1. Introduction

Nanopraticles (NPs) are of great scientific interest as they are, in effect, a bridge between bulk materials and atomic or molecular structures. NPs have unique properties that may be useful in a diverse range of applications, and consequently they have attracted significant interest. The interesting and unexpected properties of NPs are largely due to the large surface area of the material, which governs the contributions made by the small bulk of the material ^[1].

Iron oxide magnetic nanoparticles (MNPs) have attracted much attention due to their fine magnetic properties and massive fields of applications in modern science. Medical and biochemical applications of MNPs involve drug delivery ^[2], gene delivery ^[3], protein separation ^[4], magnetic resonance imaging ^[5], treatment by hyperthermia ^[5]. The magnetic properties strongly depend on their synthesis method and their surface characterization ^[8]. Surface coating with some molecules may enhance or reduce the ability of MNPs to interact with other molecules ^[9]. Therefore, synthesis of new MNPs with various surface properties is still an interesting field of study.

The protein-nanoparticle interactions involves the formation of a dynamic layer of proteins or other biomolecules adsorbed on the nanoparticle surfaces called "corona" ^[10]. Interaction of NPs with proteins and the formation of corona affect the physicochemical properties of both biomolecules and the NPs and considered as the basis of NPs bioreactivity ^[11]. It is appear that highly selective protein adsorption, added to the fact that particles can reach subcellular locations, results in significant new potential impacts for NPs on protein interactions and cellular behavior.The protein corona may influence cellular uptake, inflammation, accumulation, degradation and clearance of the NPs. Furthermore, the NPs surface can induce conformational changes in adsorbed protein molecules which may affect the overall bio-reactivity of the nanoparticle ^[12]. Among these bioactivity processes, enzyme inhibition by NPs is an important issue for study as some drugs, in fact, are enzyme inhibitors ^[13]. One of these enzymes that are targeted by inhibitors is xanthine oxidase (XO). It plays an important role in the catabolism of purines. XO catalyzes the formation of xanthine which can further catalyze the oxidation of xanthine to uric acid. Accumulation of uric acid can result in hyperuricaemia, leading to arithritis and gout ^[14]. The search for XO inhibitors is still an interesting field for study ^{[15, 16]}. The present study aimed to prepare new magnetic NPs by direct surface modification of the original magnetic Fe₃O₄ NPs using sulphadiazine and cholesterol. The prepared MNPs will used to immobilize XO enzyme on their surfaces and study the possible changes in enzyme activity and secondary and tertiary structures induced by the MNPs for future medical applications.

2. Materials and Method

Iron Oxide nanoparticles Fe₃O₄

In the present work Fe₃O₄ nanoparticles were prepared by a modified co-precipitation method ^[17]. Briefly, 0.04 mol of FeCl₃·6H₂O and 0.02 mol of FeSO₄·7H₂O were dissolved in 50 ml of 0.5 M HCl solution. Then, 500 ml of 1.5 M NaOH was added dropwise to the solution under vigorous stirring at 80 °C. The Fe₃O₄ precipitate obtained was separated using a magnet and was repeatedly washed with deionized water until the supernatant becomes neutral. Finally, the precipitant was dried at 50°C for 4h and then overnight at room temperature. For the preparation of MNP@Cholesterol or MNP@Sulphadiazine, same method was followed but before the separation of the obtained Fe₃O_4, a volume of oversaturated solution of cholesterol or folic acid at ratio 2:1 was added to MNPs suspension with continuous stirring at 80°C for 1h. Then, the suspensions were transferred to a watch glass and placed in an oven at 40°C overnight until completely dried. Transmission electron microscopy (TEM) was used to visualize the morphology and size of the prepared MNPs.

XO from buttermilk has a molecular weight of 275,000D was used in all the enzyme containing experiments. The enzyme activity was measured using Kalckar method ^[18] as follows: into a quartz cuvette pipette 2.9 ml of different concentration of xanthine (substrate) solution (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07and 0.08 mM). After zero adjustment at 290 nm, One hundred microliters of enzyme solution (0.1 U per ml of 0.05 M Sodium phosphate buffer) were added to the cuvette, mixed and the increase in absorbance at 290 nm was recorded during 5 minutes. The increase in absorbance at 290 nm, caused by the oxidation of xanthine to uric acid, is a measure of the catalytic activity of XO.

Kinetic parameters K_m, V_maxwere calculated from the Lineweaver-Burke equation(1/ʋ₀ =(K_m/V_max[S])+ 1/V_max) by plotting 1/ʋ₀vs. 1/[S].Where; ʋ₀: initial velocity μM/min, V_max: maximum velocity μM/min, K_m:Michaelis constant(μM), S:substrate concentration(μM). Then intercept=1/V_max and slope= K_m/V_max.

