Systematic elucidation of interactive unfolding and corona formation of bovine serum albumin with cobalt ferrite nanoparticles

Abstract

Nanoparticles (NPs) are extensively being used in modern nano-based therapies and nano-protein formulations. The exposures to these comprehensively used NPs lead to changes in protein structure and functionality, hence raising grave health issues. In this study, we thoroughly investigated the interaction and adsorption of bovine serum albumin (BSA) with CoFe₂O₄ NPs by circular dichroism (CD), Fourier transform infrared (FTIR), absorption, and fluorescence spectroscopic techniques, scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrating sample magnetometry (VSM), thermogravimetric analysis (TGA) and dynamic light scattering (DLS). The results indicate that CoFe₂O₄ NPs cause fluorescence quenching in BSA by a static quenching mechanism. The negative values of van't Hoff thermodynamic expressions (ΔH, ΔS and ΔG) corroborate the spontaneity and exothermic nature of static quenching. The major contributors of higher binding affinity of CoFe₂O₄ NPs with BSA were van der Waals forces and hydrogen bonding. Furthermore, BSA protein corona formation on CoFe₂O₄ NPs was confirmed by SEM, TGA, DLS and zeta potential studies. TGA, DLS and zeta potential results confirmed the formation of a thick layer of BSA on CoFe₂O₄ NPs with negative boost in zeta potential. This coating of BSA over CoFe₂O₄ NPs leads to a decrease in the magnetic saturation value from 50.4 to 46.2 emu, hence the magnetic character of CoFe₂O₄ NPs. The development of protein corona on CoFe₂O₄ NPs was further estimated by comparing the steady state fluorescence quenching and theoretical data. In addition, FTIR, UV-CD, and UV-visible spectroscopy and three dimensional fluorescence techniques confirmed that CoFe₂O₄ NP binding could induce microenvironment perturbations leading to secondary and tertiary conformation changes in BSA. Furthermore, synchronous fluorescence spectroscopy confirmed the significant changes in microenvironment around tryptophan (Trp) residue caused by CoFe₂O₄ NPs. The denaturing of BSA biochemistry by CoFe₂O₄ NPs was investigated by assaying esterase activity.

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Introduction

CoFe₂O₄ nanoparticles (NPs) with biocompatibility and excellent electrical properties make them very fascinating for use in drug delivery, gene therapy, MRI scanning¹ and in a wide range of electronics.^2,3 However, the prolonged and extensive use of these superparamagnetic NPs raises health issues such as alteration in metabolic, hormonal^3,4 and immunologic² responses. Specifically, the behavior of these NPs in altering metabolism and immunological processes is still an enigma.⁵ There is a lot of literature available elaborating nanomaterial toxicity,^2,4 while very few studies have elaborated the interactions of NPs with bio(macro)molecules such as proteins, lipids and carbohydrates. These NPs are prone to change the native configuration and hence the functionality of bio(macro)molecules.^6,7

When NPs come in physical contact with bio(macro)molecules, these biomolecules competitively bind on the surface of the NPs forming a protein corona, thus changing the fate and mechanism of nanomedicine and nanotoxicology.⁸ Previous studies also showed that corona formation depends on chemical formulation, size, culture conditions, serum concentration, surface fashioning and charge of the NPs.^9,10 Protein coronas are classified as hard protein coronas possessing an intact layer of adsorbed proteins (first monolayer) and soft protein coronas with constantly changing protein exterior. With stability and long lifetime, a hard protein corona reduces the direct interaction of native NPs with cell receptors, resulting in changed role of NPs in biological milieu.^8,11 Therefore, to study the modification and the accumulation of NPs in target cells, complex interactions of NPs with proteins and biological molecules of body fluid in which they are suspended are of high significance.¹²

There is evidence that NPs interact and induce conformational changes in serum proteins,^13,14 affecting the secondary and tertiary structures of albumins, and hence their functionality. For example, nano-carbon severely affects the transport ability of serum proteins by altering their structure and conformation.^15,16 Physical binding of silver (Ag) NPs with bovine serum albumin (BSA) not only leads to protein corona formation but also induces conformation transitions in BSA.¹⁷ Therefore, it is very important to study the interaction of NPs with BSA, and hence becomes a very important field of research in chemistry and life sciences.⁵

BSA is the most extensively studied protein, because of its low cost, easy availability, natural abundance, and homology with human serum albumin (HSA). The serum albumins also perform a wide range of physiological functions in the plasma, i.e., transport of exogenous/endogenous materials, and also maintain ∼80% of colloidal stability and osmotic pressure.^12,13 BSA is a heart-shape globular protein with molecular weight of 69 000 and 583 residual aromatic amino acids in a single polypeptide chain.^18,19 BSA is constituted of three homologous domains (I, II and III) and each domain is further divided into two subdomains, namely IA and IB, IIA and IIB, IIIA and IIIB, which are interconnected by nine (L1–L9) loops with 17 disulphide bonds. The loops in each domain are made up of a sequence of large-small-large loops forming a triplet. X-ray crystallographic investigation further reveals that BSA is fundamentally an α-helix, and the remaining polypeptide occurs in turns and extended or flexible regions between subdomains with no β-sheet.^20–23 Contrary to HSA, BSA intrinsic fluorescence is because of the presence of two Trp residues, Trp134 is in the first domain (situated at the surface) and Trp212 is in the second (present within the hydrophobic binding pocket).^24,25

Studies of binding properties of BSA with different nanomaterials (NMs) can provide very important insights for understanding the mechanism of action of those NMs in the biological milieu.²⁶ The interaction of NMs with serum albumins changes their morphology by wrapping them, resulting in decreased toxicity and target-specific ability,²⁷ but this also induces conformation transformations. In addition, measuring the fluorescence quenching of BSA is an important modern tool to reveal the interactions of NMs with BSA. What is more, such studies also reveal the possible interactions of NPs inside the body, and provide a direction for designing better NMs for future biomedical use.

