Effect of Functionalized Magnetic MnFe2O4 Nanoparticles on

Sep 23, 2014 - Gajalakshmi Sekar , Ida Florance , A. Sivakumar , Amitava Mukherjee , Natarajan Chandrasekaran. International Journal of Biological ...
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Effect of Functionalized Magnetic MnFe2O4 Nanoparticles on Fibrillation of Human Serum Albumin Shubhatam Sen,† Suraj Konar,§ Amita Pathak,§ Swagata Dasgupta,*,§ and Sunando DasGupta*,‡ †

Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur 721302, India Department of Chemical Engineering, Indian Institute of Technology Kharagpur, Kharagpur 721302, India § Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India ‡

S Supporting Information *

ABSTRACT: Pathogenesis of amyloid-related diseases is related to nonnative folding of proteins with the formation of insoluble deposits in the extracellular space of various tissues. Having the unique properties of small size, large surface area, biodegradability, and relative nontoxicity, magnetic nanoparticles have drawn a lot of attention in biomedical applications. Herein, we demonstrate the effect of bare and differently functionalized magnetic MnFe2O4 nanoparticles on fibrillation of human serum albumin in vitro. The process has been monitored using Thioflavin T fluorescence, Congo red binding assay, circular dichroism, fluorescence microscopy, and transmission electron microscopy. From our experimental results, amine functionalized MnFe2O4 nanoparticles are found to inhibit formation of fibrils more effectively than bare ones, while carboxylated nanoparticles do not have a significant effect on fibrillation. This study has explored the prospects of using specific magnetic nanoparticles with appropriate modification to control self-assembly of proteins and may act as a precursor in therapeutic applications.

1. INTRODUCTION

the presence of metal ions, sugars, surfactants, etc. for promoting, inhibiting, or disintegrating purposes.14−17 Literature indicates use of nanosized materials in various biomedical applications.18,19 Owing to their small size, nanoparticles pass through the blood-brain barrier freely.20 Nanoparticles also have enhanced surface to volume ratio which can be utilized to modify their surface properties physically or chemically thus controlling the interaction of protein with nanoparticles. Linse et al. have shown that nanoparticles such as copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes significantly enhance the rate of formation of fibrils.21 While gold nanoparticles have been reported to induce formation of protein aggregates,22 photothermal ablation of amyloid aggregates by gold nanoparticles has also been reported.23 Rocha et al. have reported that fibrillation of amyloid-β (Aβ) peptide can be significantly prevented by fluorinated nanoparticles.24 Studies also indicate the effect of DHLA-capped quantum dots and Au nanoparticles on the fibrillation pathway of HSA under varying experimental conditions.25,26 Magnetic nanoparticles (MNP) are finding increased use in biorelated applications due to their magnetic properties, biocompatibility, and relative nontoxicity. The application of

Aggregation of protein resulting into amyloid fibrils is the central reason behind many human diseases like Parkinson’s, Huntington’s, and prion diseases and Alzheimer’s disease.1−3 In these types of diseases, normally soluble proteins get transformed into toxic amyloid fibrils with high cross β-sheet content, the perpendicular arrangement of strands to fibrillar axis.4 The deposited fibrils in extracellular spaces of various tissues cause cellular damages.5 Human serum albumin (HSA), the most abundant plasma protein, may be considered as an amyloidogenic model protein due to its tendency to aggregate in vitro.6,7 HSA is a natively α-helical (>60%) globular protein consisting of 585 amino acid residues. HSA consists of three domains, each with two subdomains and 17 disulfide bridges.8,9 HSA plays a crucial role in the transportation of fatty acids, metal ions, and physiologically important compounds. Lacking any predisposition to form amyloid fibrils, HSA requires necessary solution conditions, such as low pH, high temperature, presence of chemical denaturant, metal ions, etc. that will favor partial destabilization of HSA molecules to form amyloidlike fibrils.10 In the literature, various factors have been reported to affect HSA aggregation. The influence of pH, ionic strength, and electrostatic interactions on the fibrillation process of HSA has previously been reported.11,12 The effect of solvation on the conformational change of HSA in aqueous ethanol solvent has been observed.13 Aggregation of HSA has also been studied in © 2014 American Chemical Society

Received: August 5, 2014 Revised: September 18, 2014 Published: September 23, 2014 11667

