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Surface Coating Rescues Proteins from Magnetite Nanoparticle Induced Damage

Nidhi Joshi , Anindita Mukhopadhyay , Sujit Basak , Goutam De ,* and Krishnananda Chattopadhyay *

The presence of magnetic nanoparticles (NPs) in physiological systems induces toxicity through its effects on mitochondrial function and reactive oxygen species (ROS) imbalance. Magnetic NP induced cytotoxicity has been elaborately evaluated for impending threats, however, a detailed investigation is lacking. It is shown that the interaction of Fe 3 O 4 NPs with cytochrome c can lead to different events based on the NPs to protein ratio, the solution conditions, and the type of surface protection. At low NPs concentration, rapid binding and subsequent electron transfer are the preferred events while at higher concentration slow oxidative modifi cation of the protein is initiated. The slow event of protein modifi cation yields conformational disorientation, loss of stability, and formation of amyloid-like structures with cytochrome c. The possibility that the NP induced oxidative stress and age can work in concert to compromise different aspects of cellular quality control processes is discussed. Suitable surface modifi cations of the NPs inhibit their direct binding to the protein molecules and minimize NP induced toxicity.

1. Introduction

Nanomaterials with varying metallic and non-metallic core compositions and surface modifi cations have been aggres-sively devised and characterized because of their potential biomedical and therapeutic implications. [ 1 ] However, iron NPs induced toxicity is also a major concern. [ 2 ] Signifi cant toxicity has already been established with iron containing nanoma-terials (e.g., the nanorods and nanofi bers of asbestos [ 3 ] ). The exposure to NPs from various sources like the emission from industrial establishments, combustion of diesel and wood and also nanoparticulates coming out from tire-road wear could be potentially damaging as they bypass regulatory organizations’ scrutiny. [ 4 ] Adding up to this delicate issue is the presence of

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DOI: 10.1002/ppsc.201200148

N. Joshi, S. Basak, K. Chattopadhyay Protein Folding and Dynamics Laboratory Structural Biology and Bioinformatics Division CSIR-Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road , Kolkata , 700032 , India E-mail: [email protected] A. Mukhopadhyay, G. De Nano-Structured Materials Division CSIR-Central Glass and Ceramics Research Institute 196, Raja S. C. Mullick Road , Kolkata , 700032 , India E-mail: [email protected]

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iron NPs in ambient air as well. [ 4,5 ] For example, Karlsson et al. [ 6 ] have found that the uncharacterized particles found in subways are mainly comprised of Fe 3 O 4 NPs and these particles are capable of inducing mitochondrial depolarization. NPs are known to travel throughout the body and deposit in multiple organs and trigger injurious responses. [ 7 ] There are NPs which have long residence times and hence they would remain in the circula-tory system for extended time periods. [ 8 ] These kinds of particles can potentially impose slow physiological manifestations if remained untreated or if not bio-layered.

While the toxicity of iron NPs has been studied in detail at the cellular level, [ 9 ] the molecular level information on their effects towards a protein conformation and stability is still scanty. It can be sus-pected that the interaction of a NP with

cules, such as proteins, would show typical
the biological molestructural or stability related changes depending on several fac-tors associated with both the nature of NPs and biomolecules. The NPs-protein interface, also known as the nanoparticle-pro-tein corona has been suggested to be an "inevitable entity" in in vitro and in vivo biological studies. [ 10 ] It is extremely important to study protein-NPs interactions and their biological implica-tions which would help, not only to develop innovative thera-peutic applications, but also to assess the toxicity of NPs.
It has been suggested that Fe 3 O 4 NPs may induce their tox-icity through the generation of reactive oxygen species (ROS). [ 11 ] In addition, Fe 3 O 4 NPs can act as an electron source because of available electrons at their surface. [ 12 ] It is interesting to note that both the electron transfer defects and ROS imbalance, which can be induced by the presence of Fe 3 O 4 NPs, may have signifi cant implications on mitochondrial dysfunction as well as on several neurodegenerative diseases. [ 13 ] The role of ROS in the neurodegenerative diseases, e.g., Alzheimer’s diseases, have been explored extensively. [ 14 ] Keeping these issues in mind, we wanted to choose a protein system in this study that can be used as a probe to study electron transfer defects and ROS imbalance related complications. We fi nd cytochrome c (Cytc) to be an excellent model system for these purposes because of several reasons. First, it is an integral part of electron transport chain and by virtue of its low reorganization energy Cytc can take up or release electrons easily. Second, the conformation, stability, and folding in different oxidation states of Cytc have

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been studied extensively in literature. Finally, Cytc responds to ROS levels in the cell and forms an integral component of the apoptotic cascade. [ 15 ]

We have previously reported a facile method for the synthesis of polyethylene glycol (PEG) coated Fe 3 O 4 NPs, where we have additionally shown that the interaction of uncoated bare NPs with Cytc leads to the reduction of the protein. [ 16 ] Here, using several spectroscopic and kinetic measurements, we show that bare Fe 3 O 4 NPs can bind effi ciently with Cytc and exert its elec-tron transfer and ROS related effects. The protein to NP concen-tration ratio has been found to be one of the deciding factors. For example, in the presence of low concentration of bare NP, only electron transfer occurs; but at high NPs concentration slow event of radical induced protein modifi cation begins. Although NPs induced electron transfer seems to be structurally and con-formationally benign with minimum or no effect on stability, ROS induced modifi cation of Cytc results in large alteration in the secondary and tertiary structure of the protein. In addi-tion, these ROS induced structural changes infl uence the sta-bility and aggregation propensity of Cytc signifi cantly resulting in the formation of short amyloid fi brils, an observation reported for the fi rst time with magnetite NPs. Surface coating of the Fe 3 O 4 NPs using PEG (10 kD) or dextran (15–30 kD) hinders protein binding on NP surface and inhibits both elec-tron transfer and oxidative modifi cations.