According to the best of our knowledge, this is the first report describing the detailed mechanism of pristine CoFe₂O₄ NPs' interaction, adsorption and conformation changes in BSA by quenching mechanisms, binding and thermodynamic parameters. We employed a fluorescence quenching study of BSA to calculate the protein monolayer formation (protein corona) in comparison with a theoretical model. In addition, this conformation transformation by protein corona formation was also confirmed by various analytical techniques like SEM-EDX, XRD, TGA and DLS. The biochemical inactivity of BSA as a result of adsorption on NPs and conformation transformation was further confirmed by monitoring the esterase activity.

Materials and methods

Materials

BSA and HEPES were purchased from Aladdin Industrial Inc., Shanghai. All BSA solutions were prepared in a 0.1 M HBS buffer solution (HEPES = 0.01 M, NaCl = 0.15 M, pH 7.4). BSA solutions were stored in the dark at 4 °C. CoFe₂O₄ NPs were purchased from Shanghai Future Technology, China. Milli-Q water was used to prepare all the solutions and suspensions.

Physicochemical characterization of CoFe₂O₄ NPs

Size and morphology of the CoFe₂O₄ NPs were observed by a field emission scanning electron microscope (JEOL model JSM-6301F) at an accelerating voltage of 15 kV. Powder X-ray diffraction (XRD) was used to verify the crystalline structure and size of CoFe₂O₄ NPs. Powder XRD was performed using an X'Pert PRO (PANalytical Company, Netherlands) with Cu K_α radiation to determine the structural properties and impurities. To get information about surface coatings, we obtained Fourier transform infrared (FTIR) spectra (Thermo Fischer Nicolet 6700) of the provided NPs. The magnetic properties of the investigated NPs were measured at room temperature using a vibrating sample magnetometer (VSM; 9600-1 LDJ, USA).

Circular dichroism measurements

Circular dichroism (CD) measurements were recorded with a JASCO (J-810) spectropolarimeter (190–260 nm and cell path length was 0.1 cm) by fixing BSA at (2 μM) while varying the CoFe₂O₄ NP concentration from 20 to 250 μM. The CD results were expressed in terms of the mean residual ellipticity (MRE) in deg cm² dem⁻¹ according to the following equation:

MRE = CD_obs (m deg)/C_pnl × 10 (1)

where C_p = molar concentration of the protein, n = amino acid residues (583 for BSA) and l = path-length (0.1 cm). The α-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the following equation:

α-Helix (%) = −MRE₂₀₈ − 4000/33 000 − 4000 (2)

Steady state fluorescence spectroscopy

All fluorescence spectra were recorded with a SpectraMax M5 Microplate Reader (Molecular Devices, USA). The maximal fluorescence emission of BSA after excitation at 280 nm was located at 340 nm. Steady state fluorescence spectra were recorded at 298.15, 303.15 and 308.15 K in the range of 300–420 nm. The spectrum bandwidths of excitation and emission slits were both kept at 5.0 nm. The fluorescence intensities of BSA and BSA-NPs conjugate suspensions were corrected for inner filter effect according to the following equation:²⁸where F_cor and F_obs are the corrected and observed fluorescence intensities at the emission wavelength (A_em), respectively. A_ex and A_em are the sum of the absorbance of all components at the excitation and the emission wavelength, respectively.

Time resolved fluorescence study

Fluorescence lifetimes were measured by the time correlated single photon counting technique using a HORIBA Jobin-Yvon FluoroLog® 3 with nanoLED at 280 nm (IBH, nanoLED-07) as the light source to trigger the fluorescence of BSA, and the signals were collected at the magic angle of 54.71 to eliminate contributions from anisotropy decay.²⁹ The decays were deconvoluted by Data Station v-2.5 decay analysis software provided along with the instrument. The acceptability of the fits was evaluated by the χ² criterion and visual inspection of the residuals of the fitted function to the data. The average (mean) fluorescence lifetime 〈τ_f〉 was calculated from the following equation:in which α_i denotes the pre-exponential factor corresponding to the ith decay time constant τ_i.

Synchronous and three-dimensional (3D) fluorescence spectroscopy

The synchronous fluorescence spectra of all BSA suspensions were recorded with an F97Pro spectrofluorimeter with 1 cm quartz cell (Shanghai LengGuang Industrial Co. Ltd, Shanghai, China) at different scanning intervals of Δλ (Δλ = λ_em − λ_ex) at room temperature. The Δλ values were set at 15 nm and 60 nm. Three-dimensional fluorescence spectroscopy was conducted with setting the excitation wavelength at 250–330 nm and emission wavelength at 250–450 nm with an increment of 5 nm.

UV spectrophotometry

Absorption spectra were recorded in the range of 190–350 nm, using a double beam spectrophotometer (JASCO-V550, Japan). Absorption spectra were recorded by keeping the concentration of BSA constant (2 μM) while varying the CoFe₂O₄ NP concentration from 20 to 250 μM. Absorbance values were recorded after each successive addition of CoFe₂O₄ NPs.

