Neurotoxicity of pre-incubated alpha-synuclein with …scientiairanica.sharif.edu/article_4419_f84c94416bd3cb19... · 2018-06-30 · Neurotoxicity of pre-incubated alpha-synuclein

Scientia Iranica F (2017) 24(6), 3542{3553

Sharif University of TechnologyScientia Iranica

Transactions F: Nanotechnologywww.scientiairanica.com

Neurotoxicity of pre-incubated alpha-synuclein withneutral nanoliposomes on PC12 and SHSY5Y cell lines

F. Aliakbaria,b,c, A.A. Shabanib;�, H. Bardaniad, H.A. Eslampanah Seyedic,H. Mohammad-Beigie, A. Tayaranian Marvianc, M. Nasoutic, A.A. Vafaeif,S.A. Shojaosadatig, A.A. Sabouryh, G. Christianseni and D. Morshedic;�

a. Student Research Committee, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran.b. Department & Center for Biotechnology Research, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran.c. Bioprocess Engineering Department, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic

Engineering and Biotechnology, Tehran, Iran.d. Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran.e. Interdisciplinary Nanoscience Centre (iNANO) and Department of Molecular Biology and Genetics, Aarhus University, Gustav

WiedsVej 14, DK-8000 Aarhus C, Denmark.f. Laboratory of Learning and Memory, Research Center and Department of Physiology, School of Medicine, Semnan University of

Medical Sciences, Semnan, Iran.g. Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran.h. Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran.i. Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.

Received 13 September 2016; received in revised form 4 October 2016; accepted 8 November 2016

KEYWORDSAlpha-synuclein;Amyloid-relateddiseases;Fibrillation;Nanoliposome;Neurotoxicity;Parkinson's disease.

Abstract. Alpha-synuclein (�-Syn) is a protein with high tendency to convert into toxicaggregates that are involved in Parkinson's Disease (PD). The conversion of �-Syn intotoxic aggregates may associate with bio-membranes and the interaction between �-Syn andbio-mimic liposomes can trigger the neurotoxicity. Some inhibitors of �-Syn aggregationare challengeable drugs. Using nano carriers such as nanoliposomes may overcome suchchallenges. Nevertheless, there are few studies on nanoliposomes e�ects on neuronal cellsand �-Syn. Herein, we investigated the neurotoxicity and �brinogenesis of neutral chargednanoliposomes. Thin layer evaporation method and hydration method was employed toformulate the nanoliposomes from Dipalmitoylphosphatidylcholine to the size of � 100 nm.Fibrillation and cytotoxicity of �-Syn treated with nanoliposome were measured. Neitherneurotoxicity nor �brillation was observed when �-Syn treated with di�erent concentrationsof nanoliposome. These results well indicate that nanoliposome at the concentrationsrequired for drug delivery not only is able to prevent �brillation process, but also hasno considerable toxic e�ects on the cells.© 2017 Sharif University of Technology. All rights reserved.

1. Introduction

Proteins misfolding and aggregation are pathog-nomonic for nearly all age-related neurodegenerative

*. Corresponding authors. Tel.: +98 23-33654187 (A.A.Shabani); +98 21-44878423 (D. Morshedi)E-mail addresses: [email protected] (A.A. Shabani);[email protected] (D. Morshedi)

doi: 10.24200/sci.2017.4419

diseases, such as Parkinson's Disease (PD), Alzheimer'sDisease (AD), and Lewy body with dementia, inwhich the formation of proteins aggregation has beenidenti�ed as amyloid �brils [1,2]. The aggregation of �-Syn, small (14 kDa) and highly conserved presynapticprotein [3,4], has been identi�ed to have a criticalrole in PD and other synucleinopathy diseases [5-7]. The conversion of soluble �-Syn into insolublewell-ordered beta sheet structures is associated with

F. Aliakbari et al./Scientia Iranica, Transactions F: Nanotechnology 24 (2017) 3542{3553 3543

the deposits known as Lewy bodies and Lewy neu-rites [8,9].

PD is the second most common neurodegenerativedisease, and so far, various studies have reported on therole of �-Syn in the initiation and spreading of neurode-generation in PD. The degeneration of dopaminergicneuronal cells has been shown to be connected withthe development of �-Syn �brillation [10]. Hence,there is a considerable interest in inhibiting �-Syn �brilformation, which can be a virtuous avenue to impedethe development of PD [11-13].

Nowadays, nanoparticles are immensely consid-ered a promising strategy in drug delivery systems [14].Remarkably, engineered nanomaterials not only sug-gest novel structural, optical, and electronic propertiesin diagnostics and therapy of di�erent diseases [15], butalso deliver several functionalities due to their extendedsurface area, diverse surface chemistry, and uniquepharmacokinetics [15,16]. In fact, these characteristicsbring about mighty potency to increase the solubility,concerning the hydrophobic drugs and stability of bothhydrophobic and hydrophilic drugs in the aqueousmedium of the body, and carry them to the targettissue [17].

Liposomes, as a major class of macromolecularcarriers, owing to several merits including biocompat-ibility, low immunogenicity, protecting cargo againstenzymatic degradation, and easy surface modi�cation,have been interestingly aimed at targeted delivery [18].Moreover, the nature and structure of liposome, con-taining a core of aqueous solution with two layers ofphospholipid, gives it a unique capacity to deliver bothhydrophobic and hydrophilic drugs [15]. This meansthat nanoliposomes are signi�cantly regarded as nanocarriers with high capacity for encapsulation of drugs.

Nanoliposomes may themselves possess an apti-tude to be utilized as a substance for disease treat-ment [19,20]. According to the estimations, less thanhalf of the drugs used in the treatment of humandiseases are hydrophobic with very low solubility inbody uids; thus, the requisite for a high dose andfrequency of consumption is a challenge concerning theuse of these drugs. Furthermore, sometimes, in order toraise the solubility of the drugs, chemical modi�cationis required, causing side e�ects. In addition, a drugoverdose and its high intake can a�ect non-targettissues [21,22]. Therefore, the use of nanoliposomes inovercoming such problems is applicable. So far, mostreports corresponding to Food and Drug Administra-tion (FDA) have con�rmed that liposome-based drughave been focused on cancer treatment [23,24].

To date, some studies have been reported regard-ing the use of nanoparticles in amyloid diseases, PDand AD, to alleviate toxicity in the course of �bril-lation process [25,26]. Nanoliposomes are respectablecandidates for drug delivery in amyloid diseases due to

their phospholipid-based structure, compatibility withthe body's system, and minute size, making them passacross blood brain barrier easily [27]. However, giventhe aggregated species of �-Syn transferring from cellto cell, causing cell degeneration [28,29], assessment ofnanoliposome impact on neurons cells sounds essentialbefore being used as a drug carrier.