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MNPs in magnetic resonance imaging (MRI),27 biosensing,28 drug delivery,29 bioseparation,30 catalysis,31 hyperthermia,32 etc. have already been reported. However, few studies have been performed to study the relationship of MNPs and protein aggregation. Skaat et al. have reported the use of fluorescent MNPs in the detection of amyloid-β (Aβ) plaques and their removal by magnetic field.33 They have also shown that differently functionalized MNPs affect fibril formation of insulin and Aβ40 either by inhibiting or accelerating the process.34,35 Bellova et al. have shown that magnetic Fe3O4 nanoparticles inhibit formation of amyloid fibrils in the case of hen lysozyme.36 The use of albumin-modified magnetic fluid in destruction of insulin amyloid aggregates has also been reported by Siposova et al.37 Thus, we believe specific MNPs with proper modifications can have a role in affecting the protein aggregation process and may open new routes of controlling self-assembly of proteins leading to possible use in nanomedicine. To the best of our knowledge no study has been done to probe the effect of magnetic nanoparticles on the aggregation of HSA. The aim of the present study is to investigate the effect of magnetic MnFe2O4 nanoparticles with and without functionalization on the aggregation process of HSA. We have chosen superparamagnetic MnFe2O4 nanoparticles because it has very low magnetocrystalline anisotropy and higher magnetic moment compared to other ferrites such as Fe3O4, Fe2O3 and CoFe2O4 nanoparticles.38 Synthesis of MnFe2O4 nanoparticles and their functionalization with amine group and carboxyl group, respectively, are performed following reported procedures.39−41 The structure, morphology, size, and magnetic properties of the resultant magnetic nanoparticles (NPs) are characterized by X-ray diffraction (XRD), transmission electron microscope (TEM), dynamic light scattering (DLS), and superconducting quantum interference device (SQUID). The binding of APTES and PMIDA to the magnetic nanoparticles is confirmed by Fourier transform infrared (FTIR) spectroscopy. Next we have studied the potential of the synthesized nanoparticles to inhibit the fibrillation process of HSA. For this purpose, solutions of HSA with and without the NPs at pH 7.0 are incubated at 37 °C in the presence of 50% (v/v) ethanol for 24 h. The fibrillation process is monitored by the Thioflavin T-(ThT) binding fluorescence study, Congo red (CR)-based UV study, circular dichroism (CD) spectroscopy, fluorescence microscopy, and transmission electron microscopy.

addition of ammonia and then the resultant mixture was sonicated for 1 h to obtain the black precipitates of MnFe2O4. The MnFe2O4 precipitate was then separated through centrifugation, repeatedly washed with milli-Q water and finally with ethanol, and was eventually dried (at 80 °C, 12 h) in a hotair oven before being ground to fine powders. For surface functionalization with amine groups, 0.8 mL of APTES was slowly added into the sonicated−dispersion of 200 mg of MnFe2O4 powders in 1:1 (v/v) solution mixture of ethanol and water. The entire mixture was heated at 40 °C and vortexed for 2 h under inert argon gas atmosphere to separate out the amine functionalized-MnFe2O4 (i.e., MFN). The separated mass of MFN was repeatedly washed with milli-Q water and finally with ethanol and dried in a vacuum oven at 80 °C for 12 h. The MnFe2O4-surface was also functionalized with carboxyl groups by adding 2 mmol of PMIDA into the sonicated, aqueous dispersion of 200 mg of MF of pH = 10. The resulting solution was vortexed for another 12 h and the obtained solid of carboxyl functionalized-MnFe2O4 (i.e., MFC) was separated, washed with milli-Q water then with ethanol, and finally dried in a vacuum oven at 80 °C for 12 h. The phase analysis of synthesized nanoparticles (NPs) was carried out by X-ray diffraction (XRD) using Cu−Kα radiation over the 2θ range of 20−80° at a scan rate of 3 deg min−1 with an applied voltage of 40 kV using a Bruker AXS Diffractometer D8 powder XRD. To detect the presence of functional groups in MFN and MFC, Fourier transform infrared (FTIR) spectroscopy was carried out using a PerkinElmer Spectrum RX-II (model no. 73713) within the scan range 4000−400 cm−1. The morphology and particle size of the synthesized NPs were examined by transmission electron microscopy (TEM) using TECNAI G2 20S-TWIN (Japan) with an acceleration voltage of 200 kV. The size distribution histograms were prepared by analyzing TEM images using ImageJ software (version 1.33; National Institutes of Health) over a significant number of nanoparticles from several pictures.42 The hydrodynamic radius and surface charge potential (SCP) of the resultant samples were determined by a Malvern Nano ZS instrument (Germany). The magnetic measurements were carried out by superconducting quantum interface device (SQUID) using a Quantum Design, Ever Cool SQUID VSM DC magnetometer (USA) which was fitted to a superconducting coil that produced a magnetic field of ±5 T. 2.3. Fibril Formation. The stock solution of HSA was prepared by dissolving HSA in double distilled water, and its concentration was measured spectrophotometrically at 280 nm using a molar extinction coefficient of 35 219 M−1 cm−1.43 The inhibiting activity of the NPs was investigated by adding 50 μL aliquots of MF, MFN, and MFC in buffer (1 mg/mL) separately to the soluble HSA solution before inducing fibrillation by following standard procedure.16 Briefly, HSA (50 μM) at pH 7.0 (20 mM Tris-HCl buffer) was incubated with and without NPs in the presence of 50% (v/v) ethanol at 37 °C for 24 h. For each analysis, Tris-HCl buffer of pH 7.0 (20 mM) was used for dilution of samples. 2.4. ThT Fluorescence. Thioflavin T binding assay was performed by withdrawing aliquots, incubated at 37 °C for 24 h, from the different sets of HSA solutions with and without nanoparticles. After the addition of ThT, the samples were incubated for 5 min and fluorescence emission measured using a Horiba Jobin Yvon Fluoromax 4 spectrofluorimeter. To check ThT binding, aliquots, withdrawn from the sets of solutions were diluted using 20 mM Tris-HCl buffer of pH 7.0 to achieve

