Viologen-Phosphorus Dendrimers Inhibit α‑Synuclein FibrillationKatarzyna Milowska,*,† Justyna Grochowina,† Nadia Katir,‡,§ Abdelkrim El Kadib,§ Jean-Pierre Majoral,‡

Maria Bryszewska,† and Teresa Gabryelak†

†Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska Street,90-236 Lodz, Poland‡Laboratoire de Chimie de Coordination CNRS, 205 route de Narbonne, 31077, Toulouse, France§Institute of Nanomaterials and Nanotechnology (iNANOTECH)-MAScIR (Moroccan Foundation for Advanced Science,Innovation and Research), ENSET, Avenue de I’Armee Royale, Madinat El Irfane, 10100 Rabat, Morocco

ABSTRACT: Inhibition of α-synuclein (ASN) fibril forma-tion is a potential therapeutic strategy in Parkinson’s diseaseand other synucleinopathies. The aim of this study was toexamine the role of viologen-phosphorus dendrimers in the α-synuclein fibrillation process and to assess the structuralchanges in α-synuclein under the influence of dendrimers.ASN interactions with phosphonate and pegylated surface-reactive viologen-phosphorus dendrimers were examined bymeasuring the zeta potential, which allowed determining thenumber of dendrimer molecules that bind to the ASNmolecule. The fibrillation kinetics and the structural changeswere examined using ThT fluorescence and CD spectroscopy.Depending on the concentration of the used dendrimer andthe nature of the reactive groups located on the surface, ASN fibrillation kinetics can be significantly reduced, and even, in thespecific case of phosphonate dendrimers, the fibrillation can be totally inhibited at low concentrations. The presented resultsindicate that viologen-phosphorus dendrimers are able to inhibit ASN fibril formation and may be used as fibrillar regulatingagents in neurodegenerative disorders.

KEYWORDS: α-synuclein, viologen-phosphorus dendrimers, fibrillation, circular dichroism

■ INTRODUCTION

Proteins, as one of the main components of cells, perform manyimportant functions in the body, and their properties dependon their spatial structure. It has been shown that particles with adisordered spatial structure are characterized by the presence ofthe β structure, and consequently proteins have a tendency toaggregate.1

The tendency of proteins to form insoluble fiber is animportant problem in biotechnology and the pharmaceuticalindustry, because aggregated proteins do not possess the samebiological properties as proteins in the native state. Aggregatedproteins may be immunogenic, and may even show a strongtoxicity effect.2

Inhibition of the accumulation of insoluble protein depositscan have a potential therapeutic application. Earlier studies havedemonstrated that polyamidoamine (PAMAM) and polypro-pylenoimine (PPI) dendrimers, due to their unique branchedstructure, are promising candidates for the treatment ofneurodegenerative changes. The results showed that den-drimers inhibit the formation of insoluble forms of protein, andalso remove the existing ones. This suggests that themechanism of action is not limited to blocking the monomersfrom which deposits are formed, but probably the existingaggregates are disrupted.3−6

One of the most important proteins involved in manyneurodegenerative diseases is α-synuclein (ASN). ASN is apresynaptic neuronal protein, whose normal function has notbeen yet established; however, several studies suggest that ASNplays a role in the regulation of the synaptic function,dopaminergic system, neuronal plasticity, and vesicular trans-port.7 Misfolded protein and the accumulation of its aggregatesis a key event in the pathological cascades of severalneurodegenerative disorders, e.g., it is one of the basiccomponents of Lewy bodies, whose presence has beenestablished in degenerated neurons.8 A better understandingof the biological role played by α-synuclein is a major challengefor researchers. In order to prevent aggregation of ASN, it isimportant to know which compound will effectively prevent thedegradation of this protein. Dendrimers, thanks to theirproperties, are potential candidates for the inhibition of thisprocess. The positive effect of cationic polyamidoamine(PAMAM) and phosphorus dendrimers on the process ofα-synuclein aggregation has been already demonstrated.9−11

Received: November 7, 2012Revised: January 30, 2013Accepted: February 4, 2013Published: February 4, 2013

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Specifically, PAMAM G3, G4 and G5 dendrimers inhibit theα-synuclein fibrillation process by preventing the formation ofthe β structure in its molecule. The inhibition efficiencyincreases with both the dendrimer concentration and thegeneration number.10 The mechanism of dendrimers action isnot limited only to inhibiting the growth of fibrils. Thesemacromolecules are also involved in the breaking anddegradation of the existing protein aggregates. In addition,the nature of functional peripheral groups plays a pivotal role inthe inhibition of the α-synuclein aggregation process. Forinstance, it has been demonstrated that PAMAM G3.5dendrimers decorated with carboxyl groups on the surface donot allow the inhibition of α-synuclein aggregation.9