Hence, considering that few reports are availableon the e�ects of nanoparticles on neuronal cells and�-Syn, the aim of this study is to investigate thecytotoxic e�ects of neutral charged nanoliposome indi�erent concentrations (35, 70, 140, 280, 560, 1120,1500, and 3000 �M) alone and when pre-incubatedwith �-Syn on dopaminergic cell lines, PC12 andSHSY5Y. The nanoliposome was formulated from Di-palmitoylphosphatidylcholine (DPPC) with the sizeof �100 nm, and its e�ect on the �-Syn �brillationwas assessed. Subsequently, the neurotoxicity of �-Syn in the presence of nanoliposomes was monitoredusing MTT and LDH assays. Results extracted fromthis study showed that neutrally-charged nanoliposomehas an acceptable potential as a drug carrier in theneurodegenerative disorders associated with �-Syn ag-gregation. Moreover, having high capacity to transferboth hydrophobic and hydrophilic small molecules,the neutrally-charged nanoliposome can trigger �-Syn�brillation and also decrease the cell death resultingfrom such protein aggregated species.

2. Experimental procedure

2.1. ReagentsDPPC and Mini-Extruder were prepared from AvantiPolar Lipids, Inc. (USA). Thio avin T (ThT), Congored, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). LDH was acquired from Pishtazteb Co.(Iran). PC12 and SHSY5Y cell lines were acquiredfrom Pasteur Institute (Iran). All salts and organicsolvents were purchased from Merck (Darmstadt, Ger-many). The cell culture mediums (DMEM high glucoseand DMEM-F12) and antibiotics were purchased fromGibcoBRL (Life Technologies, Paisley, Scotland). Fetalbovine serum (FBS) was from Biosera (England).

2.2. Nanoliposome formulation andcharacterization

Thin layer evaporation and hydration procedures wereemployed to formulate the liposomes [30]. In brief,DPPC was dissolved in 1 mL chloroform and the thinlayer of lipid was arranged using rotary evaporator with400 Mbar pressure at 37�C at 150 rpm shaking in around bottom ask. In the following, the lipid layer washydrated with Phosphate Bu�er Saline (PBS, pH 7.4)at 50�C for approximately 2 h. In order to convert theMulti-Vesicular Vesicles (MVV) and Multi-Lamellar

3544 F. Aliakbari et al./Scientia Iranica, Transactions F: Nanotechnology 24 (2017) 3542{3553

Vesicles (MLV) to smaller vesicles, including LargeUnilamellar Vesicles (LUV) and Small UnlilamellarVesicles (SUV), the material was then sonicated usingUP400S Ultrasonic processor (hielscher, Germany) forfour times (Cycle 0.5, Amplitude 60%), each for 45seconds with an internal rest of 30 seconds. Then,the Mini-Extruder set equipped by 100 nm �lter wasemployed to formulate SUV in a size of � 100 nm.In the last step, size, surface charge, and form ofthe formulated nanoliposomes were evaluated usingZeta seizer (MAlVER, UK) and Transmission ElectronMicroscopy (TEM) (JEM-1010; JEOL, Tokyo, Japan).

2.3. Transmission electron microscopyA 5 �L sample of nanoliposome was relocated to acarbon-coated, glow-discharged 400-mesh grid for 30seconds. The grids were then rinsed by two droplets ofdouble distilled water, and stained with 1% phospho-tungstic acid (pH 6.8), and blotted dry. The sampleswere processed for microscope observation (JEM-1010;JEOL, Tokyo, Japan) working with 60 kV and theimages were obtained using an Olympus KeenView G2camera.

2.4. Protein expression and puri�cationThe pET11-D plasmid, containing human �-SyncDNA, was transformed to Escherichia coli BL21(DE3) strain. The transformed cultures were grownovernight at 37�C in 1 L of LB medium (10 gr Tryptone,5 gr Yeast Extract and 10 gr NaCl per liter) containing100 �g/mL of kanamycin. The sub-culture of the grownbacteria was prepared and protein expression was car-ried out using auto-induction according to the protocoldescribed elsewhere [31]. To extract protein, cellswere centrifuged for 20 min at 3500 rpm at 4�C. Thepellet obtained by 1 L culture was then resuspended in100 mL osmotic shock bu�er (30 mM Tris-HCl, 40%sucrose, 2 mM EDTA, pH 7.2) and incubated for 10min at room temperature followed by centrifugationfor 30 min at 9000 g at 20�C. The acquired pellet wasresuspended in 90 mL ice-cold deionized water followedby adding 40 �L of saturated MgCl2 and incubatedon ice for 3 min. Then, the material was centrifugedfor 20 min at 9000 g at 4�C, and the supernatant wascollected. Precipitation of the supernatant was carriedout by titration with 1 M HCl to pH 3.5 and incubatedfor 5 min. The supernatant was then collected bycentrifugation for 20 min at 9000 g at 4�C and titratedto pH 7.5 with the drop-wise addition of 1 M NaOH.The extracted protein was �ltered through 0.45 �m�lter and loaded onto a Q-Sepharose column (HiTrapQ H P), which was pre-equilibrated with 20 mM Tris-HCl (pH 7.5). The column was rinsed with 0.1 M NaClin bu�er with three column volumes, �-Syn was elutedwith a NaCl gradient from 0.1-0.5 M, and the collectedpuri�ed protein was dialyzed against deionized water.

SDS - Polyacrylamide Gel Electrophoresis (with 13%resolving gels) was employed to analyze the fractions.Finally, the puri�ed �-Syn was freeze dried and storedat �20�C for the next steps.

2.5. �-Syn �brillation assay�-Syn was dissolved in PBS bu�er (pH 7.2) in a�nal concentration of 70 �M supplemented by di�erentconcentrations of neutral charged nanoliposomes (35,70, 140, 280, 560, 1120, 1500, and 3000 �M) in a�nal volume of 150 �L, added to each well of a 96-well plate with a 3 mm diameter glass bead. Asample of �-Syn without nanoliposome was consideredas control. Plates were sealed with a crystal clearsealing tape (Hampton Research, Aliso Viejo, CA).The �brillation process was conducted at 37�C with300 rpm orbital shaking. After a period of time, the�bril formation with or without nanoliposomes wasassessed by standard tests including ThT and Congored.