FTIR spectroscopy

The FTIR spectra of BSA in the presence and absence of CoFe₂O₄ NPs were recorded in the range of 500–1800 cm⁻¹. BSA concentration was fixed at 2.0 μM while that of CoFe₂O₄ NPs was 2.0 μM in the presence of HBS buffer.

SEM-EDX and XRD

SEM-EDX data of CoFe₂O₄ NPs and BSA@CoFe₂O₄ NPs suspension were obtained with a field emission scanning electron microscope coupled with an energy dispersive X-ray spectrometer (JEOL model JSM-6301F). A drop of aqueous dispersion of CoFe₂O₄ NPs and BSA@CoFe₂O₄ NPs suspensions were placed onto two separate carbon-coated copper grids, and the solutions were evaporated under ambient conditions. The samples were gold-sputtered and examined at an accelerating voltage of 15 kV. Powder XRD patterns of CoFe₂O₄ NPs and BSA@CoFe₂O₄ NPs were acquired by using an X'Pert PRO (PANalytical Company, Netherlands) with Cu K_α radiation.

Thermogravimetric measurements

Thermogravimetric analysis (TGA) of BSA@CoFe₂O₄ NPs samples was performed using a PerkinElmer Simultaneous Thermal Analyzer STA6000 (Waltham, PA). Weight loss of CoFe₂O₄ NPs, BSA and BSA@CoFe₂O₄ NPs samples was recorded from 25 °C to 700 °C at a heating rate of 10 °C min⁻¹, under a constant nitrogen gas flow (50 mL min⁻¹).

Saturation magnetization

A VSM (9600-1 LDJ, USA) was employed to determine the superparamagnetic property of CoFe₂O₄ NPs and BSA@CoFe₂O₄ NPs. The hysteresis loop of samples was obtained by applying a maximum magnetic field strength of 3 T. The saturation magnetization (M_s) was determined from M_s versus applied field plots.

Dynamic light scattering and zeta potential measurements

Dynamic light scattering and zeta potential analyses were carried out to examine the hydrodynamic diameter and surface charge of CoFe₂O₄ NPs in the absence and presence of BSA at room temperature (25 °C) using a Malvern Zetasizer Nano ZS with 633 nm He–Ne laser, equipped with an MPT-2 Autotitrator. The measured data are the average of at least 10 runs. The average hydrodynamic diameter and mean zeta potential of each sample were analyzed according to the software provided by the manufacturer.

Esterase activity measurements

The influence of CoFe₂O₄ NPs on the esterase activity of BSA was examined with a p-nitrophenyl acetate (pNPA) substrate (5.00 × 10⁻⁴ mol dm⁻³) followed by formation of p-nitrophenol (pNP) at 405 nm. The esterase activity of BSA and BSA@CoFe₂O₄ NPs was determined by using pNPA as substrate in HEPES, pH 7.40, at 298 K. The concentration of pNP was determined by absorption measurements using a molar extinction coefficient value of 17 700 dm³ mol⁻¹ cm⁻¹ at 405 nm.⁸

Results and discussion

Physicochemical characterization of CoFe₂O₄ NPs

The physicochemical properties of employed CoFe₂O₄ NPs have been discussed in detail in our previous publication.³

UV-visible absorption spectroscopy

Protein conformation change and complex formation can easily be explored by using simple UV/visible spectroscopy techniques.²⁷ BSA showed (ESI Fig. 1†) two absorption peaks, one strong absorption peak at 210 nm revealing the framework conformation and a weak peak at 278 nm reflecting the aromatic amino acids (Try, Trp and Phe) of the protein.²⁹ The shift in chromophore absorption is affected by changes in the microenvironment, indicating the characteristic π → π* transition due to changes in the polarity of the solvent.³⁰ The rise in absorption at 210 nm is also an indication of extension of polypeptide strand causing a decrease in hydrophobicity, hence the increased unfolding.^31,32 At this stage we can compare the changes in UV spectra with the results of CD. The results of CD indicated the unfolding in the polypeptide chain upon interaction with CoFe₂O₄ NPs. Therefore, amide residues in the hydrophobic core of native BSA are more exposed to the solvent molecules following CoFe₂O₄ NP binding and undergo low energy π–π* transition.³³ It is clear from ESI Fig. 1† that the observed peak around 278 nm increased with the addition of NPs with slight bathochromic shift, indicating that NP-induced unfolding of BSA results in exposure of the aromatic amino acids leading to possible conformational changes to the microenvironment of the protein, hence probably perturbing the tertiary structure of BSA.⁸

Effect of CoFe₂O₄ NPs concentration on steady state fluorescence

Diminution of fluorescence (FL) emission intensity by intrinsic or extrinsic factors is called FL quenching. FL quenching may involve excited state reactions, energy transfer, molecular rearrangements, complex formation and dynamic molecular interactions.³³ A notable increase in FL quenching (Fig. 1) of BSA tryptophan (Trp) fluorophore was observed by successive addition of CoFe₂O₄ NPs, illustrating that CoFe₂O₄ NPs can bind with BSA. Additionally, a slight shift in maximum emission from 340 nm to 342 nm (2 nm) at higher concentrations of CoFe₂O₄ NPs suggests the change in microenvironment.^23,25 The FL quenching data were analyzed by the well-known Stern–Volmer eqn (5)³⁴ and Stern–Volmer quenching (K_SV) values at different temperatures are provided in Table 1:F₀ and F denote FL intensities of BSA in the absence and presence of CoFe₂O₄ NPs, respectively, K_SV is the Stern–Volmer quenching constant and [Q] is the concentration of CoFe₂O₄ NPs. It is very crucial to identify the mechanism of intrinsic FL quenching of Trp residue in BSA, which may be of either dynamic or static quenching.⁷ Dynamic and static quenching can be differentiated by the change in FL and change in viscosity with temperature. Higher temperature promotes the dynamic quenching by enhancing the diffusion and limits the static quenching because of extensive dissociation of weak complexes.³⁵ The decrease in K_SV (Table 1) value with rise in temperature corroborates the dominance of static quenching processes, which are most probably initiated by ground state complex formation.⁵