2. Results

2.1. The Interaction of Bare Fe 3 O 4 with Cytc Leads to Electron Transfer or Oxidative Modifi cation Depending on the NP Concentration

We have recently reported a facile synthesis method and detailed characterization of PEG surface modifi ed Fe 3 O 4 NPs. [ 16 ] Interaction of bare Fe 3 O 4 NPs with Cytc leads to the electron transfer to the oxidized Cytc leading to instantaneous reduc-tion of the protein. The reduction of Cytc is characterized by the appearance of two prominent absorption bands at 520 nm and 550 nm ( Figure 1 a). Figure 1 b shows the absorption intensity at 550 nm, which measures the extent of reduction, in the pres-ence of different protein to NPs ratios. At low NPs concentra-tion (for example, Cytc:NPs of 1:0.2) the extent of reduction is not pronounced whereas in the presence of high concentration of NPs (for example, Cytc:NPs of 1:1) signifi cant reduction has been observed (Figure 1 b).

An additional change in the absorption spectra has been observed with time when the protein is incubated at room temperature in the presence of high concentration of bare Fe 3 O 4 NPs. A slow loss in the heme absorption has been found to occur with time at and beyond a Cytc:NPs ratio of 1:1 (Figure 1 c–e). Loss of heme absorbance of Cytc of this kind has also been observed with the addition of hydrogen peroxide (Figure S1, Supporting Information), although the extent of the absorbance loss is much higher with hydrogen peroxide. It has been shown that the peroxide induced heme loss observed in Cytc is a result of the oxidative damage to the protein. [ 17 ] The addition of excess sodium azide, a known free radical quencher,

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inhibits the bare Fe 3 O 4 NPs induced loss of heme absorbance (Figure S2, Supporting Information). This is to be noted that the presence of sodium azide has no effect on the bare NP induced reduction mentioned above (Figure S2, Supporting Information). The formation of hydroxyl and superoxide radi-cals by oxygen reduction induced by surface activity of iron minerals has been reported. [ 18 ] Spin-trapping EPR measure-ments have been used to monitor surface mediated produc-tion of hydroxyl radicals directly as a mechanism of iron oxide induced biotoxicity. [ 19 ] Magnetic Fe 3 O 4 NPs have been shown to generate ROS by EPR, [ 20 ] resulting in oxidative cellular stress and DNA damage. [ 21 ]

2.2. Surface Protection Using Biocompatible Agents Inhibits Both the Reduction and Oxidative Modifi cation Events

The interaction of bare magnetic NPs with Cytc results in two events. First, at a relatively low NPs concentration, rapid reduction of Cytc takes place at the NP surface. Second, Cytc is modifi ed by a slow oxidative process in the presence of rela-tively high concentration of NPs. Experiments carried out in Dulbecco’s modifi ed Eagle medium (DMEM), supplemented with fetal bovine serum (FBS) show the occurrence of both the events, although their extents are found to be less (Figure S3, Supporting Information). This observation may be attributed to the competitive binding of the serum proteins on to the NPs surface, thus leading to a decreased overall effect on Cytc.

In order to understand, how surface protecting agents infl u-ence these two events, UV-visible experiments have been car-ried out in the presence of Fe 3 O 4 NPs surface modifi ed with 10 kD polyethylene glycol (PEG10 kD) and dextran (15–30 kD). Surface modifi cations using PEG10 kD or dextran inhibit both the processes (Figure 1 f). Whereas a detailed characterization of the PEG10 kD NPs has been shown previously, [ 16 ] the char-acterization of the dextran modifi ed NPs has been included as supporting information (Figure S4, Supporting Information)

2.3. Binding of Cytc with NPs takes Place Prior to NP Induced Reduction or Oxidative Modifi cations

We hypothesize that rapid reduction observed with Cytc on addition of bare NPs needs an initial binding of the protein onto the NP surface. To monitor the binding between the NPs and the protein directly, we have used steady state fl uorescence spectroscopy. Since the intrinsic fl uorescence of the tryptophan residue (Trp-59) is completely quenched by heme (and the pro-tein is non-fl uorescent) in horse heart Cytc, we choose tetrame-thyl rhodamine 5 maleimide (TMR) labeled yeast Cytc for the binding measurements. Yeast Cytc contains a surface exposed cysteine which can be readily labeled with an extrinsic dye. Oth-erwise, horse heart Cytc and yeast Cytc are very similar with respect to their sequence and have analogous structures (Figures S5,S6, Supporting Information). Also, the conformation and stability of the TMR labeled Cytc has been shown to be similar to the unlabeled protein. [ 22 ] Figure 2 a–c shows the interaction of TMR labeled Cytc with an increasing concentration of bare and PEG10kD coated NPs. The fl uorescence intensity of TMR