2. MATERIALS AND METHODS 2.1. Materials. All the chemicals used were of analytical grade and purchased from commercial sources without further purification. Human serum albumin (HSA), Thioflavin T (ThT), and Congo red (CR) were purchased from Sigma Chemical Co. (St. Louis) and used as received. (3Aminopropyl)triethoxysilane (APTES) and N(phosphonomethyl)iminodiacetic acid hydrate (PMIDA) were purchased from Sigma-Aldrich. Ferric chloride (FeCl3) was purchased from Merck. Manganese chloride (MnCl2) was purchased from Loba Chemie Pvt. Ltd., Mumbai, India. 2.2. Synthesis and Characterization of Magnetic Nanoparticles (MF, MFN, and MFC). Magnetic MnFe2O4 nanoparticles (MF) were synthesized by the chemical coprecipitation method under alkaline condition. In brief, 1 mmol of MnCl2 and 2 mmol of FeCl3 were mixed in 50 mL of milli-Q water under continuous stirring for 1 h to obtain a clear solution. The pH of the solution was maintained at 10 by slow 11668

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final protein and dye (ThT) concentrations of 2 μM and 10 μM, respectively. Samples were scanned in a quartz cell of 1 cm path length at an excitation wavelength of 450 nm with a scanning range of 470−600 nm. Slit widths (for both excitation and emission) and integration time were kept at 5 nm and 0.3 s, respectively. All spectra acquired were corrected with respect to corresponding blank spectra. No ThT fluorescence was observed in the control sets that comprised native HSA and nanoparticles separately. Each measurement was done in triplicate and the error bars represents standard deviation from the mean value. 2.5. Congo Red (CR) Binding Study. Difference in Congo red absorption due to binding with HSA fibrils in presence and absence of NPs was measured using a UV−vis-NIR spectrophotometer, Shimadzu-2450 in the scanning range of 400−650 nm. In the final working solutions, the protein and dye concentrations were kept at 4 μM and 10 μM, respectively, using 20 mM Tris-HCl buffer (pH 7.0). Each spectrum was corrected with respect to the corresponding blank. 2.6. Circular Dichroism (CD) Spectroscopy. Far-UV CD spectra were recorded on a JASCO-810 automatic recording spectrophotometer at room temperature. A quartz cuvette with a 0.1 cm path length was used. CD spectra were accumulated at a scan rate of 50 nm/min between a wavelength range of 190− 240 nm. The final concentration of protein for each solution was kept at 5 μM. The protein secondary structure content was determined using the online DICHROWEB server.44 Each measurement was done at least three times, and the error bars represent standard deviation from the mean value. Using CD, the change in free energy of folding (ΔG) of HSA in the presence of NPs as a function of temperature has been estimated using the standard method.45 αs =

