Synthesis and characterization of fluorinated magnetic core–shell nanoparticles for inhibition of insulin amyloid fibril formation This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2009 Nanotechnology 20 225106 (http://iopscience.iop.org/0957-4484/20/22/225106) Download details: IP Address: 129.89.24.43 The article was downloaded on 14/03/2013 at 14:31 Please note that terms and conditions apply. View the table of contents for this issue, or go to the journal homepage for more Home Search Collections Journals About Contact us My IOPscience
Transcript
Synthesis and characterization of fluorinated magnetic core–shell nanoparticles for inhibition
of insulin amyloid fibril formation
This article has been downloaded from IOPscience. Please scroll down to see the full text article.
Synthesis and characterization offluorinated magnetic core–shellnanoparticles for inhibition ofinsulin amyloid fibril formationHadas Skaat1, Georges Belfort2,3 and Shlomo Margel1,4
1 Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel2 Howard P Isermann Department of Chemical and Biological Engineering, RensselaerPolytechnic Institute, Troy, NY 12180, USA3 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute,Troy, NY 12180, USA
Received 12 February 2009, in final form 2 April 2009Published 12 May 2009Online at stacks.iop.org/Nano/20/225106
AbstractMaghemite (γ -Fe2O3) magnetic nanoparticles of 15.0 ± 2.1 nm are formed by nucleationfollowed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Uniformmagnetic γ -Fe2O3/poly (2,2,3,3,4,4,4-heptafluorobutyl acrylate) (γ -Fe2O3/PHFBA) core–shellnanoparticles are prepared by emulsion polymerization of the fluorinated monomer2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) in the presence of the maghemite nanoparticles.The kinetics of the insulin fibrillation process in the absence and in the presence of theγ -Fe2O3/PHFBA core–shell nanoparticles are elucidated. A significant direct slow transitionfrom α-helix to β-sheets during insulin fibril formation is observed in the presence of theγ -Fe2O3/PHFBA nanoparticles. This is in contradiction to our previous manuscript, whichillustrated that the γ -Fe2O3 core nanoparticles do not affect the kinetics of the formation of theinsulin fibrils, and to other previous publications that describe acceleration of the fibrillationprocess by using various types of nanoparticles. These core–shell nanoparticles may thereforebe also useful for the inhibition of conformational changes of other amyloidogenic proteins thatlead to neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, mad cowand prion diseases.
1. Introduction
Human diseases characterized by insoluble extracellulardeposits of proteins have been recognized for almost twocenturies [1]. Amyloidoses have traditionally been defined asdiseases in which normally soluble proteins accumulate in theextracellular space of various tissues as insoluble deposits ofca 10 nm diameter fibrils [2]. In contrast to the secondarystructure of the native proteins, which is dominated by α-helical and random coil conformations, these amyloid fibrilscommonly consist of polypeptide chains organized mainly intocross β-sheets [1–8]. The formation of amyloid aggregates
4 Author to whom any correspondence should be addressed.
in tissues is a pathological feature of many neurodegenerativediseases such as Alzheimer’s, Parkinson’s, Huntington’s, madcow and prion diseases. There are many examples of secretedcirculating proteins that can, under abnormal circumstances, beconverted in part to highly stable extracellular fibrils. Theseinclude immunoglobulins in primary systemic amyloidosis,multiple myeloma, amylin in the diabetic pancreas, and smallsoluble proteins of uncertain function such as amyloid β-peptide (Aβ) in Alzheimer’s disease [1]. However, themechanism for normal and soluble proteins to assemble intofibrillar and insoluble aggregates still remains unresolved. Ithas been suggested that unfolded amyloid protein can forma partly unfolded helix-containing intermediate, from whichfibril formation is accelerated. Amyloid fibrillation entails
the creation of soluble oligomers, which grow to higher orderaggregates such as protofibrils and fibrils [9, 10].
In this study, insulin was chosen as a model amyloidogenicprotein. The formation of insulin fibrils is a physical processin which non-native (or unfolded) insulin molecules interactwith each other to form structure aggregates. Amyloid fibrildeposits of insulin have been observed in patients with typeII diabetes as well as after insulin infusion and repeatedinjection [1, 3]. Insulin fibrils are known to deposit in arterialwalls and membrane surfaces. Insulin aggregation is a majorproblem in production, storage and delivery of the protein.
One of the main research directions for preventing theprogression of amyloid-related diseases is to find efficientinhibitors for the α-to-β and random-to-β structural transitionsthrough stabilization of the normal state. Fluorinatedcompounds such as trifluoroethanol and hexafluoroisopropanolhave been reported to induce α-helical conformation in fibril-forming peptides [11, 12]. This behavior of the fluorinatedalcohols is interpreted to be the result of alterations of thehydration shell of the amyloidic sequence, due to the stronghydrophobic character of these fluorocompounds.