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Figure 1. a) UV-vis spectra of Cytc in the presence of bare and PEG coated Fe 3 O 4 NPs. The reduction of Cytc occurs in the presence of bare Fe 3 O 4 NPs (red) but is absent in the presence of PEG10kD coating of the NPs (black). b) Absorbance at 550 nm in the presence of varying concentrations of bare (red), PEG10kD (black), and dextran coated Fe 3 O 4 NPs (blue). For this and all other fi gures, the Cytc:NP numbers are shown in a single number; for example, Cytc:NPs of 1:0, 1:1 and 1:5 are shown as 0, 1, and 5 respectively. c–e) The changes in UV-vis absorbance spectra of Cytc with time in the presence of bare NPs with Cytc:NPs of 1:0.5 (c), 1:1 (d), and 1:5 (e). The insets in (c–e) show the respective absorbance values at 413 nm plotted with time. f) The absorption spectra in the presence of PEG10kD and dextran-coated NPs (1:5).

decreases sharply with increase in the concentration of bare Fe 3 O 4 NPs and saturates at a Cytc:NPs ratio of 1:0.6 (Figure 2 a,b). Compared to bare NPs, PEG10kD and dextran coated NPs show signifi cantly less change in the fl uorescence intensity (Figure 2 a,c). At pH 4.5, no NPs induced decrease in fl uorescence intensity has been observed even with the bare NPs suggesting complete absence of binding at low pH (Figure 2 a).

Assuming a fast binding event of Cytc molecules at the sur-face of the magnetic NPs which is needed for the subsequent events to take place, the binding constant between Cytc and bare NPs has been determined ( K b = 2.7 × 10 9 M −1 , Table 1 ). Figure S7a,b (Supporting Information) shows the linear fi ts to Equation 1 for the determination of binding constant (Experi-mental Section). The fi t also suggests that two molecules ( n = 2) of Cytc bind to each molecule of bare NPs (Table 1 ). The

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observed binding constant (Table 1 ) suggests that the binding between bare Fe 3 O 4 NPs and Cytc at pH 7.4 is slightly stronger compared to that of Fe 3 O 4 NPs with BSA although the binding stoichiometry has been found to be identical. [ 23 ] In contrast, the binding constant, K b , for the dextran coated Fe 3 O 4 NPs were found to be 6.3 × 10 3 M −1 (Table 1 ) which is signifi cantly less than that of the bare NPs. In the presence of PEG10kD sur-face modifi cation or at low pH, the initial binding is either very weak to be determined (in the presence of PEG10kD) or completely absent (at pH 4.5) (Table 1 ). Figure 2 d shows a com-parison between the binding event (Figure 2 a) and the reduc-tion event (Figure 1 b). This comparison clearly indicates that the protein binding on to the NPs surface occurs effi ciently and immediately, while the process of reduction begins only after a threshold NP concentration.

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Figure 2. a) The interaction between Cytc and bare NPs at different solution conditions. b) Rep-resentative emission spectra which shows the extent of fl uorescence quenching of TMR-labeled Cytc in the presence of different concentrations of bare NPs at pH 7.4. c) Representative emis-sion spectra of TMR labeled Cytc in the presence of different concentrations of PEG10kD coated Fe 3 O 4 NPs. d) The comparison between the fl uorescence and absorbance data at 550 nm in the presence of Cytc:bare NPs of 1:5.

2.4. The Slow Oxidative Modifi cation and Not the Rapid Elec-tron Transfer Leads to Altered Secondary and Tertiary Structures

To obtain further insights into the implications of the above two events on the structure and conformational integrity of the protein, far and near UV CD experiments have been car-ried out. Figure 3 a,b show the far UV CD spectra obtained with Cytc in the presence of different concentrations of bare and PEG10kD coated Fe 3 O 4 NPs respectively. The far UV CD data obtained with the dextran coated NPs are shown in Figure S8 (Supporting Information). The far UV spectrum of Cytc at pH 7.4 in the absence of NPs (Figure 3 a) indicates the presence of two prominent minima at 209 nm and 222 nm indicating the protein to be predominately α -helical. The far UV CD spectrum changes signifi cantly in the presence of high concentration of bare Fe 3 O 4 NPs (Cytc:NPs of 1:1 or higher) (Figure 3 a).

The ellipticity at 222 nm, which is commonly used as a measure of the helical content of a protein, has been plotted with the protein to NPs ratio for the bare, PEG10 kD and

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Table 1. Binding constants and the number of bound protein molecules.