(θs − θf ) (θn − θf )

ThT under the experimental conditions used herein as no ThT binding species were present. 2.8. Transmission Electron Microscopy (TEM). The sample solutions were diluted 10-fold and applied to carbon coated TEM grids. Samples were negatively stained with an aqueous solution of uranyl acetate [1% (w/v)], air-dried, and examined in a TECNAI G2 20S-TWIN transmission electron microscope operating at an accelerating voltage of 80 kV.

3. RESULTS AND DISCUSSION 3.1. Characterization of Nanoparticles. The XRD pattern (Figure S1 in the Supporting Information) of all three samples match with the standard JCPDS file (card no. 100319)46,47 and hence can be indexed to the cubic spinel structure of MnFe2O4. The average crystalline sizes, calculated using the Debye−Scherrer’s formula,48 of MF, MFN, and MFC are found to be 21, 30, and 28 nm, respectively. The FTIR spectra of all three samples (i.e., MF, MFN, and MFC), shown in Figure S2 in the Supporting Information, reveal a strong and sharp band around 580 cm−1, possibly contributed by Fe−O bond vibration49 arising from MnFe2O4. The bands around 1000 and 1119 cm−1 in the FTIR spectra of MFN can be assigned to the SiO−H and Si−O−Si linkages,50 possibly from APTES on the MnFe2O4 surface. The spectra also show peaks at around 2917 and 1635 cm−1, which may be, respectively, assigned to the asymmetric C−H stretching vibration from the propyl groups and NH2 bending vibrations from the free amine groups of APTES.50 For MFC sample, the bands centered around 1722 and 1050 cm−1 may be, respectively, attributed to the presence of carboxylic acid (−CO2H) and PO groups as a result of PMIDA41 incorporation on the MnFe2O4 surface. We have also examined FTIR spectra of the samples obtained after incubating MFC and MFN separately with 50% ethanol for 24 h followed by centrifugation and drying. No changes in the appearance of spectra from those obtained for MFN and MFC (Figure S2 in the Supporting Information) are found which confirms that 50% ethanol has no effect on the APTES and PMIDA which are used for the functionalization of the magnetic nanoparticles. The mean particle diameters of MF, MFN, and MFC from TEM studies are found to be around 34, 42, and 38 nm, respectively (Figure S3 in the Supporting Information). The average hydrodynamic diameters of MF, MFN, and MFC from dynamic light scattering (Figure S4 in the Supporting Information) studies are higher than their respective mean particle diameters from TEM, which may be attributed to the solvation of the particles in aqueous medium. However, their trends in size remain consistent from both the studies. The maximum magnetization versus an applied magnetic field of 5 T, and a temperature of 300 K is found to be 62, 27, and 46 emu/g for MF, MFN, and MFC (Figure S5 in the Supporting Information), respectively. The magnetization curves for all three samples do not show any hysteresis, indicating their superparamagnetic nature.49 The reduction of maximum magnetization in the case of functionalized MF (i.e., MFN and MFC) can be attributed to the presence of the corresponding organic layers on their surfaces.41,51 Earlier studies have indicated that an increase in the thickness of the coating layer causes a greater reduction in the maximum magnetization.52,53 A similar trend is displayed in the case of MFN and MFC as well as where MFN exhibits a lower value of magnetization compared to that of MFC. This result is supported by the difference of thickness of APTES and PMIDA coatings in MFN (∼8 nm) and MFC (∼4 nm),

(1)

where αs represents the fraction of folded HSA in the presence of NPs, θs is the observed mdeg value at 208 nm for HSA-NP solutions, θf is the mdeg value at 208 nm for HSA fibrillar solution, θn is the mdeg value at 208 nm for native HSA solution. K=

αs F = U (1 − αs)

(2)

K stands for the folding constant, F and U are the concentrations of folded and unfolded forms of HSA, respectively. ΔG = −RT ln K

(3)