Recent literature concerning novel biomaterials indicatesan increasing interest in developing nanoparticles to detect,prevent and treat protein-misfolding diseases [13–16]. Theirpotential to influence protein fibrillation is a functionof both the nanoparticle surface interfacial properties,including charge, and its enormous surface area/volume.Various nanoparticles, such as copolymer particles of N-isopropylacrylamide/N-tert-butylacrylamide, cerium oxide,quantum dots, carbon nanotubes and titanium oxide, havebeen reported to promote protein assembly into amyloid fibrilsin vitro, by assisting the nucleation process [17, 18]. Veryrecently Linse et al reported on the inhibition of the amyloidβ fibril formation by modified nanoparticles [19].
Based on the above results with fluoroalcohols, Brezesin-ski et al recently demonstrated in preliminary studies that fluo-rinated nanoparticles (a complex of polyampholytes and do-decanoic and perfluorododecanoic acid) induce α-helix-richstructure in B18 peptide (a short peptide sequence with astrong tendency to self assemble and form amyloid fibrils).Their alkylated analogs, however, do not have this effect, andmay even enhance the rate of fibril growth [12]. Fluorinatednanoparticles are therefore proposed as potential candidates forthe inhibition and reversal of conformational changes of pro-teins that lead to amyloid fibril formation [12, 20].
Iron oxide magnetic nanoparticles are considered to benon-toxic and biodegradable.
For example, iron oxide nanoparticles such as Feridex(SPIO; Feridex-USA; Endorem-Europe) have already beenFDA-approved as magnetic resonance imaging (MRI) contrastagents for cells of the reticulo-endothelial system [21]. The useof iron oxide magnetic nanoparticles for various biomedicalapplications e.g., hyperthermia, diagnostic, cell-labeling andsorting, DNA separation, MRI contrast agents and drugdelivery has already been demonstrated [22–37].
The present paper describes a method to prepare newfluorinated γ -Fe2O3 core–shell magnetic nanoparticles, whichinduce a significant direct slow transition from α-helix to β-sheets during insulin fibril formation. It should be noted,
however, that our previous manuscript illustrated that theγ -Fe2O3 core nanoparticles do not affect the lag time andhence the kinetics of the formation of the insulin fibrils [38].
These core–shell nanoparticles were prepared by emulsionpolymerization of the fluorinated monomer HFBA in thepresence of γ -Fe2O3 nanoparticles [32, 33]. Human insulinamyloid fibrils were formed by incubating the monomericinsulin dissolved in an aqueous continuous phase at pH1.6 and 65 ◦C. The kinetics of the insulin fibrillationprocess in the absence and the presence of differentconcentrations of the γ -Fe2O3/PHFBA nanoparticles wereelucidated. In contrast to the previous work, which indicatedthat various nanoparticles may be used as catalysts for theprotein fibrillation process [17, 18], the γ -Fe2O3/PHFBAnanoparticles significantly delay the lag time and hence thekinetics of the nucleation and oligomer formation prior to theformation of the fibrils.
2. Materials and methods
2.1. Materials
The following analytical-grade chemicals were purchased fromcommercial sources and used without further purification:ferrous chloride tetrahydrate, hydrochloric acid (1 M),sodium hydroxide (1 M standard solution), sodium chloride,sodium nitrite, gelatin from porcine skin, human insulin,potassium persulfate (PPS) and sodium dodecylsulfate (SDS)from Sigma; 2,2,3,3,4,4,4-heptafluorobutyl acrylate fromFluorochem (UK); water was purified by passing deionizedwater through an Elgastat Spectrum reverse osmosis system(Elga, High Wycombe, UK).
2.2. Synthesis of the γ -Fe2O3 nanoparticles
γ -Fe2O3 magnetic nanoparticles of 15.0 ± 2.1 nm diameterwere prepared according to previous publications [32, 33].Briefly, 240 mg of gelatin was dissolved in 80 ml of waterat 60 ◦C. Then, 160 μl of Fe2+ solution (10 mmol in 5 ml0.1N HCl) and 57.6 μl of sodium nitrite solution (7.27 mmolin 5 ml H2O) were added to the shaken gelatin solution.For nucleation, titration with sodium hydroxide (1 M) untilpH of 9.5 was performed. This procedure was repeatedsuccessively four more times. The reaction mixture was thenshaken at 60 ◦C for an additional 1 h. The formed γ -Fe2O3
magnetic nanoparticles were then washed from non-magneticwaste with water by the high gradient magnetic field (HGMF)technique [32].