Also, phosphorus containing dendrimers may be potentialinhibitors of α-synuclein fibrillation, however, their effectivenessdepends on the used concentration and the size of dendrimers.These studies showed that phosphorus dendrimers at lowerconcentrations inhibited the aggregation of α-synuclein. Thehigher concentrations did not inhibit the fibril formationprocess. The phosphorus dendrimer G3 was a better inhibitorof the aggregation process than G4. It is suggested that theseresults are related to the large size of the particles ofdendrimers.11

In this study, as potential factors in preventing fibrillation ofASN in vitro, we selected two viologen-phosphorus containingdendrimers, new, highly branched polymers, whose biologicalproperties are still insufficiently known. These types ofcompounds exhibit antimicrobial properties, and their behaviordepends on the number of viologen units, the size and thenature of the surface groups.12 The dendrimers containingviologen demonstrated antiviral activity against the humanimmunodeficiency virus (HIV) and, to a lesser extent, againstother viruses (herpes simplex virus, Reovirus and respiratorysyncytial viruses).13

In the present work, we investigated whether polycationicviologen-phosphorus dendrimers (two positive charges perviologen unit) form complexes with ASN and can inhibit ASNfibrillation. These compounds are characterized by the presenceof a hexafunctionalized core (P3N3)(NCH3N)6, viologenlinkages as branches and either phosphonate groups orpolyethylene glycol (PEG) groups on their surface. The

chemical structures of the generation 0 dendrimers are shownin Figure 1.

■ EXPERIMENTAL SECTION

Materials. Viologen-phosphorus containing dendrimerswere synthesized according to the protocol previouslydescribed.12

Human ASN was purchased from Sigma-Aldrich (USA). Forall experiments, ASN and dendrimers were dissolved inphosphate-buffered saline (1.9 mM NaH2PO4, 8.1 mMNa2HPO4, pH = 7.4).All the other chemicals were of analytical grade. All solutions

were made with water purified by the Milli-Q system.Zeta Potential. The measurements of the zeta potential

(electrokinetic potential) were performed with Zetasizer NanoZS from Malvern, which uses electrophoresis and LDV (laserDoppler velocimetry) techniques. Applying a combination ofthese two techniques allowed measuring the electrophoreticmobility of the molecules in the solution. The zeta potentialvalue was calculated directly from the Helmholtz−Smoluchow-ski equation using the Malvern software.14

The measurements were performed at 37 °C with threerepetitions. Increasing concentrations of the dendrimer in therange 0.1−10 μM were added to α-synuclein of a concentrationof 0.2 μM, and zeta potential was measured.

Thioflavin T Fluorescence Measurements. The processof fibrillation was monitored using dye thioflavin T (ThT),whose fluorescence depends on the presence of aggregates.The samples of ASN alone and ASN in the presence of

viologen-phosphorus dendrimers were incubated at 37 °C for 0,8, 24, 32, 48, and 72 h. The concentration of ASN was 2 μM,and the concentrations of viologen-phosphorus dendrimerswere 1, 2, 3, and 4 μM. After incubation, ThT at a finalconcentration of 15 μM was added to the samples with theprotein and the protein with dendrimers. Fluorescencemeasurements were performed at 37 °C using a Perkin-ElmerLS-50B fluorimeter with the excitation at 450 nm, and theemission at 490 nm.Before examining the fluorescence with ThT, it was made

sure that viologen-phosphorus dendrimers did not bind ThTand did not emit fluorescence.

Figure 1. Viologen-phosphorus dendrimer structures.

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Circular Dichroism (CD) Spectroscopy. The CD spectra(195−260 nm) were recorded for ASN in the presence/absence of dendrimers on a Jasco J-815 CD spectropolarimeter,in 10 mm path length quartz cuvettes, with a wavelength step of1 nm, a response time of 4 s and a scan rate of 20 nm/min,thermostatted at 37 °C. Each spectrum was the average of threerepetitions. Viologen-phosphorus dendrimers alone at theconcentration used in the experiments did not produce anydiscernible features in their CD spectra and were used as blanksfor ASN-dendrimer complex spectra.The changes in the secondary structure of ASN in the

presence and absence of dendrimers were tested immediatelyand after 24, 48, and 72 h of incubation at 37 °C. In theseexperiments, we used ASN at a concentration of 2 μM anddendrimers at the concentrations of 1, 2, 3, and 4 μM.Statistical Analysis. The results of the fluorescent study

and zeta potential are presented as mean ± SD from sixindividual experiments. Each experiment was repeated threetimes. Statistical evaluation of the difference between thecontrol and the treated group was performed using Student’s ttest. P < 0.05 and below was accepted as statistically significant.