ΔG represents the free energy of folding, R is the gas constant (8.314 J K−1 mol−1), and T the incubation temperature 310 K (37 °C). 2.7. Fluorescence Microscopy. For fluorescence imaging, 10 μL of protein solutions in the absence and presence of NPs were incubated with 5 μL of 1 mM ThT to achieve the required staining. Images were captured using a Leica DM 2500 M microscope equipped with a fluorescence attachment. Filter cube no. 2 (Leica I3 11, 513, 878, BZ: 01) was used for ThT excitation and emission. The images were obtained with a Leica DFC 310 FX camera attached with the microscope. All images were acquired at 10×/0.25 (N PLAN EPI). All the possible controls, native HSA, NPs, APTES, and PMIDA, separately did not exhibit any detectable fluorescence upon incubating with 11669

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To gain further information regarding progress of fibril growth with time in the absence and presence of nanoparticles, ThT binding assay is performed by extracting aliquots at different time intervals (Figure 2). During the incubation

respectively, as obtained from TEM images. Figure S6 in the Supporting Information shows the plot of the surface charge potential (SCP) versus pH for the three NPs. The isoelectric point (IEP) of MF is found to be 7.1. In comparison, the IEP of MFN is shifted to a higher value of 8.8 due to the presence of protonated amine groups on the MF surface, while for MFC it is shifted to a lower value of 3.9 due to deprotonated carboxylic acid surface groups. 3.2. ThT Binding Study. Thioflavin T (ThT) is a fluorescent dye that specifically binds to cross β-sheet structures of amyloid fibrils and thus enables one to characterize and detect the presence of amyloid fibrils. It displays significant increase in fluorescence intensity with an emission maximum at around 485 nm, when excited at 450 nm due to binding with amyloid fibrils.54,55 Though it has been reported that ThT is able to bind in the hydrophobic pocket of HSA,56 it does not show significant fluorescence with native HSA when excited at 450 nm under the experimental conditions used herein. The ThT fluorescence intensities with and without NPs have also been checked to ensure there is no effect of NPs on ThT binding. The corresponding ThT intensities of HSA fibrils in absence and presence of NPs have been shown in Figure 1.

Figure 2. Change in ThT fluorescence intensity of HSA at 37 °C with time in the absence and presence of NPs. Error bars represent standard deviation from the mean value estimated from at least three individual measurements.

process, a time dependent increase of ThT fluorescence (no discernible lag phase) with no significant change in profile of fibrillation kinetics of HSA, HSA-MF, and HSA-MFC solutions is observed. However, HSA-MFN solution shows a sigmoidal transition with an initial lag phase followed by increase in intensity attaining a quasi-plateau region. This observation indicates that presence of MFN leads to introduction of lag phase (during which ThT-detectable species are unavailable) followed by exponential growth (elongation phase) of fibrils to achieve the plateau region of which the fluorescence intensity is significantly lower than the values of corresponding regions of other solutions. It may also be noted that even with longer incubation, the fluorescence intensity of the plateau regions for the solutions does not change significantly, indicating that final fibrillar state is achieved. 3.3. Congo Red Binding Study. Congo red (CR), a hydrophobic azo dye binds specifically with the β-sheet structure of amyloid fibrils due to electrostatic interaction between the negatively charged sulfonic groups of CR and positively charged amino acids of protein. CR gives the maximum absorption peak at 498 nm, and upon binding with amyloid fibrils the absorption maxima shows a characteristic red shift of about 35−45 nm.57 Figure 3 displays the CR difference spectra for HSA fibrillar solutions with and without NPs. As expected only HSA fibrillar solution shows a characteristic red shift of about 41 nm signifying formation of well-defined amyloid fibrils. In the case of HSA-MF and HSA-MFC, we observe a red shift of about 38−40 nm which indicates the formation of amyloid fibrils, whereas in case of HSA-MFN the shift is of about 33 nm. This indicates all the solutions except HSA-MFN have produced well-ordered amyloid fibrils while HSA-MFN solution is lagging in complete development of fibrillar aggregates.25 Thus, Congo red binding study is consistent with the results obtained from the ThT fluorescence study, namely, the observation that the presence of MFN in the solution inhibits the formation of HSA fibrils. 3.4. Circular Dichroism Study. Circular dichroism spectroscopy can be used to monitor the change in secondary structure content of protein associated with fibrillation which

Figure 1. Representative ThT fluorescence spectra of HSA solutions in the absence and presence of NPs after 24 h of incubation. Inset showing ThT spectra in the absence and presence of native HSA.

