0 0

c)

Comparative binding (nano-EGCG (1)/molecular EGCG)

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


20 EGCG (µg/mL)

200

30

lysozyme 20

Aβ1-40

10

0 0

50 100 150 EGCG (µg/mL)

200

Figure 10. a) Binding of ThT with matured lysozyme fibrils in the presence of molecular EGCG and nano-EGCG(1), b) binding of ThT with matured Aβ fibrils in presence of molecular EGCG and nano-EGCG(1) and c) comparative binding of nano-EGCG(1) with respect to equivalent molecular EGCG. Typically, same concentration of lysozyme/Aβ fibrils are incubated with EGCG/nano-EGCG(1) followed by addition of excess ThT. Next, fluorescence of ThT is measured and binding % is estimated from ThT fluorescence, assuming 100 % for control sample in absence of any EGCG.

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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

Scheme 2. Summary of the mechanism of action of nano-EGCG(1). It involves multivalent interaction of nano-EGCG with aggregating protein either at extracellular space or inside cell. This interaction leads to the inhibition of fibrillation, disintegration of matured fibrils and lowering of neurotoxicity due to effective isolation of fibrils and/or insufficient formation of toxic aggregates.

Extracellular space

Inside cell

(oligomer) poly Q X (matured fibril) (fibril inhibition)

poly Q aggregates

(disintegrated fibril) nano-EGCG(1)

EGCG dopamine

fibril/aggregate forming monomer

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Page 39 of 39

TOC

OH O

25 nm

OH O O HO

O

N

NH

OH OH OH

untreated

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


X

polyQ aggregates

39


Efficient Inhibition of Protein Aggregation, Disintegration of Aggregates

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