Binding Parameters Cytc in the presence of bare NPs at pH 7.5

BSA in the presence of bare NP

Cytc in the presence of dextran coated NP

K b (M −1 ) 2.7 × 10 9 1.24 × 10 8,a) 6.3 × 10 3

N 1.98 1.95 a) 1

a) Data from ref. [ 23 ]; b) The binding in this solution condition is too weak to be determined.

dextran coated Fe 3 O 4 NPs (Figure 3 c). In the presence of low concentration of bare NPs (for example, at Cytc:NPs of 1:0.2) the change in ellipticity at 222 nm is insignifi cant. How-ever, large decrease in the ellipticity has been observed beyond a threshold Cytc:NPs of 1:1 (shown using a red arrow in Figure 3 c). In contrast, no signifi cant change in the ellip-ticity has been observed in the presence of different concentration of PEG10kD and dex-tran coated magnetic NPs (Figure 3 c).

A comparison (Figure 3 d) between the change in the absorbance at 550 nm, which probes the electron transfer from the NP to Cytc (Figure 1 b), and the far UV CD, which probes the change in the secondary struc-ture of Cytc (Figure 3 c), with Cytc:NPs ratio is interesting. For both absorbance and CD, the changes occur beyond a threshold pro-tein to NPs ratio, although the threshold ratio is considerably less for the absorbance (1:0.4 for the absorbance and 1:1 for the far UV CD). Consequently, beyond Cytc:NPs of 1:1, the protein undergoes a large decrease in the secondary structure due to the oxida-tive modifi cation (the right hand side of the blue vertical line, Figure 3 d). In contrast, below Cytc:NPs of 1:0.4, the protein is bound at the NP surface, oxidized with native like secondary structure (the left hand side of the

e, Figure 3 d). Between Cytc:NPs of 1:0.4 and
black vertical lin1:1, the protein is reduced with native like secondary structure and without any oxidative modifi cation (the area between the black and blue vertical lines, Figure 3 d).
To understand further the time evolution of these processes, we have carried out manual mixing kinetics measurements of the UV-visible absorbance at 550 nm ( Figure 4 a) , and far UV CD at 222 nm (Figure 4 b) at Cytc:NPs of 1:5. The kinetics of the absorbance change at 550 nm contains at least two phases. The fi rst phase is an early increase in the absorbance (Figure 4 a, red), which occurs too rapidly to be analyzed by the manual mixing technique. The second phase, which is the loss in the absorbance (Figure 4 a, red) is relatively slow occurring with a rate of 0.25 min −1 . The rate is measured by calculating the slope at the initial linear region. In the presence of sodium azide, the absorbance kinetics has only one phase of early reduction and the second slow phase is not observed.

The kinetics of the change in far UV CD at 222 nm indi-cates the presence of only one kinetic phase accompanying a large decrease in the secondary structure of the protein

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Cytc in the presence of bare NP, pH 4.5

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Figure 4. a) Manual mixing kinetics of Cytc-bare NPs interaction as monitored by UV-visible spectrophotometer at 550 nm. A prominent reduction-oxidation phase can be observed where the ratio between Cytc and bare NPs is 1:5 (red). The data of Cytc without NPs is used as a control (black). b) Manual mixing kinetics of Cytc-bare NPs interaction as monitored by ellipticity at 222 nm (red). A control Cytc data without NP is also shown (black). c) Near UV CD observed with Cytc in the absence (black) and presence (red) of bare Fe 3 O 4 NPs (Cytc:NPs of 1:5). d) Near UV CD of Cytc in the absence (black) and presence (red) of PEG10kD coated Fe 3 O 4 NPs.

Figure 3. a) Far UV CD spectra of Cytc in the presence of different concentrations of bare Fe 3 O 4 NPs. The ratio between the protein and bare Fe 3 O 4 NPs was varied between Cytc:NPs of 1:0.2 and 1:10. b) Far UV CD spectra obtained with Cytc in the presence of different concentrations of PEG10kD coated Fe 3 O 4 NPs. c) The values of ellipticity at 222 nm of Cytc in the presence of bare (black), PEG10kD (red) and dextran (blue)-modifi ed NPs. The data are represented in log scale in x-axis. d) Comparison between the ellipticity of Cytc at 222 nm and absorbance of Cytc at 550 nm in the presence of bare NPs with Cytc:NPs of 1:5.


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Figure 5. a) The temperature induced unfolding transitions of Cytc in the presence of bare NPs with Cytc:NPs of 1:0, 1:0.6, 1:2, 1:5. b) The temperature induced unfolding transitions of Cytc in the presence of PEG10kD coated NPs. To identify clearly the presence of the intermediate states and to determine the amplitudes and mid-points of different steps of the thermal unfolding transitions, double derivatives of the unfolding data have been shown in the presence of dif-ferent concentrations of c) bare and d) PEG10kD coated NPs. Panels (c,d) clearly show the presence of an intermediate state in the presence of bare NPs at high NPs concentrations while the unfolding data can be fi t to an ideal two state transition model in the presence of PEG10kD coating. e) Light scattering experiment of Cytc in the presence of varying concentrations of bare (black) and PEG10kD coated (red) NPs monitored at 350 nm. f) Light scattering experiments with Cytc in the absence and presence of bare and PEG10kD coated Fe 3 O 4 NPs (Cytc:NPs of 1:5) at different temperatures in sodium phosphate buffer at pH 7.5.