While MF is found to be able to reduce ThT intensity by ∼27%, MFN causes the reduction in ThT fluorescence (∼56%) significantly as compared to HSA fibrillar solution without NPs. However, MFC causes only ∼14% reduction in ThT intensity. The reduction in ThT intensity is indicative of lesser content of fibrillar species present in the solution. It is unlikely that any free APTES will remain in MFN as a result of several washings with milli-Q water and ethanol. Even then the effect of APTES alone on fibrillation of HSA has also been investigated by ThT fluorescence. At 0.8% (v/v) concentration of APTES, used for synthesis of MFN, only ∼7% reduction in ThT fluorescence intensity compared to HSA fibrillar solution without APTES is observed. Therefore, it may be concluded that the effect of APTES alone at the concentration levels used herein on HSA fibrillation is insignificant. This observation indicates that formation of HSA fibrils is inhibited in the presence of NPs, with the amine functionalized MnFe2O4 nanoparticles (MFN) found to be more effective in the prevention of HSA fibrillation followed by MF and then MFC. 11670

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Figure 3. Difference in the absorption spectra for Congo red for various HSA fibrillar solutions incubated at 37 °C for 24 h in the presence of 50% (v/v) ethanol at pH 7.0 (a) HSA, (b) HSA-MF, (c) HSA-MFN, and (d) HSA-MFC.

Figure 4. (a) Representative far-UV CD spectra and (b) histogram of % β-sheet content of HSA solutions in the absence and presence of NPs after incubation at 37 °C for 24 h in the presence of 50% (v/v) ethanol at pH 7.0. Error bars represent standard deviation from the mean value.

primarily involves α-helix to β-sheet conversion.58 Figure 4a represents the CD spectra of the formed HSA fibrils in the absence and presence of NPs. Before the commencement of fibrillation process, the CD spectrum of HSA shows two minima at 208 and 222 nm, characteristic of the natively α-helical protein.59 The presence of negative absorption band at 208 nm is due to the π → π* transition of carbonyl groups in polypeptide chains and the other band at around 222 nm is due to the n → π* transition of the carbonyl group.60 After being treated to form fibrils, the CD spectrum of HSA shows that there is a gradual loss in intensities of the 222 and 208 nm bands (becomes less negative), which indicates the loss of α-helicity with a concomitant increase of βsheet structure.7 On the other hand, the presence of NPs results

in an increase in mdeg value at 208 nm (becoming more negative) which is indicative of increase in helicity. Further, to determine the free energy of folding (ΔG) of HSA after incubation at 37 °C in the presence of NPs, eqs 1−3 are used. From the large negative value of ΔG (folding), it is apparent that among the three NPs, MFN causes the most stabilization in the structure of HSA (Table 1). However, the positive ΔG values in the presence of MF and MFC indicate that they do not have any effect in stabilization of HSA. Thus, from the thermodynamics of folding, it is evident that MFN stabilizes the more stable (folded) structure of HSA and thus prevents unfolding leading to formation of fibrils. For quantitative estimation of secondary structures, the online DICHROWEB server is used. Figure 4b shows the 11671

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intensity is observed in the case of MFN (Figure 5c) due to a substantial decrease in the HSA fibrillar content. TEM is used to further assess the morphological feature of various HSA fibrillar solutions. The HSA fibrillar solution in absence of NPs displays an abundance of fibrillar networks (Figure 6a). In accordance with other studies, the presence of

Table 1. Free Energy of Folding Calculated for Various HSA Solutions in the Presence of NPs Using Circular Dichroism Study solution

ΔG (kJ/mol)