Fig. 1 Fluorescence spectra and Stern–Volmer plots. (A) FL spectra of BSA (2 μM) in the presence of CoFe₂O₄ NPs: (0) 0, (1) 20, (2) 50, (3) 100, (4) 150, (5) 200 and (6) 250 μM. (B) Modified Stern–Volmer plots at different temperatures. The inset shows the unmodified Stern–Volmer curves of F₀/F vs. concentration of CoFe₂O₄ NPs at different temperatures.

Table 1 The Stern–Volmer quenching constant (K_SV), quenching rate constant (k_q), binding constant (K_b), number of binding sites (n) and the thermodynamic parameters (ΔH, ΔS and ΔG) for BSA–CoFe₂O₄ NPs interaction at different temperatures

T (K)	KSV × 10^3 (L mol^−1)	kq × 10^12 (L mol^−1 s^−1)	Kb × 10^3 (L mol^−1)	n	ΔG (kJ mol^−1 K^−1)	ΔH (kJ mol^−1)	ΔS (J mol^−1 K^−1)
298.15	2.56	0.374	2.40	1.74	−2.17	−17.94	−52.88
303.15	1.80	0.263	2.13	1.62	−1.90
308.15	1.60	0.234	1.28	1.10	−0.63

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In addition, the confirmation of the aforementioned mechanism can be derived by calculating the quenching rate constant (k_q) according to eqn (6) by considering the FL lifetime (τ₀) of biopolymer of 10⁻⁸ s:^5,13

The observed k_q value is 2.56 × 10¹² L mol⁻¹ s⁻¹ at 298.15 K, which is much higher than the maximum quenching rate constant (2.0 × 10¹⁰ L mol⁻¹ s⁻¹) of the biopolymer. The data presented in Table 1 show progressively decreased quenching constant (k_q) with rise in temperature which is still higher than the threshold value (2.0 × 10¹⁰ L mol⁻¹ s⁻¹), indicating static quenching.³³

To further validate the static quenching, which should decrease with a rise in temperature, we investigated the static binding constant (K_b) and number of binding sites (n) of BSA with CoFe₂O₄ NPs (free and bound) which were measured by the Lineweaver–Burk equation:^33,36

The values of n and K_b decreased with a rise of temperature (Table 1). This signified that temperature effectively decreased the number of binding sites, hence the stability of the complex. In addition, these aforementioned results also substantiate the predominance of static quenching and ruling out the contribution of dynamic quenching in the presently investigated BSA–CoFe₂O₄ system.

Calculation of thermodynamic parameters ΔH, ΔS and ΔG

In general, the interactions among proteins and nanoparticles can be of various categories and magnitudes, like electrostatic, hydrogen bonding, van der Waals, steric, hydrophobic/hydrophilic within the binding sites and so forth.³³ The magnitudes and signs of thermodynamic parameters give us an extensive amount of information about the involvement of crucial binding forces.^37,38 From the thermodynamic point of view, ΔS > 0 and ΔH < 0 indicate an electrostatic force, ΔS > 0 and ΔH > 0 indicate a hydrophobic interaction, and ΔS < 0 and ΔH < 0 indicate van der Waals forces or hydrogen bond formation. The enthalpy of the reaction of CoFe₂O₄ NPs and BSA can be treated as a constant, if there is no remarkable change in temperature. We calculated the enthalpy change (ΔH), free enthalpy change (ΔG), and entropy change (ΔS) according to the following thermodynamic eqn (8)–(10):

ΔG = −RT ln K_b (9)

ΔG = ΔH − TΔS (10)

where R is the gas constant (8.314 J mol⁻¹ K⁻¹) and T is the temperature (K). K_b is the equilibrium binding constant at the corresponding temperature. The values of K_b, ΔH, ΔS, and ΔG are presented in Table 1. The negative values of ΔG and ΔH revealed spontaneous and exothermic processes, respectively. The negative values (Table 1) of ΔH and ΔS were −17.94 kJ mol⁻¹ and −52.88 J mol⁻¹ K⁻¹ indicating the interaction of CoFe₂O₄ NPs with BSA is mostly enthalpy driven, while the entropy is not favorable for it. Moreover, in this study the hydrogen bonding and van der Waals interactions are the key binding players in ground state complex formation.^23,33

Fluorescence lifetime analysis

Analysis of fluorescence lifetime is the state of the art technique used for identification of the local environment of a fluorophore,³⁹ and differentiation and confirmation of static and dynamic fluorescence quenching mechanisms.^28,40 To confirm the static mechanism involved in the BSA fluorescence quenching by addition of various concentrations of CoFe₂O₄, fluorescence lifetime analysis was performed and a representative fluorescence lifetime graph is shown in Fig. 2.