2.3. Synthesis of the γ -Fe2O3/PHFBA core–shellnanoparticles
γ -Fe2O3/PHFBA nanoparticles were formed by emulsionpolymerization of HFBA in the presence of the γ -Fe2O3
nanoparticles. In a typical experiment, 0.1 g SDS and 7 mgPPS were added to 10 ml of nitrogen bubbled γ -Fe2O3
nanoparticle water dispersion (4 mg ml−1). The mixturewas shaken at room temperature to dissolve the solids,giving concentrations of 1 and 0.07% (w/v) SDS and PPS,
2
Nanotechnology 20 (2009) 225106 H Skaat et al
respectively. Then, 0.25 ml HFBA (2.5% v/v) was added.The mixture was then shaken at 73 ◦C for 12 h. Theformed magnetic γ -Fe2O3/PHFBA core–shell nanoparticleswere washed from non-magnetic waste by using the HGMFtechnique with deionized water. Core–shell nanoparticles ofdifferent shell content were formed similarly by changing theHFBA concentration.
2.4. Synthesis of PHFBA
0.1 g SDS and 7 mg PPS were added to 10 ml of nitrogenbubbled deionized water. The mixture was shaken at roomtemperature to dissolve the solids, giving concentrations of 1and 0.07% (w/v) SDS and PPS, respectively. Then, 0.25 mlHFBA (2.5% v/v) was added. The mixture was then shakenat 73 ◦C for 12 h. Excess SDS was then removed from theaqueous dispersion by extensive dialysis against water. DriedPHFBA was then obtained by lyophilization.
2.5. Preparation of the human insulin amyloid fibrils in theabsence and presence of the γ -Fe2O3/PHFBA nanoparticles
Several tubes, each containing 2 mg of human insulin dissolvedin 1 ml of 0.025 M HCl aqueous solution containing 0.1 MNaCl (pH 1.6), were incubated at room temperature. Forinitiating the insulin fibrillation process, the temperature ofthe insulin solutions was quickly raised to 65 ◦C. Fibrillationkinetics were measured by decreasing the temperature ofeach chosen tube to room temperature at each time interval.All the insulin samples were freshly prepared immediatelyprior to each experiment in order to minimize the possibleformation of fibril nuclei in the solution, which would affectthe kinetics of the fibril formation. The concentration ofthe dissolved monomer and other oligomers was obtainedfrom the calibration absorbance curve at 280 nm versus aseries of known insulin concentrations. The kinetics ofthe disappearance of monomeric insulin were elucidated bymeasuring the absorbance at 280 nm after removal of theinsulin fibrils by centrifugation. The kinetics of the insulinfibril formation were obtained using the absorbance at 600 nm.
A similar process to that described above was alsoperformed in the presence of different concentrations of theγ -Fe2O3/PHFBA nanoparticles. Briefly, different volumes,7.5–70 μl, (0.03–0.28 mg, or 1.5–14.0 wt% from the insulin)of the γ -Fe2O3 nanoparticles aqueous dispersion (4 mg ml−1)were added to 1 ml aqueous solution at pH 1.6 containing 2 mghuman insulin, as described above. The formation of the fibrilswas then initiated by quickly raising the temperature of theaqueous mixture from room temperature to 65 ◦C for differenttime intervals.
2.6. Characterization
F analysis was performed by the Microanalysis Lab., Instituteof Chemistry, The Hebrew University of Jerusalem, Jerusalem.The reported values are an average of measurements performedon at least three samples of each tested sample, and have amaximum error of about 2%.
Figure 1. Low-resolution TEM and HRTEM images of the γ -Fe2O3
nanoparticles ((A) and (C), respectively) and the γ -Fe2O3/PHFBAcore–shell nanoparticles ((B) and (D), respectively).
Fourier transform infrared (FTIR) analysis was performedwith a Bomem FTIR spectrophotometer, model MB100,Hartman and Braun. The analysis was performed with 13 mmKBr pellets that contained 2 mg of the examined particles and198 mg KBr. The pellets were scanned over 200 scans at a4 cm−1 resolution.
Low-resolution transmission electron microscopy (TEM)pictures were obtained with a JEOL-JEM 100SX electronmicroscope with 80–100 kV accelerating voltage. High-resolution TEM (HRTEM) images were obtained by employ-ing a JEOL-3010 device with 300 kV accelerating voltage.Samples for TEM and HRTEM were prepared by placing adrop of a diluted sample on a 400-mesh carbon-coated coppergrid. The dry particles’ average size and distribution were de-termined by measuring the diameter of more than 100 particleswith the image analysis software AnalySIS Auto (Soft ImagingSystem GmbH, Germany).