2.6. Cell culturePC12 and SHSY5Y cell lines were cultured in DMEMhigh glucose and DMEM-F12, respectively, which werecomplemented with 100 u/mL penicillin, 100 �g/mLstreptomycin, and 10% FBS, and incubated at 37�Cin humidi�ed atmosphere with 5% CO2 and 90%humidity. Then, the cells were subcultured in 96-wellmicroliter plates for the toxicity assays.

2.7. Toxicity assaysIn order to assess the cytotoxicity of di�erent con-centrations of nanoliposomes alone and when pre-incubated with �-Syn on the cell lines, MTT and LDHassays were employed as follows:

2.7.1. Cell viability assayTo conduct MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide, as a colorimetric assay forevaluating cell metabolic activity revealing cytotoxicityand cell viability, the cells were seeded with density of3 � 104 and 6 � 104 cell/ml for PC12 and SHSY5Y,respectively, and incubated in a CO2 incubator for 24h followed by treatment with di�erent concentrationsof nanoliposomes alone and when pre-incubated with�-Syn (10% v/v). After that, the cultures wereincubated in the same condition for more 24 h. Then,the medium was removed, the wells were washed byPBS, a freshly medium complemented with 10% (v/v)MTT stock solution (5 mg/mL in PBS) was addedto each well, and the plates were incubated for 4 hin the dark. In the following, the insoluble purpleformazan crystals, formed because of reducing thetetrazolium dye through cellular oxidoreductase, weredissolved in DMSO. The purple formazan product wasthen measured at 570 nm by Eliza reader (Expert96, AsysHitch, Ec Austria) to re ect the number of


Figure 1. Schematic procedure to formulate neutrally-charged nanoliposome from DPPC using thin layer evaporation andhydration methods followed by sonication and extrusion.

viable cells present. The percentage of control cellswas regarded as 100%, and the percentage of viablecells was considered as follows:

Cell viability of the treated sample (%)

=(Abs570(Treated cells))(Abs570(Control cells))

� 100: (1)

2.7.2. Lactate dehydrogenase release assayLactate dehydrogenase (LDH) is a cytosolic enzymepresent in most live cells, and this experiment quan-titatively measures LDH released into the media fromdamaged cells as an indication of cytotoxicity. Here,according to the LDH cytotoxicity assay kit, extra-cellular LDH in culture media was measured usingan enzymatic reaction that results in the reduction ofsubstrate. In brief, following the treatment of cells andfurther incubation for 24 h, a total of 100 �L sampleof growth media was collected and added to 1 mLof the arranged substrate from the kit. In the �nalstep, the absorbance was measured at 340 nm usingspectrophotometer (T80+ UV/VIS, PG instrumentsLtd, England) at 37�C. The LDH reaction acts asfollows:�

Pyruvate + NADHA +H+LDH! Lactate

+NAD+�: (2)

2.8. Statistical analysisStatistical analysis was conducted using SPSS version16.0. All experiments were carried out for three times,and the results were provided as means �SD. One-wayANOVA and Unpaired Student's t-test were used toconsider the statistical signi�cance within the groupsand between the groups, respectively. P Values < 0:05were regarded as signi�cant.

3. Results and discussion

3.1. Nanoliposome preparation andcharacterization

The neutrally-charged nanoliposome in a size of �100 nm diameter was formulated from DPPC using thinlayer evaporation and hydration methods followed bymeans of sonication and mini-extruder set, as shown inFigure 1.

To characterize the formulated nanoliposome,DLS was employed to analyze the size and Polydispersity Index (PdI). The particle size evaluatedby DLS (z-average) based on number of particleswas determined for nanoliposomes prepared from theneutral charged phospholipid as 71.37 nm (PdI 0.234),as shown in diagram (Figure 2(a)). Furthermore, thesurface charge of nanoliposome was measured by �potential assessment. Results indicated � potentialvalue of �1:795 � 0:545 mV for nanoliposome. As aresult, we con�rmed that nanoliposomes are neutrallycharged. The outcomes of TEM analysis (Figure 2(b))con�rmed what was achieved by DLS analysis, andrevealed the spherical and unilamellar morphology ofthe nanoliposomes. In addition, the results obtainedfrom TEM analysis demonstrated that the sizes ofnanoliposomes were nearly in the range of 60-90 nm.

The stability of nanoliposomes was assessed byevaluating the changes in the size of nanoliposomesthroughout 21 days at 4�C. No considerable evidencewas observed in the change of nanoliposomes size wherestored at 4�C over a period of 21 days (Table 1).

3.2. E�ect of the neutral charge nanoliposomeon �-Syn �brillation

The molecular basis of PD appears to be tightlyassociated with the aggregation of �-Syn into amyloid�brils, which is believed to play an important role inPD and some other amyloid-related disorders [6, 32-34]. Results obtained from the research on various cells


Figure 2. Size, charge, and morphology of nanoliposomeformulated from DPPC were evaluated using DLS andTEM: (a) Size distribution by number for neutral chargenanoliposomes measured by dynamic light scattering, and(b) transmission electron microscopy image fornanoliposome (Scale bar, 200 nm).

Table 1. The stability assessment of the nanoliposomesize throughout 21 days at 4�C using DLS.

Storage time(day)

Nanoliposome derivedfrom DPPC

Size (nm) PDI0 71.37 0.237 76.85 0.2414 98.52 0.2321 103.74 0.22

and animal models o�er that the prevention of amyloidaggregation is worthwhile to alleviate the symptomsof amyloidosis [35,36]. Accordingly, numerous stud-ies have been reported concerning the compounds toinhibit and/or disaggregate �-Syn aggregation, whichcould be considered a therapeutic strategy towardsthese neurodegenerative amyloid disorders. Someof the anti-aggregating components, which have sofar been identi�ed to have a potential use in thetreatment of amyloidosis, include antibodies, syntheticpeptides, chemically-synthesized compounds, small or-ganic molecules, and heat shock proteins [37-41].

Recently, nanoparticles have been signi�cantlyconsidered in not only the study of protein aggregaterecognition and destruction [42-43], but also in the

realm of protein �brillation [44-46] and inhibition of�bril formation [47]. According to previous studies,the interaction of nanoparticles with proteins can leadto perturbation of both protein structure and func-tion. Nanoparticles' biophysicochemical properties,containing size, morphology, chemical composition,surface charge, and hydrophobicity, possess importantroles in the �brillation process through their impacton proteins conformation [46]. Therefore, a generaltrend of response in the �brillation is not expected bydissimilar nanoparticles. It means that the interactionof nanoparticles and amyloid proteins might result indi�erent outcomes, including inhibition or facilitationof amyloid formation, or even be ine�ective.