■ RESULTS

Zeta Potential. The changes in the zeta potential of ASNupon addition of viologen-phosphorus dendrimers in the molarratio range 0−25 for vpd/ASN were studied. The results arepresented in Figure 2. The zeta potential of α-synuclein has avalue of −21 mV. The addition of viologen-phosphorusdendrimers led to significant changes in the values of the zetapotential but did not change the sign of the potential, it was stillnegative. The values of the zeta potential after adding

dendrimers changed from −21 mV to −6 mV for vpd-1 and−3 mV for vpd-2 (Figures 2A and 2B). The analysis of possiblechanges of the zeta potential as a function of the molar ratio canbe approximated by the number of dendrimer molecules thatcan attach to one molecule of α-synuclein. The number ofbinding centers is the point (X,O) corresponding to the pointof intersection of two straight lines tangent to the curve of thezeta potential as a function of the molar ratio. The calculated nis 1.5 for vpd-1 and 1 for vpd-2.

Thioflavin T Fluorescence. Fibril formation was moni-tored using the fluorescent dye thioflavin T (ThT). ThT is ahighly selective dye whose fluorescence depends on theformation of protein fibrils. Figure 3 demonstrates how

viologen-phosphorus dendrimers affect α-synuclein aggrega-tion. A control curve (ASN without dendrimer) shows a typicalfibrillation process, where fibrils were systematically created,and a plateau was obtained after 48 h of incubation. After theincubation of ASN with viologen-phosphorus dendrimer vpd-1at concentrations 1 and 2 μM, the fibrillation process wascompletely inhibited. For the two higher concentrations ofvpd-1 (3 and 4 μM) and all concentrations of dendrimer vpd-2a slight increase in ThT fluorescence intensities starting from32 h of incubation was observed. However, independently ofthe elongation of the incubation time the fluorescence intensitywas at the same level.

Circular Dichroism (CD) Spectroscopy. To correlate theamount of formed fibrils, which were observed in ThTmeasurements, with the changes in a secondary structure ofASN, CD experiments were carried out. ASN CD spectra arepresented in Figure 4. α-Synuclein is a natively unfoldedprotein and exhibits a random coil structure under physiological

Figure 2. α-Synuclein zeta potential in the presence of viologen-phosphorus dendrimers (A) vpd-1, (B) vpd-2.

Figure 3. Thioflavin T fluorescence during the fibrillation process of α-synuclein in the presence of dendrimers (A) vpd-1, (B) vpd-2.

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conditions. The minimum was observed at 200 nm. After theincubation, we observed a change in the ASN spectrum shape,which indicated changes in its secondary structure. After 48 h ofincubation, a positive signal was obtained in the range 195−205nm in the CD spectrum, which may indicate the conversion ofthe disordered structure into the β form.CD spectra of α-synuclein alone and α-synuclein incubated

with dendrimers for 48 h are shown in Figures 5A and 5B. Inthe case of vpd-1 dendrimer a slight decrease in negative signalintensity (from −10 to −8) occurred after 48 h of incubation atconcentrations of 3 and 4 μM. However, no significant changesin the shape of α-synuclein spectra were observed, suggestingthat the structural changes in α-synuclein were inhibited by the

dendrimer. In the case of the second dendrimer (vpd-2), minorchanges were observed at the minimum, but these changes didnot lead to the conversion into the β form. These resultsindicated that the structure of ASN was still disordered.Summarizing, after 48 h of incubation, the CD spectra of ASNin the presence of dendrimers were very similar to the spectrumof native ASN, indicating that the used dendrimers inhibitedα-synuclein fibrillation. The results after 24 and 72 h ofincubation are not presented because there are no significantdifferences compared to the data obtained after 48 h.

■ DISCUSSIONProtein aggregates formed in the central nervous systemcontribute to many neurodegenerative diseases.15,16 Fibrilformation and the presence in the brain of deposits ofpathological protein are characteristic of neurodegenerativediseases such as Parkinson’s, Alzheimer’s or dementia withLewy bodies.17 Nowadays many research groups are looking forfactors or agents that could prevent or inhibit the aggregationprocess.Unique properties of dendrimers and the possibility of their