Fig. 2 Fluorescence lifetime decays of BSA (λ_ex = 280 nm, λ_em = 340 nm) at minimum (20 μM) and maximum (250 μM) concentrations of CoFe₂O₄ NPs in HBS buffer, pH 7.4. The concentration of BSA was fixed at 2 μM.

A mean fluorescence lifetime of 6.83 ns was calculated for free BSA from eqn (4), while a minimal decrease in fluorescence lifetime 6.73 ns was observed at concentration of CoFe₂O₄ NPs of 250 μM indicating the perturbations in the microenvironment.³⁹ The data presented in Table 2 show the nominal changes in the fluorescence lifetime of BSA in the presence of various concentrations of CoFe₂O₄ NPs. In addition, the data also corroborate the formation of a non-fluorescent ground state complex between BSA and CoFe₂O₄ NPs. It is also a well-established fact that the formation of a static ground state complex does not decrease the decay time of the uncomplexed fluorophore because only the lifetimes of unquenched fluorophores are observed in time resolved spectroscopic analysis.⁴¹ This corroborates the results of the steady state fluorescence quenching study. Normally, Trp lifetimes are known to be reduced when exposed to polar environments. The fluorescence characteristics, such as the average fluorescence lifetime and the components of the lifetime of protein fluorophore Trp, are sensitive to the environment and hence indicative of the protein conformational alterations during protein–ligand interactions.³⁹

Table 2 Fluorescence lifetime decay data of BSA in various concentrations of CoFe₂O₄ NPs. The excitation wavelength used was 280 nm and emission wavelength was 340 nm

Sample	τ1 (ns)	τ2 (ns)	τf (ns)	χ^2
BSA	3.54 (23%)	6.66 (77%)	6.83	1.08
BSA + 20 μM CoFe2O4 NPs	3.54 (14%)	6.63 (86%)	6.76	1.16
BSA + 50 μM CoFe2O4 NPs	3.53 (22%)	6.55 (78%)	6.71	1.19
BSA + 100 μM CoFe2O4 NPs	3.51 (15%)	6.62 (85%)	6.78	1.14
BSA + 150 μM CoFe2O4 NPs	3.53 (20%)	6.57 (80%)	6.71	1.08
BSA + 200 μM CoFe2O4 NPs	3.54 (18%)	6.55 (82%)	6.75	1.20
BSA + 250 μM CoFe2O4 NPs	3.54 (19%)	6.59 (81%)	6.73	1.12

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Circular dichroism studies

CD spectroscopy is a sensitive analytical technique used for quantitative investigation of conformation of proteins in solutions.⁴² The CD spectra (Fig. 3) display two negative peaks at 208 and 222 nm, representing the π → π* and n → π* electronic transitions in α-helix structure, respectively.^43,44 The fractions of α-helix and β-sheets are shown in Table 3. Compared to the native BSA, the α-helical content of BSA@CoFe₂O₄ NPs decreased from 55.9% to 48.1%, and the β-sheet content increased from 37.3% to 39.9%. A reduction in the percentage of α-helix (∼14%) and an increase in that of the β-sheets (∼7%) were observed, suggesting strong interactions among CoFe₂O₄ NPs and BSA. The decreased α-helix and increased β-sheet content in BSA indicated that CoFe₂O₄ NPs are bound to the aromatic amino acid residues (Trp and Tyr) of the main polypeptide chain of BSA and destroy the hydrogen bonding networks leading to unfolding of protein.^45,46

Fig. 3 CD spectra of BSA in the absence and presence of CoFe₂O₄ NPs at 250 μM (T = 298 K). The concentration of BSA was fixed at 2 μM in HBS buffer with pH = 7.4. The blue line represents the CD spectrum of CoFe₂O₄ NPs alone in HBS buffer, pH = 7.4.

Table 3 Changes in the secondary structure of BSA in the absence and presence of CoFe₂O₄ NPs^a

System	α-Helix (%)	β-Chain (%)
a *Concentration of BSA was fixed at 2 μM in HBS buffer, pH = 7.4. **Concentration of CoFe2O4 NPs was 250 μM.
BSA	55.9	37.3
BSA + CoFe2O4 NPs	48.1	39.1

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Synchronous fluorescence spectroscopy (SFS)

Synchronous fluorescence spectroscopy (SFS) introduced by Lloyd has been used for fingerprinting of complex samples.³³ Some of the hallmarks of SFS are sensitivity, spectral simplification, reduced bandwidth without any perturbations. SFS also provides information about the change in the molecular microenvironment of the fluorophore (aromatic amino acid residues) functional groups of BSA.⁴⁷ In SFS, the Δλ values (Δλ = λ_em − λ_ex) fixed at 15 or 60 nm, present the characteristic information of tyrosine (Tyr) and tryptophan (Trp) residues, respectively.⁴⁸ The SFS spectra elucidating the effect of CoFe₂O₄ NPs on BSA microenvironment-specific perturbations are shown in Fig. 4. As can be seen from Fig. 4, the fluorescence of Tyr residues was weak and the position of maximum emission wavelength had no effect when Δλ was 15 nm. While the fluorescence of Trp residue was strong and the maximum emission wavelength moderately shifted toward longer wavelength (red shift) when Δλ was 60 nm. This reflects the perturbation of the microenvironment of the Trp residue by CoFe₂O₄ NPs binding. It is reported that the maximum emission wavelength (λ_max) located at 330–332 nm indicates that Trp resides are located in the non-polar region, they are buried in a hydrophobic cavity in BSA; λ_max located at 345–352 nm shows that Trp residues are located in the hydrophilic region.⁴⁹ The red shift suggested a less hydrophobic environment of Trp residue and ground state complex formation.^50,51 These results also corroborate that CoFe₂O₄ NPs very possibly bind to Trp residue of BSA, resulting in a change in conformation and enhanced polarity around the Trp residues with increased hydrophilicity.⁵² Hence the probable interaction site of CoFe₂O₄ NPs is in close proximity of Trp amino acid. A slight red shift in SFS spectra for Δλ = 60 nm indicates some conformation changes in the proximity of Trp residue and enhanced hydrophilicity. These results are in good agreement with previous studies.^33,48,53 In addition, very minor changes of SFS spectra at Δλ = 15 nm elucidates the insignificant changes in the microenvironment of Tyr residue in the presence of increasing concentration of CoFe₂O₄ NPs.⁵⁴ Such disturbances in tertiary structure can be revealed by utilization of three-dimensional fluorescence spectroscopy.⁵³