HSA-MF HSA-MFN HSA-MFC

0.454 ± 0.02 −1.708 ± 0.03 2.64 ± 0.01

percentage of β-sheet content of HSA in the absence and presence of NPs. As expected, fibrillation leads to an increase in the β-sheet content along with a simultaneous decrease in helicity with respect to its native state. The HSA fibrillar solution in the absence of NPs shows ∼12% α-helix and ∼34% β-sheets compared to ∼50% α-helix and ∼10% β-sheets in the native state. However, in the presence of NPs, a substantial change in the secondary structural content of HSA is observed. The relative % decreases in β-sheet content in the presence of MF, MFN, and MFC are ∼36%, ∼58%, and ∼16%, respectively, with respect to HSA fibrillar solution without NPs. This result indicates that HSA fibrillation is significantly inhibited in the presence of MFN followed by MF, whereas MFC is not able to inhibit to that extent. Thus, aminated MnFe2O4 nanoparticles have been found to be the most effective in preventing fibrillation of HSA. The CD study is in agreement with the results obtained from other studies like the ThT fluorescence study and CR binding study. 3.5. Morphological Evolution: Microscopic Study. The formation of HSA fibrils are monitored using fluorescence microscopy. The fluorescence microscopic image of HSA fibrillar solutions in the absence of NPs (Figure 5a) shows an intense fluorescence suggesting the presence of a large quantity of HSA fibrillar moieties. The presence of MF and MFC shows a slight decrease in fluorescence attributed to a lesser amount of fibrils (Figure 5b,d) as compared to HSA fibrillar solution without NPs. However, the maximum reduction in fluorescence

Figure 6. TEM images of HSA solution in the absence and presence of NPs after incubation at 37 °C for 24 h in the presence of 50% (v/v) ethanol at pH 7.0: (a) HSA fibrils, (b) HSA-MF, (c) HSA-MFN, and (d) HSA-MFC (scale bar for panels a−d is 200 nm). Red circles are indicative of the presence of corresponding nanoparticles.

MFN results in the maximum decrease in the fibrillar network which is due to the collapse of fibrillar species into nonfibrillar aggregates (Figure 6c). On the other hand, the presence of MF and MFC does not reduce the fibrillar network effectively (Figure 6b,d). Thus, results obtained from fluorescence microscopic and TEM studies are in agreement with the other biophysical studies indicating that MFN is most effective in hindering the process of association of HSA monomers to form fibrils. 3.6. Inhibiting Ability of NPs. The development of aggregates with fibrillar morphology from protein monomers involves a number of intermediate steps including nucleation, oligomerization, and fibril formation.61 The inhibiting activities of the NPs can be explained on the basis of adsorption of the HSA molecules on the surface of nanoparticles. Some earlier studies have shown that strong interactions of the proteins with NPs reduce the protein concentration in solution available to proceed to fibrillation and thus inhibiting the aggregation process.36,62,63 This is also reflected in our results as obtained from the various biophysical techniques. HSA is a globular protein with the approximate dimension of 80 Å × 80 Å × 30 Å.64 Because of its large surface to volume ratio, the adsorption efficiency of NPs is much higher than bulk materials with the same composition.65 During the fibrillation process, NPs can adsorb the protein monomers efficiently, which leads to decreasing concentration of the free HSA monomers in bulk. We suppose that this adsorption causes hindrances in the

Figure 5. Fluorescence microscopic images of HSA solution in the absence and presence of NPs after incubation at 37 °C for 24 h in the presence of 50% (v/v) ethanol at pH 7.0: (a) HSA fibrils, (b) HSAMF, (c) HSA-MFN, and (d) HSA-MFC. Scale bars represent 100 μm. 11672

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Figure 7. UV spectra of HSA solutions in the absence and presence of NPs at pH 7.0. Inset showing absorption spectra in the range of 270−290 nm.

nucleation process, the preliminary stage in fibrillation thereby obstructing the self-assembly of large number of HSA molecules to develop fibrils. As is evident from the ThT binding assay of fibrillar growth of HSA with time in the absence and presence of NPs, the presence of MFN influences the aggregation process by introducing an initial lag phase and thus affecting the nucleation step. We used a UV based spectrophotometric method to estimate HSA adsorption capacity of NPs by measuring the change in concentration of HSA in the solution after adsorption. The UV absorbance value at 280 nm was recorded for HSA solutions with and without NPs (Figure 7). The adsorptive capacity of HSA by 1 mg nanoparticles is calculated by eq 4,66 η=

protein solutions to release the protein molecules through disruption of the electrostatic interaction between HSA and MFN, and Trp fluorescence intensity of the corresponding solutions was measured with excitation at 295 nm with the emission spectra collected from 305 to 500 nm (Figure 8). As a

mHSA (AHSA − A mag ) mmag AHSA

(4)