Here, the formulated neutrally-charged nanolipo-somes (as described in previous section) were appliedto investigate the nanoliposomes impact on �-Syn�brillation using ThT uorescence intensity, Congo redabsorbance, and uorescents microscopy images.

To determine the e�ect of nanoliposome on theprotein �brillation, �-Syn was treated with di�erentconcentrations of nanoliposome and incubated for 24and 48 h at 37�C. Rate of �-Syn �brillation in the pres-ence or absence of nanoliposome was measured basedon the level of ThT uorescence emission (Thio avinT-binding assay) (Figure 3(a)). The emission of ThT uorescence is relevant to �-sheet structure and �brilformation. The results indicated that nanoliposomealmost permanently decreased �-Syn �brillation in aconcentration-independent manner. In addition, formore control samples, the monomer of �-Syn, monomerincubated with 3000 �M of nanoliposome for 1 min,3000 �M of nanoliposomes alone, 3000 �M of nanoli-posomes with ThT were assessed by the uorimetryto evaluate whether they raise ThT emission andcould interfere with the results or not. No emissionwas taken at 480 nm for each sample, indicatingno interference of ThT with nanoliposome (data notshown). Congo red analysis also con�rmed ThTresults. As elucidated from Figure 3(b), a detectablered shift was observed in the �-Syn incubated withoutnanoliposomes rather than the protein treated withnanoliposomes.

To analyze the morphology of �-Syn aggregateswith or without nanoliposomes, uorescent microscopyimages were taken after 24 h of incubation (Fig-ure 3(c)). The noticeable �-Syn �bril extensions weredetected in the �-Syn sample alone (Figure 3(c.1)),while the protein treated with nanoliposomes displayedlower �brillation (Figure 3(c.2-9)).

The neutral charge nanoliposomes formulatedhere demonstrated to have no inducing e�ect on the�bril formation. In fact, they were shown to have thepotential to decrease such a phenomenon. However,there are still vague features in the �eld of interactionsbetween nanoparticles and amyloid proteins. More


Figure 3. �-Syn �bril formation assay in the absence or presence of di�erent concentrations of nanoliposome using ThT uorescence intensity, Congo red absorbance and uorescence microscopy images: (a) ThT uorescence intensity for �-Synincubated alone or with di�erent concentrations of nanoliposome after 24 and 48 h of incubation, (b) Congo red spectra(400-600 nm) for monomer of �-Syn and �-Syn incubated alone or with di�erent concentrations of nanoliposome after 24 hof incubation, and (c) uorescence images taken from �-Syn incubated alone or with di�erent concentrations ofnanoliposome after 24 h of incubation: (c.1) �-Syn alone, (c.2-9) �-Syn incubated with 35, 70, 140, 280, 560, 1120, 1500,and 3000 �M of nanoliposomes, respectively. Results of �-Syn incubated with di�erent concentrations of nanoliposome aresigni�cantly dissimilar to those of controls (�-Syn incubated alone) (mean � SD, n = 3; �p � 0:05).

studies are required to elucidate the mechanism of �brilformation at the molecular level.

3.3. Neurotoxicity of nanoliposomes andnanoliposomes pre-incubated with �-Syn

To investigate nanoliposomes toxicity as well as capa-bility of nanoliposomes to hamper �-Syn aggregates-mediated cellular toxicity, MTT and LDH assays wereperformed using PC12 and SHSY5Y cell lines.

MTT, a tetrazolium salt, is reduced into insolubleformazan crystals by mitochondrial dehydrogenase inliving cells. At �rst, the cytotoxic potential of nano-liposome on PC12 and SHSY5Y cells was assessed.

Results indicated that nanoliposome had no consider-able cytotoxic impact on the two kinds of cell linesin di�erent concentrations ranging from 35-3000 �M(Figure 4(a)). Nonetheless, the susceptibility of thecell lines seems to be slightly di�erent from each otherwhile being treated with nanoliposomes. In SHSY5Ycells, at the concentrations of 1500 and 3000 �M ofnanoliposomes, a slight enhancement was observed incell proliferation. It can be presumed that this increasemight be correlated to nanoliposomes' role as a sca�old,which leads to cell growth or as a nutrition source forcells. With this interpretation, it has been recentlyidenti�ed that oxidation of fatty acids is essential for


Figure 4. Neurotoxicity of varied concentrations of nanoliposome on the PC12 and SHSY5Y cell lines after 24 h ofincubation: (a) The percentage of cell viability treated with nanoliposomes using MTT assay, and (b) measurement ofreleased lactate dehydrogenase from cells (an indicator of cell membrane integrity) treated with nanoliposomes after 24 hof incubation.

malignant glioma cells (Glioma is of primary malignantbrain tumor in adult) in which targeting this metabolicpathway leads to decreasing energy, and consequentlyreducing cell proliferation. In other words, besidesglucose, the cancer cells use lipid for their respiratoryactivity [48].

In the following, the neurotoxicity of �-Syn pre-incubated with varied concentrations of nanoliposomewas evaluated on both PC12 and SHSY5Y cell lines.�-Syn aggregates showed to reduce the cell viabilityof PC12 and SHSY5Y cells with di�erent rates, whichvalidated the dissimilar susceptibility of two kindsof cell line (Figure 5(a) and Figure 6(a) and (b)).However, pre-incubation of �-Syn with nanoliposomeled to modulating the �-Syn aggregates-mediated neu-rotoxicity towards lower cytotoxic e�ect.

Simultaneously, the LDH assay supported theresults obtained from MTT for both experiments,including the neurotoxicity of nanoliposome alone (Fig-ure 4(b)) and �-Syn pre-incubated with nanoliposome(Figure 5(b)). LDH is assessing the integrity of cellmembrane, indicating live cells. The cell membraneis the �rst part dealing with external agents andthe main objective of toxic �brils. The aggregatesinvolve membrane disruption, and so the cytotoxicityis more observable in here. Results obtained here wellhighlight the safety of the formulated nanoliposomeson dopaminergic cell lines.