clinical use have led to studies on the interaction betweendendrimers and cellular components, mainly proteins. Vio-logen-phosphorus dendrimers, which were used in this work,contain in their framework viologen units (derivative of 4,4′-bipyridine salt) and phosphonate groups or polyethylene glycol(PEG) groups on their surface. The presence of phosphonategroups on the surface of one of these dendrimers is veryimportant due to their antiinflammatory properties,18 and NKcell amplification,19 etc. The results obtained in earlier researchalso demonstrated that polycationic phosphorus dendrimersexhibit anti-HIV activity,20 can be used as transporters of drugsand genetic material21,22 and can prevent aggregation ofproteins associated with neurodegenerative processes, namely,prion,23 Alzheimer’s and Parkinson’s diseases,3,11,24 whereas thepresence of PEG groups on the surface of the seconddendrimer may reduce toxicity and increase the solubility inwater.It was also demonstrated that viologen dendrimers have

antiviral properties, especially against HIV.13

Interactions between viologen-phosphorus dendrimers andα-synuclein have been studied by measuring the zeta potential.The zeta potential characterizes the electric double layer, whichis formed on the molecule−liquid phase boundary. Zetapotential value can be related to the dispersion stability orthe ability to maintain the particles in a colloidal dispersion.Unstable dispersions undergo sedimentation or coagulationprocesses.25 The measurement of the zeta potential also allowsdetermining the number of dendrimer molecules bound toprotein and the potential of this complex. As shown in Figure 2,the addition of the dendrimer to α-synuclein resulted inchanges in the zeta potential of approximately −21 mV to −6mV for vpd-1 and −3 mV for vpd-2.Distribution of the charge in α-synuclein is dependent on the

solution pH. The net charge is −9 at the neutral pH, but it isdifferent for different regions.26

The used dendrimers have a positive charge (+12), but theviologen groups, which are positively charged, are located insidethe molecule. This may explain why the zeta potential of thecomplex does not reach positive values.Analysis of the results allowed determining the approximate

number of dendrimer molecules that can attach to onemolecule of α-synuclein. α-Synuclein combined with vpd-1

Figure 4. CD spectra of α-synuclein depending on the incubationtime.

Figure 5. CD spectra of α-synuclein alone and in the presence ofdendrimers (A) vpd-1, (B) vpd-2, after 48 h of incubation. Thedendrimers were added to α-synuclein at time 0 h.

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dendrimer in a 1:1.5 ratio, and one-dendrimer molecule vpd-2falls on one molecule of α-synuclein. Based on these results, theratios of dendrimers to α-synuclein which were used in furtherstudies were selected. In addition to the calculated molar ratios,a shortage and excess of dendrimer were used.The influence of viologen-phosphorus dendrimers on the

fibrillation of ASN was examined using circular dichroismspectroscopy and the fluorescence methods with ThT. Circulardichroism spectroscopy allows investigating of the secondarystructure of proteins and peptides.Alterations in the shape of α-synuclein CD spectra, which

show the structural changes, were observed after 24 hincubation at 37 °C. After 48 h of incubation a positive signalindicating the conversion of the α-synuclein disorderedstructure to the β form was observed.After the addition of viologen-phosphorus dendrimers to α-

synuclein and incubation for 48 h, no significant changes in theshape of the spectrum in comparison to nonincubated ASNwere noticed. Based on the obtained results, it is concluded thatboth dendrimers used in all concentrations inhibit theformation of the β structure. The ability to inhibit thefibrillation process of α-synuclein has been demonstrated bythe use of cationic PAMAM dendrimers9,10 and phosphorusdendrimers.11 However, the final result of the inhibition ofaggregation is dependent on the ratio of phosphorusdendrimers to protein. The results show that dendrimers atlower concentrations are thermodynamic inhibitors, becausethe final number of fibrils is reduced.11 The work by Klajnert etal.3 demonstrates that phosphorus dendrimers are also able tointerfere in the process of peptide Prp 185−208 aggregation,thus reducing the final number of amyloid fibrils.α-Synuclein fibrillation was studied by monitoring the

changes in thioflavin T fluorescence intensity, which isdependent on the amount of protein aggregates. The curveof the ASN fibrillation is characterized by the sigmoidal shape,which can be divided into (i) an initiation phase “lag”, whichcorresponds to the formation of a critical nucleus, (ii) a rapid

growth phase, which corresponds to fibril elongation, and (iii)the final phase, which corresponds to amount of formedfibrils.27 Two main types of inhibitors of the aggregationprocess can be distinguished: kinetic and thermodynamic.These types of inhibitors can be identified by analyzing theshape of kinetics curves compared to a control curve. Kineticinhibitors are characterized by differences in lag time, but thefinal amount of fibrils is unchanged. In the case ofthermodynamic inhibitors, the final amount of fibrils is reduced,but there is no significant effect on the elongation phase.28