(Figure 4 b, red). The initial rate of this slow decrease is 0.2 min −1 which is similar to the slow kinetics of the loss in absorb-ance (Figure 4 a). The decrease in the far UV CD is absent in the presence of sodium azide (Figure S9a, Supporting Informa-tion). These results in combination with the fact that reduced Cytc contains identical helical content (Figure S9b, Supporting Information) clearly suggest that the initial binding and the early reduction do not lead to any signifi cant change in the sec-ondary structure. The slow decrease in the secondary structure takes place due to the oxidative modifi cation of Cytc.

Near UV CD (between 250 nm and 300 nm) measure-ments have been carried out with Cytc in the presence of bare and PEG10kD coated Fe 3 O 4 NPs and the results are shown in Figure 4 c,d. The near UV CD of a protein probes the asymmetric environment of the aromatic amino acids. Near UV CD of Cytc

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in its native condition (Figure 4 c, black) is characterized by two prominent minima at 282 nm and 288 nm which are attributed to the tertiary structures near tryptophan 59, the sole tryptophan residue present in the pro-tein. [ 24 ] Although no change in the tertiary structure is observed at low NP concentra-tion, these two peaks at 282 and 288 nm dis-appear completely above Cytc:NPs of 1:1 sug-gesting complete loss of the tertiary structure (Figure 4 c, red). In contrast, considerable ter-tiary structure of the protein is retained even with high concentrations of PEG10kD coated Fe 3 O 4 NPs (Figure 4 d).

2.5. Thermal Unfolding of Cytc is a Two-State Process: An Intermediate is Observed for the Oxidatively Modifi ed Protein

Thermal unfolding experiments with Cytc in the presence of different concentration of bare and PEG10kD coated Fe 3 O 4 NPs have been carried out to fi nd out if the Fe 3 O 4 NP-induced electron transfer (occurring at low NPs concentration) and the slow oxidative modifi cation (occurring at higher NPs con-centration) have any effects on the stability and refolding effi ciency (RE) of the pro-tein. The temperature induced unfolding of Cytc in the absence of Fe 3 O 4 NPs results in a sharp transition with high co-operativity. This is accompanied by a large decrease in the ellipticity at 222 nm ( Figure 5 a). The data could be well approximated by a two-state transition model (with the mid-point of 82.7 °C) (Figure 5 c). This two-state transi-tion model assumes an “all or none” transi-tion between the folded (the protein at room temperature) and the unfolded state (the pro-tein at high temperature) without signifi cant population of any intermediate state. At rela-tively low concentrations of bare Fe 3 O 4 NPs (with Cytc:NPs of less than 1:1 in Figure 5 a),

the nature of unfolding does not change. The unfolding transi-tions alter signifi cantly in the presence of high concentration of bare NPs. For Cytc:NPs of 1:5 (Figure 5 a,c) , the temperature induced unfolding is no longer a two state transition and the presence of an intermediate state is observed (Figure 5 c). The mid-point of the fi rst transition (the native to the intermediate state) is about 59 °C while the mid-point of the second transi-tion (the intermediate to unfolded state) has been found to be 78 °C (Figure 5 c). The mid-points of the unfolding transitions in the presence of different protein to NPs ratios are provided in Table 2 .

In the presence of PEG10kD and dextran coated NPs of dif-ferent concentrations, in contrast, the unfolding transitions are superimposable to that observed with the native protein in sodium phosphate buffer at pH 7.5 (Figure 5 b,d). Also,

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Table 2. Stability parameters and refolding effi ciency of Cytc in the presence of differently coated NPs.

Type of Fe 3 O 4 NPs Cytc:NPs T m1 [°C] T m2 [°C] RE [%]

No nanoparticle, Cytc in sodium phosphate buffer, pH 7.5 1:0 82.7 82.3

Bare 1:2 60 80 a) 14

1:5 59 78 a) 7

PEG10kD coated NPs 1:2 82.5 49

1:5 82 49

Dextran coated NPs 1:2 82 79

1:5 79 62

a) The unfolding transition data of Cytc in the presence of high concentration of bare NPs show the presence of two transitions.

the mid-points of transitions are found identical (Table 2 ) and no intermediate state is found even at high concentrations of PEG10kD and dextran coated magnetic NPs (Figure 5 b,d).

One of the parameters which defi ne the reversibility of the transition is its refolding effi ciency (RE). RE depends upon the competition between correct refolding and misfolding/aggregation of the protein when it is allowed to refold from its unfolded state. While high RE is important to establish a reversible unfolding transition, poor RE may suggest the pres-ence of off-pathway intermediates and irreversible aggregation. The values of RE for different protein samples obtained by this method are shown in Table 2 .The RE of Cytc unfolding is about 80% in the absence of NP. The RE does not change in the presence of low concentration but decreases signifi cantly in the presence of high concentration of bare NPs. Only 7% of the protein has been found to refold with Cytc:NPs of 1:5 (Table 2 ). In contrast, in the presence of even high concentration of PEG10kD and dextran coated NPs, the RE remains relatively high (Table 2 ).