The toxic e�ects of nanoparticles on human healthhave been one of the chief concerns of nanoparticlesapplications [49]. The studies have well demonstratedthat nanoparticles can in uence biological behavior atthe cellular and protein levels. Nanoparticles report tohave carcinogenic e�ects, which lead to DNA impair-ment, and induce in ammatory lesions [50]. Therefore,there is an essential requisite to assess the toxicity ofnanoparticles before applying them as safe componentsin medical and pharmaceutical industries. The cy-totoxicity of nanoparticles is mainly associated withparticle parameters containing size, shape, chemical

composition (purity, electronic properties, or crys-tallinity), surface structure (surface coatings-inorganicor organic), charge, hydrophobicity, and aggregationbehavior [51,52]. Nevertheless, biological factors, in-cluding type of cells, and culture circumstances, suchas temperature, culture medium components and celldensity, can a�ect cytotoxicity [52].

The surface charge seems to possess more com-plexity related to cytotoxicity compared with otherfactors such as size [53]. The cytotoxicity of liposomeshas been shown to be connected with their chargeand size [54]. Considering the similar size, neutralliposomes have been shown to have less toxicity thancharged liposomes, whereas the positive types/cationicliposomes are more toxic than negative ones in cells [54-57]. Cationic liposomes involved with reactive oxygenintermediates destroy plasma-membrane integrity, mi-tochondria, and lysosomes and increase the numberof autophagosomes in contrast to anionic particles,resulting in intracellular damage [52,56]. Therefore, thetoxicity of some formulated liposomes, especially thatincorporating cationic agent is noticed as the majordisadvantage in the medical application. However,in some cases, cationic liposomes have, in turn, beensuggested to be an e�cient tool for the selectivedelivery of antiangiogenic drugs to target tissue [58].

Since it has well been recognized that the termsof nanoliposomes formulation interfere with the cellcytotoxicity, the parameters, including kind of lipid,nature and amount of the charged component, andthe liposome size, are vital to be considered to achievenanoliposomes formulated with minimum toxicity [59].Moreover, the amount and type of nanoliposome withthe ratio of lipid to protein are involved in protein�brillation [19,20]. Given that DPPC has been shownto have less toxic e�ect than other phospholipids, suchas DMPC [59], in the present study, DPPC was utilizedto formulate neutrally-charged nanoliposomes in orderto assess nanoliposomes e�ect on the protein �brillationand neuron toxicity.


Figure 5. Neurotoxicity of �-Syn pre-incubated withdi�erent concentrations of nanoliposomes on the PC12and SHSY5Y cell lines after 24 h of incubation using MTTand LDH. In the control samples, 0.005% of NaOH wasadded to cells as a damaging reagent: (a) The cellviability percentage while treated with �-Synpre-incubated with altered concentrations of nanoliposomeafter 24 h of incubation using MTT assay, and (b) Cellmembrane integrity evaluation using LDH assay for thecells treated with pre-incubated �-Syn along with di�erentconcentrations of nanoliposome. Cytotoxicy percentages of�-Syn pre-incubated with di�erent concentrations ofnanoliposome were normalized to that of �-Synpre-incubated alone, and the outcome was signi�cantlydi�erent (�p � 0:05).

There are some controversial debates concerningthe toxicity of di�erent aggregate species of proteins.Numerous studies have been reported that the presenceof early aggregate species, known as oligomers, wouldlead to cell death. With respect to this fact, some areadvocates for a theory that inhibition of the �brillationprocedure can lead to the prevention of cell death anddisease progress [11], whereas others believe that in-duction of oligomers towards mature �brils modulatescell degeneration. Based on the latter, nanoparticles,especially those that possess an ability to accelerate

Figure 6. (a) Images of PC12, (b) SHSY5Y cells.Morphology of the cells (left) and crystals of formazan(right) is shown in the �gure. In each part, the upper andlower images are indicated for control cells and the cellstreated with incubated �-Syn for 24 h, respectively.

�brillation, would be proper candidates to reduceoligomers, and consequently hinder cell death [26]. Fur-thermore, a recent study has suggested that the trendof �brillation and aggregation process plays a crucialrole in cell toxicity, and inhibiting �bril growth andseeding aptitude may act as a viable strategy to protectcells from degeneration [10]. Finally, according to theexisting con icts, more investigations are required toelucidate the mechanism of �-Syn pathologic speciesand its impact on cell death while being treated bynanoparticles.

4. Conclusion

At present, engineered nanoparticles are recognized aspromising and mighty tools to control or treat neurode-generative diseases if their safety, biocompatibility, andbiodegradability can be addressed. In this report, wehave evaluated the toxicity of nanoliposomes formu-lated with neutral charge towards two dopaminergic


cell lines including PC12 and SHSY5Y. The e�ect ofnanoliposomes on �-Syn aggregation has been alsoassessed. Our results showed that the nanoliposomesformulated from DPPC with neutral charge have noneurotoxic e�ect on the cell lines applied here and canadditionally prevent �-Syn �brillation. Nanoliposomespre-incubated with �-Syn could, in turn, decreasethe cell toxicity, con�rming nanoliposomes bene�ts incuring amyloid-related disease. It is clear from thecurrent study that nanoliposomes can be perceived asa potent nano carrier with no neurotoxicity to transfersmall molecules, which have the potential to inhibit�bril formation in the treatment of PD and othersynucleinopathy diseases.

Acknowledgements

This work was �nancially supported by the StudentResearch Committee of Semnan University of MedicalSciences, Semnan, Iran (Grant No. 832, Code of EthicsCommittee: IR.SEMUMS.REC.1394.24), and the Na-tional Institute of Genetic Engineering and Biotech-nology, Tehran, Iran (Grant No. 832, Code of ethicsCommittee: IR.SEMUMS.REC.1394.24). We wouldlike to thank the Research Center of Biotechnologyof Semnan University of Medical Sciences Institute aswell as Industrial and Environmental Biotechnology ofNational Institute of Genetic Engineering and Biotech-nology for their cooperation and providing facilities tothis work.

Abbreviations

�� Syn Alpha-SynucleinPD Parkinson's DiseaseDPPC DipalmitoylphosphatidylcholineAD Alzheimer's DiseaseThT Thio avin TMTT 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromideMVV Multivesicular VesiclesMLV Multilamellar VesiclesLUV Large Unilamellar VesiclesSUV Small Unlilamellar VesiclesTEM Transmission Electron MicroscopyLDH Lactate dehydrogenase

References

1. Luheshi, L.M. and Dobson, C.M. \Bridging the gap:from protein misfolding to protein misfolding dis-eases", FEBS. Lett., 583(16), pp. 2581-2586 (2009).