The hydrodynamic diameter and size distribution of thenanoparticles dispersed in aqueous phase were measuredusing a submicron particle analyzer, model N4 Plus, CoulterElectronics, England.
Magnetic measurements were performed at room tempera-ture, using an Oxford Instrument vibrating sample magnetome-ter.
Electrokinetic properties (ζ -potential) as a function ofpH were determined by Zetasizer (Zetasizer 2000, MalvernInstruments, UK).
Circular dichroism (CD) spectra were measured on a JascoJ710 spectropolarimeter at 22 ◦C over a wavelength range 195–260 nm. All measurements in solution were recorded in a0.1 cm path length cell. Each spectrum is an average of fourmeasurements.
3. Results and discussion
Figure 1 presents low-resolution TEM and HRTEM imagesof the γ -Fe2O3 nanoparticles (A and C, respectively),
3
Nanotechnology 20 (2009) 225106 H Skaat et al
Figure 2. Hydrodynamic diameter of the γ -Fe2O3 nanoparticles (A)and the γ -Fe2O3/PHFBA core–shell nanoparticles (B).
and the γ -Fe2O3/PHFBA core–shell nanoparticles (B andD, respectively). The TEM pictures of the γ -Fe2O3
nanoparticles and of the core–shell nanoparticles (figures 1(A)and (B)) demonstrate the monodispersity and stability of thesenanoparticles against agglomeration. Careful measurementsindicate that both nanoparticles have a similar average diameterof 15.0 ± 1.4 nm. The similarity in the diameters mayindicate that the resolution and contrast of low-resolutionTEM is not sensitive enough to illustrate the PHFBA shell.HRTEM pictures of the γ -Fe2O3 and the γ -Fe2O3/PHFBAnanoparticles (figures 1(C) and (D), respectively) demonstratethe perfect arrangement of the atomic layers of the ironoxide. The HRTEM image of the γ -Fe2O3/PHFBA particles(figure 3(D)) demonstrates also the 3–4 nm amorphousPHFBA shell on the surface of the crystalline γ -Fe2O3 corenanoparticles.
Contrary to the TEM images, the size of the γ -Fe2O3
nanoparticles dispersed in the aqueous continuous phase,as determined by the light scattering technique, is 83 ±26 nm (figure 2(A)). The difference in the diameter of thenanoparticles measured by TEM and light scattering is dueto the fact that the first method measures the dry diameterof the nanoparticles while the second one measures thehydrodynamic diameter, which takes into account the surfaceadsorbed solvent molecules and the swelling of the particlesby the solvent molecules. It is rather interesting to find that,according to the light scattering technique, the hydrodynamicsize of the γ -Fe2O3/PHFBA nanoparticles is 46 ± 11 nm(figure 2(B)), which is significantly smaller than that of the
Figure 3. FTIR spectra of the PHFBA (A), the γ -Fe2O3
nanoparticles (B) and the γ -Fe2O3/PHFBA core–shellnanoparticles (C).
γ -Fe2O3 nanoparticles (83 ± 26 nm). This could be explainedby the presence of the hydrophobic PHFBA shell layer, whichsignificantly decreases adsorption of the water molecules onthe surface of the γ -Fe2O3 core nanoparticles.
FTIR spectra of the PHFBA (A), the γ -Fe2O3 nanopar-ticles (B) and the γ -Fe2O3/PHFBA core–shell nanoparticles(C) are shown in figure 3. The IR spectrum of the PHFBA(figure 3(A)) indicates stretching vibrations between 1200and 1300 cm−1, corresponding to the highly polarized –CF2
groups, and stretching vibrations from 1358 to 1410 cm−1, cor-responding to the –CF3 groups. The strong absorbance peakat 1756 cm−1 corresponds to the stretching vibration of C=Obonds, and the medium absorption peaks around 2980 cm−1
are characteristic of the stretching of C–H bonds. The IRspectrum of the γ -Fe2O3 nanoparticles (figure 3(B)) showsa typical absorbance peak at 610 cm−l, corresponding to thevibration band of Fe–O, a peak at 1650 cm−1 correspondsto the gelatin N–H stretching band and a very broad peak at3406 cm−1 belonging to the vibrational band of O–H. The lat-ter peak is probably due to the presence of surface hydroxylgroups (Fe–OH) of the iron oxide and carboxyl groups of thegelatin. The IR spectrum of the γ -Fe2O3/PHFBA (figure 3(C))nanoparticles is composed of peaks belonging both to thePHFBA (1358, 1756 cm−1, etc) and to the γ -Fe2O3 nanoparti-cles (610, 3406 cm−1), thereby demonstrating the presence ofthe PHFBA in combination with the γ -Fe2O3 nanoparticles.