Fig. 4 Representative synchronous fluorescence spectra of BSA at different concentrations of CoFe₂O₄ NPs ((0) 0, (1) 20, (2) 50, (3) 100, (4) 150, (5) 200 and (6) 250 μM).

Three-dimensional (3D) fluorescence spectroscopy

Three-dimensional (3D) emission spectroscopy provides signatures of the conformation changes induced by ligands in proteins by delineating the emission spectral characteristics of an intrinsic fluorophore by simultaneously varying the excitation and emission wavelengths.⁸ We performed 3D emission spectral analysis to investigate CoFe₂O₄ NP-induced conformational changes in BSA.³³ As shown in (ESI Fig. 2A†), BSA exhibits three peaks, peak 1, peak 2 and peak (a). Peak 1, Rayleigh scattering (λ_em = λ_ex), and peak 2 correspond to the spectral behavior of Trp and Tyr residues in BSA, and are directly related to the polarity of the microenvironment around Trp and Tyr residues, respectively.³³ Peak (a) depicts the backbone framework of polypeptide chain of BSA. As depicted in (ESI Fig. 2B†), the emission intensity of peak 2 and peak (a) is decreased when BSA is in the presence of CoFe₂O₄ NPs. The obvious change in the 3D emission intensity of peak 2 and peak (a) is pinpointing the conformational changes in secondary structure and backbone framework of polypeptide chain of BSA.⁵⁵ On the basis of the outcome of the 3D spectral studies, it is inferred that the binding of BSA to CoFe₂O₄ NPs led to considerable conformational changes leading to unfolding of the native structure of BSA, and this conclusion is in accordance with the results obtained from CD spectroscopy and FTIR spectral analysis described in the following.

FTIR spectroscopy

FTIR spectroscopy is an excellent tool for the investigation of protein–NPs interactions.⁴² Fig. 5 depicts the FTIR spectra of native BSA and BSA with CoFe₂O₄ NPs. The 585.3 and 577.4 cm⁻¹ bands were related to (tetrahedral) M–O⁵⁶ vibrations of CoFe₂O₄ NPs. The 1244.2 and 1240 cm⁻¹ bands were attributed to antiparallel β-sheet vibrations. The 1350.6 cm⁻¹ band was related to the α-helix (amide-III).⁵⁷ Two bands at 1650.7 and 1653.3 cm⁻¹ were attributed to high α-helix content arising from characteristic amide-I of BSA.⁵⁷ The α-helix structure loss was indicated by intensity drop-off of the 1350.6, 1650.7 and 1653.3 cm⁻¹ bands. The intensity diminution of 1240 and 1244.2 cm⁻¹ bands was attributed to alterations in antiparallel β-sheets by CoFe₂O₄ NPs. The decreased intensity at 1650.7 and 1653.3 cm⁻¹ indicated that α-helical content in BSA was reduced after binding to CoFe₂O₄ NPs.⁵⁸

Fig. 5 FTIR spectra of native BSA with and without CoFe₂O₄ NPs. The concentrations of BSA and CoFe₂O₄ NPs were 2 μM each.

Red-edge excitation energy shifts (REES)

For a polar fluorophore, where the solvent relaxation is not complete, the emission spectra shifts to longer wavelengths when the excitation is on the long wavelength edge of the absorption spectrum. This effect is known as red-edge excitation shifts or REES.²⁸ The REES of fluorescence can be used as a parameter for studying the photophysical and photochemical properties of isolated proteins. This phenomenon is an indication for a strongly reduced dynamic environment of a single Trp, which has a very low accessibility to the solvent and is caused by electronic coupling between Trp indole rings and neighboring dipoles.^8,59 Thus, REES is particularly useful in monitoring motions around the Trp residues in protein studies. From measurements of the fluorescence emission and REES of BSA upon interaction with CoFe₂O₄ NPs, it is possible to compare the microenvironment and mobility features of the Trp residue in the BSA–CoFe₂O₄ NPs complex.