As evidenced by the fibrillation kinetics curves, α-synucleinincubation at 37 °C contributes to the spontaneous fibrilformation. Depending on the concentration of the dendrimerand its groups on the surface, the process can be slightly oreven totally inhibited. A more effective inhibitor is the vpd-1dendrimer allowing total inhibition under the two lower usedconcentrations, whereas vpd-2 dendrimer and vpd-1 at higherconcentrations did not completely inhibit this process butlargely decreased the amount of formed fibrils. These resultsindicate that viologen-phosphorus dendrimers are thermody-namic inhibitors, because the final number of fibrils issignificantly or completely reduced. A similar effect occurredfor phosphorus dendrimers.11

Several preliminary observations can be formulated corre-sponding to the efficiency of different types of dendrimerstoward inhibition of the fibrillation of ASN (Table 1). Thepercent of inhibition is different for different types ofdendrimers. The most effective is viologen-phosphorusdendrimer. Inhibition necessitates the presence of positivelycharged dendrimers, but the number of positive charges for agiven type of dendrimer does not dramatically influence theactivity of these dendrimers since viologen dendrimers (vpd-1and -2, 12 positive charges) inhibit ASN fibrillation as PAMAM(64 positive charges) or phosphorus dendrimers (48 or 96positive charges) do. Negative charges on the surface ofdendrimers do not allow the inhibition of the ASN fibrillation.The inhibition also does not depend on the generation number

Table 1. The Efficiency of Different Types of Dendrimers towards Inhibition of the Fibrillation of α-Synucleina

dendrimers MW [Da] nd groups charge ASN/dendrimer molar ratio % of inhibition

PAMAM G3.5 12 928 −COO− −64 1:1 5.3 ± 4.31:2 3.0 ± 2.8

G4 14 214 −NH2 +64 1:1 27.2 ± 2.91:2 30.3 ± 3.2

phosphorus (pd) G3 16 280 −N+HEt2 +48 1:0.1 63.9 ± 3.01:0.5 59.7 ± 2.51:1 7.8 ± 3.41:2 6.0 ± 3.0

G4 33 702 −N+HEt2 +96 1:0.1 48.5 ± 3.31:0.5 36.3 ± 2.71:1 3.2 ± 2.71:2 1.2 ± 0.7

viologen-phosphorus (vpd) G0 3 789 −P(O)(OEt)2 +12 1:0.5 90.7 ± 3.41:1 86.7 ± 1.91:1.5 71.8 ± 1.31:2 73.7 ± 1.2

G0 14 421 PEG (M = 2000 Da) +12 1:0.5 73.9 ± 2.11:1 73.0 ± 1.71:1.5 74.0 ± 2.51:2 71.2 ± 1.9

aThT fluorescence for ASN taken as 100% fibrillation. The percentage of fibrillation was calculated using the following formula: % of inhibition =100% − (FASN+D/FASN) × 100%, where FASN+D is fluorescence of ASN in the presence of dendrimer and FASN+D is fluorescence of ASN.

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and the molecular weight since phosphorus dendrimers (G3and G4) as well as phosphorus-viologen vpd-1 (generation 0)are active species at the lower concentrations used. However,the presence of phosphonate groups on the surface of vdp-1allows this dendrimer to be more effective against the inhibitionof ASN fibrillation (90.7% of inhibition for ratio 1:0.5) than thecorresponding vdp-2 dendrimer of generation 0 bearingterminal PEG groups (≈71−74% of inhibition), thus indicatingthe influence of terminal groups on the properties ofdendrimers.There are three possible strategies of inhibiting the formation

of fibrils by dendrimers: reduction of protein concentration as aresult of binding to dendrimer, blocking the fibril growth andbreaking the existing fibrils.4 It is difficult to unequivocallyindicate which mechanism dominates. It seems that all of themare likely.

■ CONCLUSIONIn conclusion, the results obtained in this study suggest thatlow generation viologen-phosphorus dendrimers can bepotential inhibitors of ASN fibril formation. Vpd-1, whichpossesses phosphonate groups on the surface, is a moreeffective dendrimer. The fact that dendrimers can prevent ASNfibrillation in suspension is therefore important for furtherresearch because it may lead to the design of effectivepharmacological strategies.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: milowska@biol.uni.lodz.pl. Tel: (48 42) 635 43 80.Fax: (48 42) 635 44 74.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe project was supported by the COST Action TD0802,CNRS and MAScIR foundation.

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Molecular Pharmaceutics Article

dx.doi.org/10.1021/mp300636h | Mol. Pharmaceutics 2013, 10, 1131−11371137