Magnetization curves at room temperature of the γ -Fe2O3
(A) and the γ -Fe2O3/PHFBA (B) nanoparticles are shownin figure 4. It is readily observed that the M(H ) curve ofthe γ -Fe2O3 nanoparticles has no hysteresis loop. Zero fieldcooled-field cooled (ZFC-FC) measurements of the γ -Fe2O3
nanoparticles exhibit blocking temperature (TB) at ca 275 K,
4
Nanotechnology 20 (2009) 225106 H Skaat et al
Figure 4. Magnetization curves of the γ -Fe2O3 nanoparticles (A)and the γ -Fe2O3/PHFBA core–shell nanoparticles (B).
Figure 5. ζ -potential of the γ -Fe2O3 nanoparticles (A) and theγ -Fe2O3/PHFBA core–shell nanoparticles (B) as a function of pH.
which indicates a superparamagnetic behavior [39]. Figure 4also illustrates that both M(H ) plots reach saturation atfields of around 2000 Oe. The saturation magnetizationsof the γ -Fe2O3 nanoparticles (figure 4(A)) and the core–shell nanoparticles (figure 4(B)) are 54 and 20 emu g−1,respectively. The difference in the magnetization is probablyattributed to the coating of the core nanoparticles with the non-magnetic PHFBA shell.
To learn about the stability of the γ -Fe2O3 andthe γ -Fe2O3/PHFBA nanoparticles in aqueous medium, ζ -potential measurements can be employed as an indirectindicator of their surface charge. Figure 5 illustrates the ζ -potential curves of the γ -Fe2O3 (A) and the γ -Fe2O3/PHFBA(B) nanoparticles, as a function of pH. The titration curveof the γ -Fe2O3 nanoparticles, obtained by adding HCl to anaqueous dispersion of the γ -Fe2O3 nanoparticles (figure 5(A)),shows an increase of the ζ -potential from −28 to 16 mV whilechanging the pH from 11.0 to 2.0. This change in the surfacepotential can be attributed to the surface hydroxyl groups (Fe–OH) of the γ -Fe2O3 nanoparticles. In a basic environmentthe surface shows negative charge potential because of theformation of Fe–O−, whereas in an acidic environment apositive surface charge is expected due to Fe–OH+
2 formation.
Figure 6. Kinetics of the insulin fibril formation in aqueouscontinuous phase at pH 1.6 and 65 ◦C in the absence (A) and thepresence (B) of 6.0% (w/winsulin) of the γ -Fe2O3/PHFBAnanoparticles. Kinetics were measured using absorption at 600 and280 nm. The absorbance measured at 280 nm was converted toconcentration by using calibration absorbance curve at 280 nm, asdescribed in the experimental part.
Figure 7. Kinetics of the insulin fibril formation (using absorbance at600 nm) at pH 1.6 and 65 ◦C in the presence of 1.5 (A), 3.0 (B) and6.0 (C) % (w/winsulin) of the γ -Fe2O3/PHFBA nanoparticles.
The measured isoelectric point of the γ -Fe2O3 nanoparticlesis at about pH 6.4. Figure 5(B) illustrates that the coatingof the core γ -Fe2O3 nanoparticles with the PHFBA shifts theisoelectric point from about pH 6.4 to about pH 3.8. Theζ -potential behavior of the γ -Fe2O3/PHFBA nanoparticles asfunction of pH is not yet understood and will be investigatedfully in future studies.
The influence of HFBA concentration on the % coatingof PHFBA on the surface of the γ -Fe2O3 nanoparticles is
5
Nanotechnology 20 (2009) 225106 H Skaat et al
Figure 8. CD spectra of the formed insulin fibrils in the absence (A)and the presence (B) of 6.0% (w/winsulin) of the γ -Fe2O3/PHFBAnanoparticles at different times during the fibrillation process.
Table 1. Influence of HFBA concentration on the wt% of thePHFBA shell on the γ -Fe2O3 nanoparticles. (Note: γ -Fe2O3/PHFBAcore–shell nanoparticles were prepared according to the experimentalsection.)
a % PHFBA = (%F × 100)/52.3, where %F isobtained from the elemental F analysis; 52.3 is the %Fin pure PHFBA.
presented in table 1. As expected, by raising the HFBAconcentration, the % coating of the PHFBA shell in the core–shell nanoparticles is increased. For example, increasing theinitial HFBA concentration from 0.5% to 1% and 2% raisesthe wt% of the PHFBA of the γ -Fe2O3/PHFBA nanoparticlesfrom 15.7% to 24.3% and 40.5%, respectively.