where η refers to the adsorbed amount of HSA by 1 mg of NPs (mg g−1), mHSAis the total weight of HSA (mg), mmag is the dry weight of NPs used to bind HSA (mg), AHSA refers to the UV absorbance value of blank HSA solution, and Amag is the UV absorbance value of supernatant after adsorption. MFN is found to have the maximum HSA adsorptive capacity (∼157 mg g−1) followed by MF (∼115 mg g−1) and MFC (∼91 mg g−1). A plausible explanation for the greater efficiency of MFN in adsorbing HSA molecules can be that electrostatic interactions between HSA and MFN are highly favored. MFN is more positively charged (+26.5 mV) than MF (+1.3 mV) while MFC have negative charge (−19.9 mV) at pH 7.0. HSA being negatively charged at pH 7.0, since this is above the pI of HSA (4.9),67 they are able to interact favorably with positively charged MFN (pI of MFN is 8.8) through electrostatic attractions which causes the HSA monomers to come close to the MFN followed by adsorption. On the other hand, MF and MFC having slightly positive and highly negative charge on their corresponding surfaces, respectively, at pH 7.0 (pI of MF and MFC is 7.1 and 3.9, respectively) and are less effective in adsorbing negatively charged HSA monomers and thus do not affect the nucleation process to an appreciable extent. Further, salt titration experiments were performed to probe the contribution of the electrostatic interactions between MFN and HSA molecules in the adsorption process (data for other NPs not shown). Sodium chloride was titrated into MFN−

Figure 8. Fluorescence spectra of HSA (2 μM) in the presence of MFN with varied NaCl concentration (0−20 mM). Fluorescence of HSA increases with increasing concentration of NaCl.

control, fluorescence spectra of HSA alone with the same NaCl concentrations were also measured and no significant change in fluorescence intensity was found. Results demonstrate that with an increase in the NaCl concentration along with an increase in the ionic strength of the solution, a recovery of the Trp fluorescence intensity of HSA is observed. The release of the protein molecules from the surface of the nanoparticle indicates that the adsorption of HSA molecules on MFN is governed by electrostatic interactions.

4. CONCLUSIONS This study describes the successful synthesis and characterization of magnetic MnFe2O4 nanoparticles and their functionalized counterparts. The inhibiting potency of NPs is monitored using various spectroscopic and microscopic techniques. The maximum reduction in ThT fluorescence intensity by MFN (∼56%) than MF (∼27%) and MFC (∼14%) indicates formation of a lesser amount of fibrillar species. β-Sheet content of HSA after inducing fibrillation 11673

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conditions in the presence of MFN (∼14%) is found to be significantly less compared to MF (22%) and MFC (29%), as obtained from the CD study. Thus, fibrillation of HSA is found to be inhibited effectively in the presence of aminated magnetic MnFe2O4 nanoparticles and to a lesser extent in the presence of bare nanoparticles while carboxylated ones are found not to affect the fibrillation process significantly. The electrostatically more favored interaction between HSA molecules and amine functionalized MFN inhibits development of fibrils by disfavoring the nucleation step. More detailed studies must be conducted to realize the underlying molecular mechanism for the activity of the NPs. Though the suitability of these nanoparticles for in vivo studies as therapeutic agents for treatment of amyloid diseases requires more elaborate and systematic studies in the future, the evidence of in vitro interaction of these nanoparticles with HSA leading to inhibition of fibrillation is encouraging.

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ASSOCIATED CONTENT

S Supporting Information *

X-ray diffraction patterns of (a) MF, (b) MFN, and (c) MFC nanoparticles (Figure S1); FTIR spectra of (a) MF, (b) MFN, and (c) MFC nanoparticles (Figure S2); TEM images of (a) MF, (b) MFN, and (c) MFC nanoparticles (d) the size distribution histograms of nanoparticles corresponding to TEM images (Figure S3); DLS size distribution measurements of (a) MF, (b) MFN, and (c) MFC nanoparticles in pH 7.0 (20 mM) at 25 °C (Figure S4); field dependent magnetization curves (M-H) of (a) MF, (b) MFN, and (c) MFC nanoparticles at 300 K temperature (Figure S5); surface charge potentials of MF, MFN, and MFC nanoparticles in different pH solution measured at 25 °C (Figure S6). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +91 3222 283306. Fax: +91 3222 255303. *E-mail: [email protected]. Phone: +91 3222 283922. Fax: +91 3222 255303. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Department of Biotechnology, Government of India for a research grant (Grant BT/ PR13931/MED/3). The authors are also thankful to Central Research Facility, IIT Kharagpur for the instrumental facilities.

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REFERENCES

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