2. Eisenberg, D. and Jucker, M. \The amyloid state ofproteins in human diseases", Cell, 148, pp. 1188-203(2012).

3. Maroteaux, L., Campanelli, J.T. and Scheller, R.H.\Synuclein: A neuron-speci�c protein localized to thenucleus and presy-naptic nerve terminal", J. Neurosci.,8, pp. 2804-2815 (1988).

4. Jakes, R., Spillantini, M.G. and Goedert, M. \Identi-�cation of two distinct synucleins from human brain",FEBS Lett., 345, pp. 27-32 (1994).

5. Goedert, M. \Parkinson's disease and other alpha-synucleinopathies", Clin. Chem. Lab. Med., 39, pp.308-312 (2001).

6. Zarranz, J.J., Alegre, J., Gomez-Esteban, J.C., Lez-cano, E., Ros, R., Ampuero, I., Vidal, L., Hoenicka, J.,Rodriguez, O., Atares, B., Llorens, V., Gomez Tortosa,E., del Ser, T., Munoz, D.G. and de Yebenes, J.G.\The new mutation, E46K, of alpha-synuclein causesParkinson and Lewy body dementia", Ann. Neurol.,55, pp. 164-73 (2004).

7. Wu, C.K. \Parkinson's disease with dementia, lewy-body disorders and alpha-synuclein: recent advancesand a case report", Acta. Neurol. Taiwa., 20, pp. 4-14(2011).

8. Spillantini, M.G., Schmidt, M.L., Lee, V.M., Tro-janowski, J.Q., Jakes, R. and Goedert, M. \Alpha-synuclein in Lewy bodies", Nature, 388, pp. 839-840(1997).

9. Recasens, A., Dehay, B., Bove, J., Carballo-Carbajal,I., Dovero, S., Perez-Villalba, A., Fernagut, P.O.,Blesa, J., Parent, A., Perier, C., Farinas, I., Obeso,J.A., Bezard, E. and Vila, M. \Lewy body extractsfrom Parkinson disease brains trigger alpha-synucleinpathology and neurodegeneration in mice and mon-keys", Ann. Neurol., 75, pp. 351-352 (2014).

10. Mahul-Mellier, A.L., Vercruysse, F., Maco, B., Ait-Bouziad, N., De Roo, M., Muller, D. and Lashuel,H.A. \Fibril growth and seeding capacity play key rolesin �-Syn-mediated apoptotic cell death", Cell. Death.Di�er., 22(12), pp. 2107-2122 (2015).

11. Morshedi, D., Salmani Kesejini, T., Aliakbari, F.,Karami-Osboo, R., Shakibaei, M., Tayaranian Mar-vian, A., Khalifeh, M. and Soroosh, M. \Identi�cationand characterization of a compound from Cuminumcyminum essential oil with anti�brillation and cyto-toxic e�ect", RPS., 9, pp. 431-443 (2014).

12. Breydo, L., Newland, B., Zhang, H., Rosser, A.,Werner, C., Uversky, V.N. and Wang, W. \A hyper-branched dopamine-containing PEG-based polymerfor the inhibition of �-Syn �brillation", Annu. Rev.Phys. Chem., 469(4), pp. 830-835 (2016).

13. Temsamani, H., Krisa, S., Decossas-Mendoza, M.,Lambert, O., M�erillon, J.M. and Richard, T.,\Piceatannol and other wine stilbenes: A pool of in-hibitors against �-Syn aggregation and cytotoxicity",Nutrients, 8(6), p. 367 (2016).

14. Adiseshaiah, P.P., Hal, J.B. and McNeil, S.E.\Nanomaterial standards for e�cacy and toxic-ity assessment", Wiley. Interdiscip. Rev. Nanomed.Nanobiotechnol., 2(1), pp. 99-112 (2010).


15. Sajja, H.K., East, M.P., Mao, H., Wang, Y.A., Nie,S. and Yang, L. \Development of multifunctionalnanoparticles for targeted drug delivery and non-invasive imaging of therapeutic e�ect", Curr. Drug.Discov. Technol., 6(1), p. 43 (2009).

16. Nel, A.E., M�adler, L., Velegol, D., Xia, T., Hoek,E.M.V., Ponisseril Somasundaran, P., Klaessig, F.,Castranova, V. and Thompson, M. \Understandingbiophysicochemical interactions at the nano-bio inter-face", Nat. Mater., 8(7), pp. 543-557 (2009).

17. Lehner, R., Wang, X., Marsch, S. and Hunziker,P. \Intelligent nanomaterials for medicine: carrierplatforms and targeting strategies in the context ofclinical application", Nanomedicine: N.B.M., 9(6), pp.742-757 (2013).

18. Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S.,Woo Joo, S., Zarghami, N., Hanifehpour, Y., Samiei,Kouhi, M. and Nejati-Koshki K. \Liposome: classi�-cation, preparation, and applications", Nanoscale ResLett., 8(1), p. 102 (2013).

19. Veiga, G.C. \Liposomes as versatile tools: nanoreac-tors, membrane models and drug delivery carriers",Doctoral Thesis Department of Chemical Engineering,University Rovira i Virgili Tarragona, Spain. T, pp.154-2015 (2015).

20. Galvagnion, C., Buell, A.K., Meisl, G., Michaels, T.C.,Vendruscolo, M., Knowles, T.P. and Dobson, C.M.\Lipid vesicles trigger alpha-synuclein aggregation bystimulating primary nucleation", Nat. Chem. Biol., 11,pp. 229-234 (2015).

21. Liu, Q., Zhang, J., Sun, W., Xie, Q.R., Xia, W.and Gu, H. \Delivering hydrophilic and hydrophobicchemotherapeutics simultaneously by magnetic meso-porous silica nanoparticles to inhibit cancer cells", IntJ. Nanomedicine, 7, pp. 999-1013 (2012).

22. Vazzana, M., Andreani, T., Fangueiro, J., Faggio, C.,Silva, C., Santini, A., Garcia, M.L., Silva, A.M. andSouto, E.B. \Tramadol hydrochloride: Pharmacoki-netics, pharmacodynamics, adverse side e�ects, co-administration of drugs and new drug delivery sys-tems", Biomed Pharmacother., 70, pp. 234-238 (2015).

23. Nogueira, E., Gomes, A.C., Preto, A. and Cavaco-Paulo, A. \Design of liposomal formulations for celltargeting", Colloids. Surf. B. Biointerfaces, 136, pp.514-526 (2015).

24. Suzuki, R., Omata, D., Oda, Y., Unga, J.,Negishi, Y. and Maruyama, K. \Cancer therapy withnanotechnology-based drug delivery systems: appli-cations and challenges of liposome technologies foradvanced cancer therapy", Nanomater in Pharmacol.,pp. 457-482 (2016).