The kinetics of the insulin fibrils formation in the aqueouscontinuous phase at pH 1.6 and 65 ◦C in the absence (A) andthe presence (B) of 6.0% (w/winsulin) of the γ -Fe2O3/PHFBAnanoparticles are shown in figure 6. In the absence of thenanoparticles (figure 6(A)), the main growth of the insulinfibrils (as measured at 600 nm) occurred approximately 3.0 hafter initiating the fibrillation process and was completedafter approximately 5.0 h. It is important to note that thisbehavior was also observed in the presence of 6.0% (w/winsulin)of the uncoated γ -Fe2O3 nanoparticles [38]. On the other
Figure 9. Secondary structure of the formed insulin fibrils in theabsence (A) and the presence (B) of 6.0% (w/winsulin) of theγ -Fe2O3/PHFBA nanoparticles at different times during thefibrillation process.
hand, the kinetics of the insulin fibrils growth in the presenceof 6.0% (w/winsulin) of the γ -Fe2O3/PHFBA nanoparticles(figure 6(B)) was significantly delayed, and was initiated after22.0 h only. As a consequence, the insulin fibrillation processin the presence of the γ -Fe2O3/polyHFBA nanoparticles wasinitiated much later. The presence of the perfluorinated carbongroups (–CF2 and –CF3) on the surface of the γ -Fe2O3
nanoparticles probably stabilizes the α-helix structure byhydrophobic interactions, slowing down the α-helix to β-sheettransition during the insulin fibril formation [11, 12]. Theloss of ordered water coating around the peptide, indirectlyinduced by structuring of water around the perfluorinatedcarbon groups and by the presence of perfluorinated carbongroups near the peptide, causes the –NH and –C=O groupsof the backbone to interact on short distances with each other,favoring the α-helix. Short-range interactions between nearbyamino-acid residues stabilize the α-helix structure in contrastto long-range interactions stabilizing the β-sheet structure [11].Figure 6 also demonstrates an opposite correlation between theabsorbance measured at 600 nm, which relates to the insulinfibril formation, and that measured at 280 nm, which relatesto the decrease in the insulin monomeric concentration. Itis obvious that as the absorbance of the fibrils increases intime the concentration of the monomeric insulin decreases.For example, in the absence of nanoparticles, after 5.0 hthe absorbance of the formed fibrils is around 1.2, while theconcentration of the insulin is almost zero. This indicates
6
Nanotechnology 20 (2009) 225106 H Skaat et al
Figure 10. TEM pictures showing the effect of the γ -Fe2O3/PHFBA nanoparticles on the insulin fibril formation. In the absence ((A1)and(A2)) and the presence (B) of: 6.0% (w/winsulin) of the non-coated γ -Fe2O3 nanoparticles, 9.0 h after initiation of the fibrillation process, or6.0% (w/winsulin) of the γ -Fe2O3/PHFBA nanoparticles, 9.0 h (C) and 50.0 h ((D1) and (D2)) after the initiation of the fibrillation process.(A2) and (D2) represent higher magnification of the circled regions shown in (A1) and (D1), respectively.
that after 5.0 h all the monomeric insulin (2 mg) had beenconverted to insulin fibrils. This correlation was also observedin the presence of 6.0% (w/winsulin) of the γ -Fe2O3/PHFBAnanoparticles, but only after 92.0 h.
Typical sigmoidal curves in figure 7 present the kineticsof the formation of insulin fibrils at pH 1.6 and 65 ◦C in thepresence of increasing concentrations of the γ -Fe2O3/PHFBAnanoparticles ((A)–(C)). As expected, with increased loadingof the γ -Fe2O3/PHFBA nanoparticles, the insulin fibrillationprocess becomes much slower. For example, instead ofinitiating the fibrillation process in the absence of the core–shell nanoparticles after 3.0 h, as described above, increasingthe concentration of the nanoparticles to 1.5% (A), 3.0% (B),and 6.0% (C) (w/winsulin) leads to fibril formation only after9.0, 15.0, and 22.0 h, respectively. It should be noted thatat concentrations higher than 6.0% (w/winsulin), no furthersignificant initiation effect was observed.