Two mg/mL of the prepared magnetic nanoparticles (MNP, MNP@cholesterol, and MNP@sulfadiazine was ultrasonicated for 20 minutes then cooled. The following concentration of XO enzyme were prepared in phosphate buffer: 0.2, 0.4, 0.6, 0.8, 1, 1.4 and 1.6 U/ml. Two and a half milliliters of the MNPs solution were mixed with 2.5 ml of XO solution. The mixture was mixed for 60 minutes at room temperature (22-25°C). The mixture then was centrifuged at (7,500rpm) for 25 minutes at 20˚C and the supernatant then separated. The concentration of XO (as protein) was estimated spectrophotometrically using Barford’s method at 595nm ^[19]. the isotherm were constructed between xanthine oxidase concentration at equilibrium (C_e) and amount of xanthine oxidase that adsorbed on the MNPs (Q_e), The isotherms were studied to find out the best adsorption isotherm that can explain the adsorption isotherm.

CD spectra of free xanthine oxidase, XO-MNP@Cholesterol, and XO-MNP@Sulfadiazinecomposites after adsorption were obtained by using the following concentrations: 0.2 U/ml free xanthine oxidase, 50mM potassium phosphate, pH=7.5. The free XO were put in 0.l cm Spectrosil quartz cuvette (Starna Cells) and the cuvette was put in the Jasco CD Spectrometer at 20°C. Three scans were averaged by using Chirascan Pro-Data Viewer software. Equipment parameters consisted of a 20.0–99.0°C gradient with stepped ramping, 10°C per step, with a 60 s equilibration time, and a 20 s read at each step subsequent to equilibration. Ellipticity profiles were fitted to a two-state model.

The instrument was adjusted to zero using phosphate buffer 50mM, then the spectra of free xanthine oxidase solution (0.1U/ml) was measured, also the spectra of XO-MNPs, XO-MNP@cholesterol, and XO-MNP@sulfadiazine-XO were measured.

3. Result and Discussion

The size and morphology of the synthesized magnetic nanoparticles were confirmed via TEM, which demonstrates the shape varies from spherical to cubic or truncated cubic (octahedral) structures for large particles with broad size distribution (20–100 nm) The formed MNP were ball shape). as shown in Figure 1.

The images showed that the MNP-Cholesterol & MNP-Sulafadiazine are larger than the MNPs and the larger size leads to transformation in the shape from small ball shape into larger cubic shape ^{[20, 21]}. Furthermore, the (Cholesterol &Sulafadiazine) layers appear as lighter than the cubic core of magnetic nanoparticles indicating the formation of new nanoparticles.

Figure 1. TEM images of the prepared MNP under very high resolution A-MNP, B-MNP@Cholesterol and C-MNP@Sulphadiazine

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According to TGA plots that presented the weight loss percentage per degree of MNPs plotted versus the increase in temperature as in Figure 2. It is clear that the most weight loss percentages occur at 100°C, due to loss of water that bound weakly to the structure of NPs. There is a gradual increase in the weight loss percentage as temperature increases due to loss of water that tightly bound to the structure of the MNP. The weight loss from MNP@Cholesterol and MNP@Sulphadiazineoccurs at lower temperature than the original MNPs. This results indicating the higher adsorption of water for the two new NPs than the free MNPs. The change in surface properties is expected after coating with cholesterol and sulphadiazine. Availability of various functional groups including hydrophilic and hydrophobic groups coating the surface of MNPs should affects the physicochemical properties of the surface including its ability to adsorb or absorb water molecules ^[22].

Figure 2. Weight loss percentage per degree of MNP as a function of increasing temperature

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After adsorption of XO`s on the MNPs, MNP@Cholesterol, and MNP@Sulafadiazine, the formed composites were separated and visualized by TEM. Figure 3 showed the TEM images of the composites of XO`s with MNPs, MNP@ Cholesterol, and MNP@Sulafadiazine, respectively. These images showed cubic particles larger than the original (uncoated) particles surrounded by a lighter layers of the protein corona.

Much research showed that the interaction between proteins and NPs surface leads to the formation of proteins “corona” around NPs that largely defines their biological identity as well their potential toxicity and other vital properties ^{[23, 24]}.

The proteins that readily occupy the NPs surface should either have high concentrations and high association rate constants or have lower concentrations but a higher affinity ^[12]. However in the presence of one type of proteins in the solution in contact with NPs, the factors affecting the interaction properties include the protein characteristics such as electrostatic interactions, protein stability, and kinetic parametersin addition to the properties of the surface active sites.NPs have significant adsorption capacities due to their relatively large surface area, therefore they are able to bind or carry other molecules such as chemical compounds, drugs, and proteins attached to the surface by covalent bonds or by adsorption. Hence, the physicochemical properties of NPs, such as charge and hydrophobicity, can be altered by attaching specific chemical compounds, peptides or proteins to the surface ^{[25, 26]}.

Figure 3. A-Xanthine Oxidase-MNP compositeB-MNP@Cholesterol-XOcomposite and C-MNP@ Sulfadiazine-XO composite

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The changes in the secondary structure of xanthine oxidase after adsorption on MNP`s was analyzed via CD spectroscopy, which is particularly efficient in determining the protein folding and in characterizing the secondary structure of protein and denaturant stabilities ^[27]. The secondary structure can be determined via CD spectroscopy in the far-UV spectral region (200–260 nm), and the chromophore at this wavelength is the peptide bond. Figure 4 showed the CD diagrams of the XO adsorbed on MNPs, MNP@Cholesterol and MNP@Sulphadiazine, respectively in comparison with free enzyme.