2.6. Measurement of Protein Aggregation in the Presence of Bare and Surface-Coated Fe 3 O 4 NPs

It is likely that the decreased RE is a result of irreversible aggregate formation of the protein occurring at high tempera-ture. Also, the presence of the intermediate may have direct relevance to aggregation. To investigate this, we have studied the aggregation behavior of Cytc in the absence and presence of different concentration of bare and PEG10kD coated Fe 3 O 4 NPs using fl uorescence light scattering as a tool and the data are shown in Figure 5 e. The protein samples in the presence of different concentrations of bare and PEG10kD coated Fe 3 O 4 NPs have been incubated for an hour at room temperature and the scattering intensity has been measured at 350 nm. As shown in Figure 5 e (the black points) the scattering intensity at 350 nm shows a sharp increase around Cytc:NPs of 1:1 and saturates at high NPs concentration. The overall change in the scattering intensity shows a sigmoidal profi le. PEG10kD coated Fe 3 O 4 NPs, in contrast, does not cause any signifi cant aggregation even at very high concentration (the red points in Figure 5 e).

In a separate experiment, the protein solutions in the pres-ence of Cytc:bare NPs of 1:5 are incubated for fi ve minutes at

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different temperatures and the extent of aggregation is meas-ured (Figure 5 f). While scattering is not signifi cant between 25 °C and 45 °C, a large increase in the scattering intensity (and aggregation) is observed at around 60 °C (Figure 5 f). It is important to note that the onset temperature of the aggregation (60 °C) is similar to the mid-point temperature of the interme-diate formation (59 °C).

Subsequently, the effect of concentration of the bare Fe 3 O 4 NPs on the temperature induced aggregation of Cytc has been studied. For these measurements, Cytc has been incubated in the absence (control) and presence of bare and PEG10kD coated NPs at 60 °C (the temperature where appreciable scattering is observed, Figure 5 f) for one hour at the protein to NPs ratio of 1:1 and 1:5. After an hour, the samples were removed from the incubator, whereupon precipitates could be seen with naked eyes in the presence of bare NPs (with Cytc:NPs of 1:5), but are absent in the presence of PEG10kD coated NPs (Figure S10a, Supporting Information). The samples have been centrifuged and the supernatant is subjected to UV-visible spectroscopy for the presence of any protein. The absorbance scan of the super-natant containing Cytc:NPs of 1:5 (Figure S10b) does not show the characteristic Soret peak and Q-band. The supernatant of the sample with Cytc:NPs of 1:1 contains little but not all of the protein.

The presence of protein in the precipitated sample is con-fi rmed by running SDS-PAGE with the precipitate (Figure S11, Supporting Information). In order to understand the nature and morphology of the heat induced aggregates further, the aggre-gates have been subjected to TEM imaging at different days ( Figure 6 ). TEM analyses of the fi rst day samples (Figure 6 a) suggest the formation of amorphous aggregates. However, the presence of proto-fi bril-like species (dimensions were 4 nm ± 1 nm (width) and 50 nm ± 5 nm (length)) could be seen when the aggregated samples are incubated for seven days (Figure 6 b). Figure 6 c,d show selected zoomed areas of Figure 6 b. On the other hand, PEG10kD coating prevents the formation of any amorphous or fi brillar aggregates even after 7 days of incuba-tion (Figure 6 e,f). The energy dispersive X-ray (EDX) analysis of the grids (Figure S12, Supporting Information) has also been carried out, which shows the presence of iron, which confi rms the presence of Fe 3 O 4 NPs in the samples. Copper in the EDX analysis comes from the carbon coated copper grid. Uranium represents uranyl acetate, which has been used for the negative staining of the protein.

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Figure 6. Bright fi eld TEM images of Cytc heat treated at 60 °C for one hour in the presence of bare and PEG10kD coated Fe 3 O 4 NPs a) on day 1, the presence of amorphous aggregates has been observed with samples containing bare Fe 3 O 4 NPs and b) on day 7, the heat-treated samples containing bare Fe 3 O 4 NPs shows the formation of short proto-fi bril like aggregates. Selected areas of the image chosen from (b) have been mag-nifi ed at different resolutions and shown in (c) and (d). The selected area in (b) has been marked with green circle. e,f) Images of the day 7 protein samples heat treated in the presence of PEG10kD coated NPs at two different resolutions.

3. Discussion

The data shown in the present paper has been discussed sche-matically in Figure 7 . In Figure 7 , Fe represents the magnetic Fe 3 O 4 NPs, Fe PEG is the PEG10kD coated Fe 3 O 4 NPs, N Ox is the native protein in its oxidized state (in the absence of NPs). The addition of Fe 3 O 4 NPs to Cytc leads to rapid binding with high effi ciency (forming FeN Ox ), which is then followed by fast elec-tron transfer and reduction of the protein (FeN Red in Figure 7 ). At high NPs concentration, a slow event of oxidative modifi ca-tion takes place resulting in the formation of FeN Mod . It is not clear whether the formation of FeN Red and FeN Mod take place through parallel or sequential pathways.