The results for the REES investigation of the BSA–CoFe₂O₄ conjugate are provided in Table 4. As can be seen, the value of the REES for the BSA–CoFe₂O₄ NPs system was 7. This is higher than the REES value for BSA, indicating that CoFe₂O₄ NPs induce the unfolding of BSA exposing the intrinsic fluorophore by initial complex formation. This indicates binding of CoFe₂O₄ NPs to the hydrophilic domains of BSA, which leads to the motional restriction in the BSA microenvironment. This is in accordance with the results of previous studies.^29,59

Table 4 REES effects of CoFe₂O₄ NPs (λ_ex = 280 nm and λ_ex = 295 nm)

System	λem-max (nm)		REES Δλem-max (nm)
	λex = 280 nm	λex = 295 nm
BSA	340	344	4
BSA + 250 μM CoFe2O4 NPs	343	350	7

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Effects of BSA@CoFe₂O₄ NPs corona formation

NPs' interaction with proteins is the foundation of NP bio-reactivity and leads to the formation of a dynamic NP–protein corona.⁶⁰ This so-called protein corona formation results in altered surface properties as well as cellular uptake, accumulation, biodegradation and excretion of NPs, hence the fate of nanomedicine.^61–63 Moreover, surfaces of NPs may provide sites for adsorption of proteins and induce conformation changes, which may also affect the overall bio-reactivity of NPs and flexible proteins are comparatively more vulnerable.^64–67 So, by characterizing the protein corona formation at CoFe₂O₄ NPs also provides valuable information about adsorption-induced conformational transformation and FL quenching. Moreover, FL quenching results corroborate the conformation transformation of BSA after interaction with CoFe₂O₄ NPs. Therefore, we utilized various analytical techniques, SEM-EDX, XRD, VSM, TGA, DLS and zeta potential, for confirming any possible change in physicochemical properties of CoFe₂O₄ NPs and vice versa. The results (Fig. 6) clearly showed that adsorption of BSA@CoFe₂O₄ NPs notably changed the physicochemical properties of CoFe₂O₄ NPs. As shown in Fig. 6A and B, SEM-EDX data showed the adsorption and agglomeration of CoFe₂O₄ NPs in the presence of BSA. While XRD (Fig. 6C) showed the weaker peaks of BSA@CoFe₂O₄ NPs compared to the pristine CoFe₂O₄ NPs. This indicates a reduction in spinel character of CoFe₂O₄ NPs, when BSA was bound at the surface of the NPs.⁶⁸ The decrease in signal intensity or spinel character was most probably due to the adsorption of BSA on CoFe₂O₄ NP surfaces.¹⁸ Protein corona can change the magnetic saturation (M_s) leading to a change in magnetization, magnetic hyperthermia and magnetic resonance imaging efficiency of NPs. The M_s value of superparamagnetic CoFe₂O₄ NPs readily decreases from M_s = 50.4 emu (Fig. 6D) to M_s = 46.2 emu. This is due to the surface disorder and modified BSA protein corona. The decrease in M_s is attributed to the BSA corona, preventing the core spins from aligning along the field direction.^10,68 Furthermore, to investigate the presence of bovine serum proteins in BSA@CoFe₂O₄ NPs, TGA was performed. Thermograms at 700 °C reveal that CoFe₂O₄ NPs (Fig. 6E, red thermogram) exhibit lowest weight loss due to cobalt iron oxide core, while bovine serum (Fig. 6E, black thermogram) displayed maximum weight loss due to excessive protein degradation at higher temperatures. The weight loss of all BSA@CoFe₂O₄ NPs (Fig. 6E, blue thermogram) was between those of the CoFe₂O₄ NPs and BSA samples.

Fig. 6 Physicochemical analysis of BSA@CoFe₂O₄ NPs confirms the presence of bovine serum proteins bound on CoFe₂O₄ NPs. (A) SEM-EDX micrograph for pristine CoFe₂O₄ NPs. (B) SEM-EDX micrograph showing the adsorption of BSA on CoFe₂O₄ NPs. (C) X-ray diffraction patterns of CoFe₂O₄ NPs and BSA@CoFe₂O₄ NPs. (D) Vibrating sample magnetometry indicates the change in magnetization and hyperthermia by adsorption BSA on CoFe₂O₄ NPs. (E) Weight loss thermograms of BSA, CoFe₂O₄ NPs, and BSA@CoFe₂O₄ NPs. Excessive weight loss of BSA@CoFe₂O₄ NPs indicates bovine serum protein degradation in thermograms.

Dynamic light scattering and zeta potential measurements

The change in size and zeta potential of CoFe₂O₄ NPs was measured with and without BSA. Adsorption of BSA on the surfaces of CoFe₂O₄ NPs leads to an increase in size of up to 14%. This illustrates the densely packed protein corona formation on the surface of NPs. Adsorption of BSA@CoFe₂O₄ NPs also leads to an induced structural transformation in the conformations of protein.⁶⁹ We also know that the number of adsorbed proteins also depends on the concentration of protein present in the system. Therefore, an increase in the negative charge is an indication of adsorption of BSA, because the serum albumins have negative charge at pH = 7.4.¹⁰ This indicates the establishment of electrostatic forces among CoFe₂O₄ NPs and BSA molecules. In addition, the van der Waals forces and steric contact are also contributing factors in BSA and CoFe₂O₄ NP interaction and decrease of charge.⁷⁰ Overall, the results of SEM-EDX, XRD, VSM, TGA, DLS and zeta potential measurements all showed that interactive binding between BSA and CoFe₂O₄ NP surfaces occurred. This adsorption of BSA@CoFe₂O₄ NPs also changes the surface chemistry and behavior within the physiological environment.