25. Klajnert, B., Wasiak, T., Ionov, M., Fernandez-Villamarin, M., Sousa-Herves, A., Correa, J., Riguera,R. and Fernandez-Megia, E. \Dendrimers reduce toxi-city of A� 1-28 peptide during aggregation and accel-erate �bril formation", Nanomedicine, 8(8), pp. 1372-1378 (2012).

26. Mohammad-Beigi, H., Shojaosadati, S.A., Marvian,A.T., Pedersen, J.N., Klausen, L.H., Christiansen, G.,Pedersen, J.S., Dong, M., Morshedi, D. and Otzen,D.E. \Strong interactions with polyethylenimine-coated human serum albumin nanoparticles (PEI-HSANPs) alter �-synuclein conformation and aggregationkinetics", Nanoscale, 7(46), pp. 19627-19640 (2015).

27. Chopra, D., Gulati, M., Saluja, V., Pathak, P. andBansal, P. \Brain permeable nanoparticles", RecentPatents CNS Drug Discov., 3, pp. 216-225 (2008).

28. Masuda-Suzukake, M., Nonaka, T., Hosokawa, M.,Oikawa, T., Arai, T., Akiyama, H., Mann, D.M. andHasegawa, M. \Prion-like spreading of pathologicalalpha-synuclein in brain", Brain, 136, pp. 1128-1138(2013).

29. Reyes, J.F., Olsson, T.T., Lamberts, J.T., Devine,M.J., Kunath, T. and Brundin, P. \A cell culturemodel for monitoring alpha-synuclein cell-to-cell trans-fer", Neurobiol Dis., 77, pp. 266-275 (2014).

30. Dua, J.S., Rana, A.C. and Bhandari, A.K. \Liposome:methods of preparation and applications", Int. J.Pharm. Stud. Res., 3, pp. 14-20 (2012).

31. Lorenzen, N., Lemminger, L., Pedersen, J.N., Nielsen,S.B. and Otzen, D.E. \The N-terminus of �-synucleinis essential for both monomeric and oligomeric interac-tions with membranes", FEBS Let., 588(3), pp. 497-502 (2014).

32. Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide,S.E., Dehejia, A., Dutra, A., Pike, B., Root, H.,Rubenstein, J., Boyer, R., Stenroos, E.S., Chan-drasekharappa, S., Athanassiadou, A., Papapetropou-los, T., Johnson, W.G., Lazzarini, A.M., Duvoisin,R.C., Di Iorio, G., Golbe, L.I. and Nussbaum, R.L.\Mutation in the alpha-synuclein gene identi�ed infamilies with Parkinson's disease", Science, 276, pp.2045-2047 (1997).

33. Bellotti, V., Nuvolone, M., Giorgetti, S., Obici, L.,Palladini, G., Russo, P., Lavatelli, F., Perfetti, V. andMerlini, G. \The workings of the amyloid diseases",Ann. Med., 39, pp. 200-207 (2007).

34. Petrucci, S., Ginevrino, M. and Valente, E.M. \Phe-notypic spectrum of alpha-synuclein mutations: newinsights from patients and cellular models", Park.Relat. Disord., 22, pp. 16-20 (2016).

35. Jucker, M. \The bene�ts and limitations of animalmodels for translational research in neurodegenerativediseases", Nat Med., 16, pp. 1210-1214 (2010).

36. Jucker, P.M. and Walker, L.C. \Self-propagation ofpathogenic protein aggregates in neurodegenerativediseases", Nature, 501, pp. 45-51 (2013).

37. Glabe, C.G. \Conformation-dependent antibodies tar-get diseases of protein misfolding", Trends BiochemSci., 29, pp. 42-547 (2004).

38. Wang, S.S., Chen, Y.T. and Chou, S.W. \Inhibition ofamyloid �bril formation of beta-amyloid peptides viathe amphiphilic surfactants", Biochim. Biophys. Acta.,1741, pp. 307-313 (2005).


39. Morshedi, D., Salmani Kesejini, T., Aliakbari, F.,Karami-Osboo, R., Shakibaei, M., Tayaranian Mar-vian, A., Khalifeh, M. and Soroosh, M. \Identi�cationand characterization of a compound from Cuminumcyminum essential oil with anti�brilation and cytotoxice�ect", Res. Pharm. Sci., 9(6), pp. 431-443 (2014).

40. Temsamani, H., Krisa, S., Decossas-Mendoza, M.,Lambert, O., M�erillon, J.M. and Richard, T.\Piceatannol and other wine stilbenes: A pool ofinhibitors against �-synuclein aggregation and cyto-toxicity", Nutrients, 8(6), p. 367 (2016).

41. Kampinga, H.H. and Bergink, S. \Heat shock proteinsas potential targets for protective strategies in neu-rodegeneration", The Lancet Neurol., 15(7), pp. 748-759 (2016).

42. Bastus, N.G., Kogan, M.J., Amigo, R., Grillo-Bosch,D., Araya, E., Turiel, A., Labarta, A., Giralt, E.and Puntes, V.F. \Gold nanoparticles for selectiveand remote heating of �-amyloid protein aggregates",Mater. Sci. Eng. C., 27, pp. 1236-1240 (2007).

43. Choi, J.-S., Choi, H.J., Jung, D.C., Lee, J.-H. andCheon, J. \Nanoparticle assisted magnetic resonanceimaging of the early reversible stages of amyloid [smallbeta] self-assembly", Chem. Commun., pp. 2197-2199(2008).

44. Linse, S., Cabaleiro-Lago, C., Xue, W.-F., Lynch, I.,Lindman, S., Thulin, E., Radford, S.E. and Dawson,K.A. \Nucleation of protein �brillation by nanopar-ticles", Proc. Natl. Acad. Sci., 104, pp. 8691-8696(2007).

45. Fei, L. and Perrett, S. \E�ect of nanoparticles onprotein folding and �brillogenesis", Int J Mol Sci.,10(2), pp. 646-655 (2009).

46. Zaman, M., Ahmad, E., Qadeer, A., Rabbani, G. andKhan, R.H. \Nanoparticles in relation to peptide andprotein aggregation", Int. J. Nanomedicine., 9, p. 899(2014).

47. Jones, O.G. and Mezzenga, R. \Inhibiting, promoting,and preserving stability of functional protein �brils",Soft. Matter., 8, pp. 876-895 (2012).