Next, CD measurements were performed to determine thesecondary structure changes during the insulin fibril formationin the absence and the presence of the core–shell nanoparticles.Figure 8 presents CD spectra of the formed insulin fibrils inthe absence (A) and in the presence (B) of 6.0% (w/winsulin)of the γ -Fe2O3/PHFBA nanoparticles at different times duringthe fibrillation process. In the absence of the nanoparticles(figure 8(A)) before the initiation of the fibrillation process, theCD spectrum of the insulin aqueous solution at pH 1.6 shows
a band at 195 nm and a double minimum at 208 and 222 nm,indicative of a typical spectrum of a native protein dominatedby α-helix conformation. By heating the insulin solution to65 ◦C for 5.0 h, the CD spectrum of the insulin displays a lossof intensity of the 195 and 222 nm bands accompanied by aminimum ellipticity at 216 nm, which is typical of the presenceof extensive β-sheet structures. Similar CD spectra were alsoobserved in the presence of 6.0% (w/winsulin) of the non-coatedγ -Fe2O3 nanoparticles. In the presence of 6.0% (w/winsulin)of the γ -Fe2O3/PHFBA nanoparticles (figure 8(B)), the α-helical structure remains almost constant for up to 22.0 h ofheating. Only beyond that time does the transformation to β-sheet structure start to occur. For a quantitative determinationof the secondary structure, the content of structural motifswas calculated using the SELCON software, as shown infigure 9. This figure presents the % decrease of α-helix andthe % increase of β-sheets content during the advance of theinsulin fibrillation process. In the absence of the core–shellnanoparticles (figure 9(A)), before the fibrillation and 5.0 hafter, the % α-helix decreased from 55.0% to 7.9% and the% β-sheets increased from 7.9% to 42.6%. In the presenceof 6.0% (w/winsulin) of the γ -Fe2O3/PHFBA nanoparticles(figure 9(B)), before the fibrillation and only 92.0 h after,the % α-helix decreased from 55.0% to 7.4% and the % β-sheets increased from 7.9% to 37.6%. The SELCON softwaregave percentages of secondary structure that agreed with theobservation of the CD spectra. However, it is important to
7
Nanotechnology 20 (2009) 225106 H Skaat et al
note that protein aggregates scatter light, which may reducethe CD signal. Therefore, the quantitative determination of thesecondary structure motifs at high incubation times when thefibrils have already been formed (ca 5.0 or 50.0 h after theinitiation of the fibrillation process in the absence or in thepresence of the γ -Fe2O3/PHFBA nanoparticles, respectively)may be considered as a good estimation only.
TEM photomicrographs in figure 10 illustrate theinhibition of the insulin fibril formation in the presence of6.0% (w/winsulin) of the γ -Fe2O3/PHFBA nanoparticles. Inthe absence (figures 10(A1), (A2)) or presence (figure 10(B))of 6.0% (w/winsulin) of the non-coated γ -Fe2O3 nanoparticles,the fibrils after 9.0 h of the fibrillation process had lengths ofup to several micrometers. Also, figure 10(B) reveals that thenon-coated γ -Fe2O3 nanoparticles appear to bind selectivelywith the axial external surface of the fibrils, with almost nofreely unassociated suspended nanoparticles. The obtainedmagnetic insulin amyloid fibril assemblies can then easily beremoved from the aqueous phase by using a simple magnet, aswe demonstrated previously [38]. On the other hand, wheninsulin was incubated in the presence of 6.0% (w/winsulin)of the γ -Fe2O3/PHFBA core–shell nanoparticles, a differentbehavior was observed. The TEM image taken 9.0 h afterthe initiation of the fibrillation process (figure 10(C)) revealsonly free nanoparticles in the background, which means thatthe fibrils were not yet formed. Also, figures 10((D1), (D2))shows that 50.0 h after the initiation of the fibrillation processthe obtained fibrils were still much shorter than those observedin the presence of 6.0% (w/winsulin) of the non-coated γ -Fe2O3,and were not marked at all by the core–shell nanoparticles.
4. Conclusions
This paper describes the synthesis and characterization ofnovel magnetic γ -Fe2O3 /PHFBA core–shell nanoparticles, byemulsion polymerization of the fluorinated monomer HFBA inthe presence of γ -Fe2O3 core magnetic nanoparticles. Thesestudies demonstrate the significant inhibition of the insulinfibrillation process in the presence of these nanoparticles.In future studies we plan to extend the present work toother amyloidogenic proteins, e.g., prions, amyloid β , β2-microglobulin, etc.
It is not yet certain whether these core–shell nanoparticlesare suitable for in vivo studies as therapeutics, but there isno doubt that these nanoparticles can be used as an efficientmodel to study and understand the kinetics of the amyloid fibrilformation. The clinical use of these core–shell nanoparticleswill be investigated in the future. However, a main advantagenow is the possibility for in vivo tracing of these nanoparticlesby MRI.