Theoretical determination of protein corona@CoFe₂O₄ NPs

The surface modification of NPs by competitive selective adsorption of proteins in the surrounding environment of biological vicinity alters the fate of NPs compared to pristine NPs.⁷¹ So, it is an urgent need to study how the protein corona formation induces significant extrinsic responses from subcellular to organism level.^62,63 Experimental and theoretic calculations simply help us to characterize the protein corona by counting the number of adsorbed proteins on the surface of NPs under specific conditions.^72,73

We know that BSA has a triangular prism-shaped structure in solution with a height of 3.15 nm and sides of 8.4 nm.^74,75 By considering the prism shape of BSA, the average number (N) of bound BSA molecules for monolayer formation on a spherical shaped NP can be calculated by eqn (11):^8,73

here, R₀, R_N, V₀ and V_P are the radii of NP without and with protein, the volume of the NP and the volume of the bound protein molecule, respectively. The volume of a BSA molecule and CoFe₂O₄ NPs was calculated as 96.3 nm³ and 20 288 nm³, respectively. The BSA molecule can possibly bind with the CoFe₂O₄ NP surface in a “flat-on” or “end-on” fashion and increase the radius of CoFe₂O₄ NPs from 20.5 nm to 23.65 nm (flat-on) and 25.02 nm (end-on). By assuming a complete monolayer formation on CoFe₂O₄ NP surfaces, the average numbers of BSA molecules/CoFe₂O₄ NPs were approximately 113 (flat-on) and 173 (end-on). The formation of a thick layer of BSA around CoFe₂O₄ NPs was predicted by comparing the theoretical and experimental data. From the FL quenching data, it is clear that BSA molecules have direct contact with CoFe₂O₄ NP surfaces playing a major role in monolayer formation, hence the quenching.

FL quenching induced by CoFe₂O₄ NPs showed that the number of BSA molecules bound to a single CoFe₂O₄ NP surface was ∼5 × 10⁶. It is also reported that Au nanorod and Au/Ag NPs can accommodate ∼1 × 10³ to 3 × 10³ and ∼3.3 × 10⁴ HSA molecules, respectively. This large difference in the number of surface-adsorbed BSA molecules could be attributed to the fact that round shaped CoFe₂O₄ NPs are much smaller in size with higher surface area compared with Au nanorod and Au/Ag NPs.^8,72 The adsorption of a thick layer of BSA over NPs can be evidenced from the deviation of experimental size from the theoretically predicted size. In addition, beyond the hard protein corona formation, there is a continuous competitive exchange of BSA molecules on the surface of the CoFe₂O₄ NPs. Also the value of the Hill coefficient (n > 1) strengthens the idea of notable operation of cooperative binding between BSA molecules and CoFe₂O₄ NP surfaces. It is well known that the introduction of colloidal nanoparticles into a biological medium often results in the rapid formation of a protein corona around the NP surface.^62,72,73 The Hill coefficient value is in good accord with the average number of surface-bound BSA molecules, supporting the evidence that dense layers of BSA are formed on the surface of CoFe₂O₄ NPs.

Influence of CoFe₂O₄ NPs on functionality of BSA

The influence of NPs binding with BSA and on the retention of BSA activity is of prime importance regarding the applicability of NPs for biological aspects. With a view to the breakdown of the native BSA conformation upon interaction with CoFe₂O₄ NPs, we endeavored to evaluate the influence of CoFe₂O₄ NPs binding on the activity of BSA.³³ Fig. 7 shows a representative profile for the effect of CoFe₂O₄ NPs at various concentrations on the esterase activity of BSA in terms of relative esterase activity of BSA in which the amount of enzyme required to liberate 1 mmol p-nitrophenol per minute at 37 °C has been defined to be one unit of esterase activity. It is seen that binding with increasing concentrations of CoFe₂O₄ NPs was associated with a notable reduction in the esterase activity of BSA, which may be associated with the breakdown of the native protein structure.⁸

Fig. 7 Effect of CoFe₂O₄ NPs (20, 50, 100, 150, 200 and 250 μM) on the esterase-like activity of BSA (2 μM). The asterisk (*) shows a statistically significant difference from the BSA.

Conclusion

The interaction between BSA and CoFe₂O₄ NPs was investigated by different spectroscopic techniques. According to the results, we concluded that the CoFe₂O₄ NPs bind with the BSA by ground state complex formation leading to static FL quenching and the binding/adsorption process is substantially spontaneous and exothermic in nature. The analysis of FTIR, UV-visible, UV-CD and 3D fluorescence spectroscopy illustrated the dramatic changes in secondary and tertiary structures of BSA upon interaction with CoFe₂O₄ NPs. In addition, the SFS spectra at Δλ = 60 nm corroborates the perturbations triggered around the Trp residue microenvironment by the presence of CoFe₂O₄ NPs. Furthermore, TGA, DLS and zeta potential measurements confirmed the formation of a thick layer of BSA corona on CoFe₂O₄ NP surfaces. Comparison of experimental quenching and theoretical data confirmed the protein corona formation. In addition, this protein corona (adsorption) formation severely affects the physicochemical properties of CoFe₂O₄ NPs. The results confirmed that CoFe₂O₄ NPs considerably compromised the biochemical activity as well as functionality of proteins. Therefore, there is a need to get a deeper understanding of NPs' impacts on biological systems before their widespread use in health products.

Acknowledgements

The authors are very grateful to the Project of Science and Technology Department, Zhejiang Province (2012C37058) and the Key Innovation Team of Science and Technology, Zhejiang Province (2010R50018) for financial support.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02850k

‡ Farooq Ahmad and Ying Zhou contributed equally to this manuscript and will be considered as co-first authors and co-corresponding authors as well.

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