48. Lin, H., Patel, S., A�eck, V.S., Wilson, I., Turnbull,D.M., Joshi, A.R., Maxwell, R. and Stoll, E.A. \Fattyacid oxidation is required for the respiration andproliferation of malignant glioma cells", Neuro Oncol.,p. 128 (2016).

49. Savolainen, K., Alenius, H., Norppa, H., Pylkk�anen,L., Tuomi, T. and Kasper, G. \Risk assessment of engi-neered nanomaterials and nanotechnologies{a review",Toxicology, 269(2-3), pp. 92-104 (2010).

50. Loo, S.C., Moore, T., Banik, B. and Alexis, F.\Biomedical applications of hydroxyapatite nanopar-ticles", Curr. Pharm. Biotechnol., 11(4), pp. 333-342(2010).

51. Nel, A., Xia, T., M�adler, L. and Li, N. \Toxic potentialof materials at the nanolevel", Science, 311(5761), pp.622-627 (2006).

52. Fr�ohlich, E. \The role of surface charge in cellularuptake and cytotoxicity of medical nanoparticles", Int.J. Nanomedicine, 7, pp. 5577-5591 (2012).

53. Petushkov, A., Intra, J., Graham, J.B., Larsen, S.C.and Salem, A.K. \E�ect of crystal size and surfacefunctionalization on the cytotoxicity of silicalite-1nanoparticles", Chem. Res. Toxicol., 22(7), pp. 1359-1368 (2009).

54. Filion Mario, C. and Phillips Nigel, C. \Toxicityand immunomodulatory activity of liposomal vectorsformulated with cationic lipids toward immune e�ectorcells", Biochim Biophys Acta., 1329, pp. 345-356(1997).

55. Campbell, P.I. \Toxicity of some charged lipids usedin liposome preparations", Cytobios, 37, pp. 21-26(1983).

56. Dokka, S., Toledo, D., Shi, X., Castranova, V. andRojanasakul, Y. \Oxygen radical-mediated pulmonarytoxicity induced by some cationic liposomes", Pharm.Res., 17, pp. 521-525 (2000).

57. Nagahiro, I., Mora, B.N., Boasquevisque, C.H.R.,Scheule, R.K. and Patterson, G.A. \Toxicity ofcationic liposome { DNA complex in lung isografts",Transplantation, 69, pp. 1802-1805 (2000).

58. Krasnici, S., Werner, A., Eichhorn, M.E., Schmitt-Sody, M., Pahernik, S.A., Sauer, B., Schulze, B., Teifel,M., Michaelis, U., Naujoks, K. and Dellian, M. \E�ectof the surface charge of liposomes on their uptake byangiogenic tumor vessels", Int. J. Cancer., 105(4), pp.561-567 (2003).

59. Smistad, G., Jacobsen, J. and Sande, S.A. \Multivari-ate toxicity screening of liposomal formulations on ahuman buccal cell line", Int. J. Pharm., 330, pp. 14-22 (2007).

Biographies

Farhang Aliakbari has been a Research Assistant atNational Institute of Genetic Engineering and Biotech-nology (NIGEB). He is now a researcher at SemnanUniversity of Medical Sciences and NIGEB carryingout research on the interaction of �-Syn with nano-liposomes and their neurotoxicity.

Ali Akbar Shabani received his PhD in MedicalBiotechnology from Pasteur Institute (2001). He is nowan Assistant Professor and the Head of the Departmentand Center for biotechnology research at Semnan Uni-versity of Medical Sciences. His research focuses ondi�erent aspect of biological products especially BetahCG in mammalian cells.

Hassan Bardania received his PhD in Nanobiotech-nology from Tarbiat Modares University (2016). Heis now an Assistant Professor in the Cellular and


Molecular Research Center at Yasuj University of Med-ical Sciences. His research group pursues application-oriented research on drug delivery using nanoliposomesand metal nanoparticles.

Hadieh Alsadat Eslampanah Seyedi graduatedwith a master degree at Isfahan University of Tech-nology (IUT). She was a Research Assistant at NIGEB.Currently, she is a PhD student in the �eld of MolecularBiology and Biochemistry at Institute for Glycomics,Gri�th University, QUL, Australia.

Hossein Mohammad-Beigi received his PhD inChemical Engineering-Biotechnology from TarbiatModares University (2015). He is now a Guest Re-searcher at Aarhus University, Denmark. His researchfocuses on the amyloid proteins and the interactionbetween these proteins with nanoparticles.

Amir Tayaranian Marvian graduated with masterdegree from Tehran University in the �eld of Molecularand Cellular Biology. He was a Research Assistant atNIGEB and now he is PhD student at the TechnicalUniversity of Munich, Germany. His research area fo-cuses on the pathology of the amyloid protein especiallyalpha-synuclein and Tau.

Mahour Nassoti graduated with master degree atNIGEB in the �eld of Molecular and Cellular Biol-ogy. Her research was focused on the e�ect of smallmolecules on di�erentiated neuronal cells.

Abbas Ali Vafaei received his PhD in Physiologyfrom Isfahan University of Medical Sciences (2000). Heis now a Professor of Physiology at Semnan University

of Medical Sciences. His research realm is on Parkin-son's disease and the mechanism of cell death in sucha disease.

Seyed Abbas Shojaosadati received his PhD inChemical Engineering from Birmingham University(1988). He now is a Professor and the Head of ChemicalEngineering Department at Tarbiat Modares Univer-sity. His research focuses on the nano-biotechnology.

Ali Akbar Saboury received his PhD in PhysicalChemistry from Tarbiat Modares University (1996).He is now a Professor at the Institute of Biochemistryand Biophysics (IBB), University of Tehran. Hisresearch realm is on the thermodynamic aspects ofprotein denaturation with the aim of obtaining addi-tional information regarding protein structure-functionrelationship.

Gunna Christiansen passed her Internship in In-ternal Medicine and Surgery in 1969. She receivedher Doctor of Medical Science (D.M.Sc.) on Electronmicroscopy of nucleic acids in 1987. She is a Professorof Medical Molecular Biology at the Department ofMedical Microbiology and Immunology, University ofAarhus, Denmark.

Dina Morshedi received her PhD in Biochemistryfrom Institute of Biochemistry and Biophysics (IBB)(2008), University of Tehran. She is now an AssistantProfessor at NIGEB. Her research realm is proteinfolding and miss folding. She works on the role of�-Synuclein in the process of neurodegeneration inParkinson's disease and using nanoparticles for drugdelivery in such a disease.

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