It is important to understand whether the decrease in helical content observed with Cytc is the result of the initial adsorp-tion (the formation of FeN Ox ) or is it because of one or both of the two events that follow (the formation of FeN Red and

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FeN Mod ). Although, there is precedence of protein adsorption resulting in large decrease in the helical content of lysozyme and RNaseA, [ 25 ] the present results seem different. A com-parison between the formation of FeN Ox (Figure 2 a), the for-mation of FeN Red (Figure 1 b) and the decrease in secondary structure (Figure 3 a) suggests that the formation of FeN Ox and/or FeN Red do not directly contribute to the decrease in the hel-ical content of Cytc. Instead, the formation of FeN Mod , which occurs at a higher protein to NP ratio, is responsible for the loss of helical content. It can also be argued that the forma-tion of FeN Mod at high NP concentration is the result of less number of Cytc bound per NP molecule as discussed previ-ously. [ 26 ] Alternatively, at low NP concentration, the number of protein molecules adsorbed on the NP surface would increase. This effect would lead to less overall surface area of the protein molecules remaining in contact with the NPs, resulting in no or minimum conformational damage to the protein at low NP concentration. [ 26 ] However, the above effect should be sodium azide independent. Our observation of the absence of high NP induced decrease in the helical content in the presence of sodium azide (Figure S8a, Supporting Information) provides further supports for the oxidative modifi cation mechanism.

Although characterizing the sequence of events is important, it may be equally important to delineate the manifestations of reduction and oxidative modifi cation on a protein’s stability. At low concentrations of bare NPs, when the protein is reduced (FeN Red ), the conformational stability of the protein remains intact. While it is interesting that both FeN ox and FeN Red have similar stability, it is not entirely unexpected. This is because, the folding of the reduced Cytc has been extensively studied and it has been established that the reduction does not decrease the stability of the protein. [ 27 ]

In the presence of high concentration of NPs, the nature of the unfolding transition changes drastically and the presence of an intermediate (FeI) state has been observed. The interme-diate is partially unfolded and about 64% of total change in the secondary structure occurs in the fi rst transition (Figure 5 c , FeN Mod to FeI transition) while the remaining secondary struc-ture (36%) unfolds in the second (FeI to FeU) transition. The involvement of FeI in the aggregation process has been sup-ported by the light scattering data which suggests that the onset temperature of aggregation matches identically with the mid-point of the FeI formation (Figure 5 f). TEM measurements suggest predominance of amorphous featureless aggregates initially, although formation of fl exible fi bril like species has been found after seven days.

It is important to discuss the implications and potential caveats of the present study with respect to its biological rel-evance. First, the present study directly shows potential toxicity with the bare NPs and emphasizes the importance of suitable surface modifi cations to minimize the risks. While NPs with therapeutic applications are extensively studied, the present paper describes one of the rare investigations to provide molec-ular level information on the toxicity of the uncharacterized but widely abandoned NPs. The various sources of exposures and entry routes available to NPs to enter inside the cellular environment have been shown in Figure 8 . [ 28 ] The properties of a nano-sized particle, which differ from their bulk counter-parts, govern their entry routes. Second, while the present

mbH & Co. KGaA, Weinheim Part. Part. Syst. Charact. 2013, 30, 683–694

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Figure 7. Schematic representation of the interaction between Cytc and NPs in the a) absence and b) presence of surface modifi cations.

study details an overall effect of NPs on the conformation of Cytc, accurate interaction sites available for the protein still remain elusive. Recently, Li et al. established possible binding sites of NPs on a protein using cross-linking chemistry coupled with mass spectrometry. [ 29 ] Third, the present study is pos-sibly the fi rst and only example, in which the effect of Fe 3 O 4 NPs induced ROS has been shown to cause the formation of amyloid like species directly in a protein. Although it can be argued that the formation of amyloid like species in Cytc is not directly implicated in any neurodegenerative diseases, it has been recently suggested that the formation of amyloid like spe-cies may not be the cause of neuro-degeneration, but only an after-effect of other triggers. The irregularity in the ROS bal-ance in mitochondria has been suggested to be one of these causative triggers. [ 30 ] The above results along with a different but prominent observation, that amyloid formation may not be limited to a few disease related proteins and could be induced in almost any protein, [ 31 ] has made the choice of the model pro-tein a rather generic problem.

4. Conclusion

The active role of a protein’s conformational defects and the presence of misfolded intermediates in its aggregation behavior have been established beyond doubts and our results also show


direct correlation between the presence of FeI and Cytc aggre-gation. While the in vivo scenario is more complicated and the sophisticated quality control mechanisms present in the human cells are equipped to detect and destroy these conformational defects, it is nevertheless important to assume that the pres-ence of NPs may impair some of these regulatory pathways. Other important considerations may be mutational effects due to environment or age related stress factors. It is likely that the NPs related toxicity may function in concert with these factors.

5. Experimental Section Materials : Cytc from horse heart and from Saccharomyceae cerevisiae ,

sodium azide, ferrozine, neocuproine, ascorbic acid, ammonium acetate, and dextran (15–30 kD) were purchased from Sigma-Aldrich, USA. TMR was purchased from Invitrogen (USA). The concentration of Cytc was determined by reducing it in the presence of trace amount of sodium dithionite and then measuring the absorbance at 550 nm using an extinction coeffi cient of 29 000 M −1 cm −1 . [ 32 ] The synthesis and detailed characterization of bare and PEG10kD coated Fe 3 O 4 NPs was carried out using a published procedure. [ 16 ] With a slight modifi cation in the ferrozine assay, [ 33 ] the concentrations of the bare and PEG10kD coated NPs were quantifi ed. [ 16 ]

Determination of the Secondary and Tertiary Structures of Cytc : CD spectra were recorded using a Jasco J715 spectropolarimeter (Japan Spectroscopic Ltd., Japan). Far-UV CD measurements were performed with 5 μ M protein using a cuvette of 1 mm path length and a scan speed


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Figure 8. Possible sources of ambient exposure and pathways of NPs entry in to the cell and their effects.

of 50 nm/min. Near-UV CD spectra were recorded with 10 μ M protein using a cuvette of 10 mm path length and a scan speed of 2 nm/min.