Acknowledgments
These studies were partially supported by a BSF (Israel-USABinational Science Foundation) grant and by a Minerva Grant(Microscale and Nanoscale Particles and Films).
References
[1] Selkoe D J 2003 Nature 426 900[2] Sunde M and Blake C C 1998 Q. Rev. Biophys. 31 1[3] Bouchard M, Zurdo J, Nettleton E J, Dobson C M and
Robinson C V 2000 Protein Sci. 9 1960[4] Klunk W E, Debnath M L, Koros A M C and
Pettegrew J W 1998 Life Sci. 63 1807[5] Klunk W E, Wang Y M, Huang G F, Debnath M L,
Holt D P and Mathis C A 2001 Life Sci. 69 1471[6] Sethuraman A, Vedantham G, Imoto T, Przybycien T and
Belfort G 2004 Proteins-Struct. Funct. Bioinform. 56 669[7] Sethuraman A and Belfort G 2005 Biophys. J. 88 1322[8] Link C D, Johnson C J, Fonte V, Paupard M C, Hall D H,
Styren S, Mathis C A and Klunk W E 2001 Neurobiol. Aging22 217
[9] Walsh D M, Lomakin A, Benedek G B, Condron M M andTeplow D B 1997 J. Biol. Chem. 272 22364
[10] Harper J D, Wong S S, Lieber C M and Lansbury P T 1997Chem. Biol. 4 119
[11] Vieira E P, Hermel H and Mohwald H 2003 Biochim. Biophys.Acta 1645 6
[12] Rocha S, Thunemann A F, Pereira M C, Coelho M A N,Mohwald H and Brezesinski G 2005 ChemBiolChem 6 280
[13] Kogan M J, Bastus N G, Amigo R, Grillo-Bosch D, Araya E,Turiel A, Labarta A, Giralt E and Puntes V F 2006 NanoLett. 6 110
[14] Cox D L, Lashuel H, Lee K Y C and Singh R R P 2005 MRSBull. 30 452
[15] Cui Z R, Lockman P R, Atwood C S, Hsu C H, Gupte A,Allen D D and Mumper R J 2005 Eur. J. Pharm. Biopharm.59 263
[16] Ji X J, Naistat D, Li C Q, Orbulescu J and Leblanc R M 2006Colloids Surf. 50 104
[17] Linse S, Cabaleiro-Lago C, Xue W F, Lynch I, Lindman S,Thulin E, Radford S E and Dawson K A 2007 Proc. NatlAcad. Sci. USA 104 8691
[18] Wu W H, Sun X, Yu Y P, Hu J, Zhao L, Liu Q, Zhao Y F andLi Y M 2008 Biochem. Biophys. Res. Commun. 373 315
[19] Cabaleiro-Lago C, Quinlan-Pluck F, Lynch I, Lindman S,Minogue A M, Thulin E, Walsh D M, Dawson K andLinse S 2008 J. Am. Chem. Soc. 130 15437
[20] Rocha S, Thunemann A F, Pereira M C, Coelho M A N,Mohwald H and Brezesinski G 2008 Biophys. Chem. 137 35
[21] De Vries I J M et al 2005 Nat. Biotechnol. 23 1407[22] Kim D K, Zhang Y, Kehr J, Klason T, Bjelke B and
Muhammed M 2001 J. Magn. Magn. Mater. 225 256[23] Hergt R, Hiergeist R, Hilger I, Kaiser W A, Lapatnikov Y,
Margel S and Richter U 2004 J. Magn. Magn. Mater.270 345
[24] Pardoe H, Clark P R, St Pierre T G, Moroz P andJones S K 2003 Magn. Reson. Imaging 21 483
[25] Chemla Y R, Grossman H L, Poon Y, McDermott R,Stevens R, Alper M D and Clarke J 2000 Proc. Natl Acad.Sci. USA 97 14268
[26] Uhlen M 1989 Nature 340 733[27] Debuire B, Chabli A and Fast Frenoy N 1993 Clin. Chem. 39
1682[28] Yoza B, Matsumoto M and Matsunaga T 2002 J. Biotechnol.
94 217[29] Scherer F, Anton M, Schillinger U, Henkel J, Bergemann C,
Kruger A, Gansbacher B and Plank C 2002 Gene Ther.9 102
[30] Bergemann C, Muller-Schulte D, Oster J, Brassard L andLubbe A S 1999 J. Magn. Magn. Mater. 194 45
[31] Rudge S R, Kurtz T L, Vessely C R, Catterall L G andWilliamson D L 2000 Biomaterials 21 1411