Determination of Cytc Binding onto the NPs Surface : Cytc was labeled with TMR using a protocol as described elsewhere. [ 34 ] Binding of Cytc onto the NPs surface has been studied using NPs induced quenching of TMR fl uorescence at the emission wavelength of 580 nm. Protein-NPs binding has been analyzed by adopting a previously published model [ 35 ] as the binding model.

According to this approach, the relation between the fl uorescence intensity ( F ) and the NPs concentration can be represented using the following equation:

F0 − F

F − F∞=

{[N Ps ]

K diss

}n

( 1 )

where F 0 is the fl uorescence intensity of TMR labeled Cytc in the absence of NPs; F is the fl uorescence intensity of Cytc in the presence of NPs, F ∞


is the fl uorescence intensity of TMR labeled cytochrome c saturated with NPs, and n is the number of binding sites on a cytochrome c molecule. The slope of the double logarithmic plot gives the number of equivalent binding sites ( n ), and the value of log[NPs] at log[ F 0 – F ]/[ F – F ∞ ] = 0 is the dissociation constant ( K diss ). From the reciprocal of K diss , the binding constant K b is obtained.

Temperature Induced Unfolding Experiments : The thermal unfolding of Cytc was monitored by measuring the ellipticity at 222 nm. The protein samples were kept in a quartz cuvette of 1 mm path length placed in a cell holder, the temperature of which could be controlled by a computer interface. The temperature was raised at the rate of 1 °C/min from 20 °C to 90 °C. Prior to the thermal measurements, Cytc solutions of 5 μ M were equilibrated for half an hour at room temperature in the presence of varying NPs concentrations.

Data Analysis of the Thermal Unfolding Experiments : In thermal unfolding experiments, the thermodynamic parameters were calculated, assuming the unfolding to be a two state process. Fractions of the


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unfolded protein f u , at different temperatures ( T ), were calculated using 2 . [ 36 ]

fu = θ − (θn + mNT )/ (θu − mU T ) − (θn + mNT ) ( 2 )

where, θ represents the ellipticity of Cytc at a given temperature. θ n and θ u are the intercepts of the native and unfolded baselines, and m N and m U represent the slopes of the native and the unfolding baselines respectively.

Therefore, the equilibrium constant of the unfolding is given by:

Keq = fu/ (1 − fu) ( 3 )

At temperature T , the corresponding free energy change is given by:

�G0T = − RT ln(K eq) = �HT − T �S ( 4 )

where R represents the gas constant and T is the absolute temperature. Δ H T and Δ S represent changes in enthalpy and entropy at the temperature of T , respectively.

In Equation (5), the Van’t Hoff plot of ln( K eq ) against 1/ T was linearly fi tted

ln(K eq) = �S/R − �H/R(1/ T ) ( 5 ) Also,

�G 0T = � H (1− T / Tm )�C p (T − Tm − T ln (T / Tm )) ( 6 )

where at the midpoint of transition, i.e., when (�G0m) = 0 , T = T m .

Calculation of Refolding Effi ciency from the Thermal Unfolding Experiments : In order to estimate the extent of refolding of a thermally unfolded protein, the sample was cooled immediately from 90 °C to room temperature (20 °C) at the end of each thermal unfolding experiment. Subsequently, a spectrum was collected at 20 °C for the refolded protein. The refolding effi ciency, RE was calculated using the following equation:

RE =θR − θU

θN − θU× 100

( 7 )

where θ N and θ U represent the ellipticity at 222 nm of the native and fully unfolded states at 20 °C and 90 °C, respectively and θ R is the ellipticity of the refolded protein at 222 nm obtained at 20 °C.

TEM Sample Preparations and Measurements : TEM experiments were carried out using a Tecnai G2 30ST (FEI) operating at 300 kV. The samples were loaded on a carbon coated copper grid (ProSciTech, Australia) and were negatively stained with 0.5% uranyl acetate. The protein to NPs ratio (both bare and PEG10kD coated) was maintained at 1:5 and incubated at 60 °C. The above mixture was sampled and analyzed using TEM at different time points (for example, the fi rst and the seventh day of incubation).

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements N.J. and A.M. contributed equally to this work. Financial support from the Department of Science and Technology, Government of India and CSIR, India is thankfully acknowledged. K.C. acknowledges a CSIR network project grant (MIND, BSC-0115) for fi nancial supports. A.M. thanks the Department of Science and Technology for Woman Science Fellowship (grant # SR/WOS-A/CS/99/2011).

Received: December 14, 2012Revised: February 10, 2013

Published online: March 25, 2013


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