Figure 4. CD for the free XO, MNP-XO, MNP@Cholesterol- XO, MNP@Sulphadiazine-XO

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The CD results were analyzed using the K2D3 web server to determine the change in the X secondary structure from the CD spectra according to the published software ^[28].The results of the analysis are presented in Table 1.

Table 1. The percentages of secondary protein structure components of free and immobilized XO enzyme

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Conformational rearrangements in which the secondary and/or tertiary structure of the protein changes because of adsorption have been observed. These changes could even be a substantial driving force for adsorption. The significant change in the secondary structure indicates that the adsorption of XO on MNPs involves a contact with the domains rich in alpha structures on the XO molecule surface. The affected secondary structures depend on the change in orientation towards the surface active sites and the magnitude of the adsorption forces. In one study, it is found that the secondary structure of glucose oxide enzyme (protein) is changed after adsorption on NP surface and maintain its native structure when adsorbed on other type of NP ^[29].

Fluorescence spectroscopy was used to monitor the changes in the tertiary structure of the protein. The three amino acids with intrinsic fluorescence properties are phenylalanine, tyrosine, and tryptophan. However, only tryptophan was used experimentally because the quantum yields (emitted photons/excited photons) of tryptophan are sufficiently high to achieve good fluorescence signal. Therefore, this technique is limited to the proteins with tryptophan. These residues can be used to follow the protein folding because fluorescence properties (quantum yields) are sensitive to the environment, which changes when the protein folds/unfolds ^[30].

The fluorescence emission of the free and adsorbed xanthine oxidase was determined from the spectra obtained using a carry eclipse fluorescence spectrophotometer. Figure 5 showed the spectra of fluorospectrophotometry of the XO adsorbed on MNPs, MNP@Cholesterol and MNP@Sulphadiazine, respectively in comparison with free enzyme.

Figure 5. Fluorescence spectra of free XO, MNP-XO, MNP@Cholesterol-XO, and MNP@Sulphadizaine-XO

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The change in the spectra may be due to the change in the environment polarity of the protein or the environment surrounding the tryptophan residues, which may be buried in the hydrophobic core of proteins; these changes result in a shift by 10 nm to 20 nm compared with those on the surface of the protein ^[31]. Figure 5 showed a significant changes in the tertiary structure of XO enzyme after adsorption on MNPs. The fluorescence emission of XO`s residues (free and dispersion) were determined from spectra obtained on carry eclipse fluorescence spectrophotometer, a speed of scan 600 nm/min, the samples were emission at scanned from 290–400 nm at room temperature. Fluorescence emission for free XO`s and dispersion XO`s with MNP`s was at (400 nm) ^[31]. Although tyrosine and phenylalanine are natural fluorophores in proteins, tryptophan is the most extensively used amino acid for fluorescence analysis of proteins. In a protein containing all three fluorescent amino acids, observation of tyrosine and phenylalanine fluorescence is often complicated due to the interference by tryptophan by resonance energy transfer ^[32]. Fluorescence of phenylalanine is weak and seldom used in protein studies ^[32]. Hence, the term "natural protein fluorescence” is almost associated with tryptophan fluorescence.

In order to obtain a better description for the behavior of the XO`s adsorption on the surfaces of the nanoparticles, the adsorption isotherm were constructed for the adsorption of XO`s on MNPs, MNP@Cholesterol, &MNP@SulfadiazineAdsorption isotherm of XO enzyme on MNPs is presented in Figure 6.

Figure 6. Adsorption isotherm of XO enzyme on MNPs, MNP@Cholesterol, and MNP@Sulphadiazine

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Plotting Ce versus Qe produce adsorption isotherm that is applied very well with the Sips equation as seen in the above figures. The Sips model for heterogeneous adsorption requires a parameter that describes the maximum adsorption amount Qm (mg/g). The parameters could have been estimated as part of the fitting exercise, but we chose to estimate the parameters from the general shape of the molecule ^[33]. The heterogeneity factors of Sips equation, t & k, of the adsorption of xanthine oxidase on MNP`s, MNP@Cholesterol-XO and MNP@Sulphadiazine-XO were (t=0.623, Ks=0.159),(t=0.520, Ks=0.210), and (t= 0.620, Ks= 0.158), respectively. The (t) value for the adsorption process of XO on MNPs are lower than one indicating that the adsorbed XO molecules interact with neighboring XO molecules in different forces. This mechanism appears to be consistent with a low XO adsorption at low solution concentrations, followed by rapid increases (lateral effect). The low K<sub>s</sub> values for the adsorption processes suggest a small portion of the surface heterogeneity of magnetic nanoparticles <sup>[34]</sup>. The development of biocompatible nanometrials for enhancing or modifying the bio-properties is an important challenge in the biotechnology field <sup>[35]</sup>. The protein adsorption laws and principles are applicable on XO`s as a protein compounds. Proteins are not simply hard spherical colloidal particles. Because of this, several authors have considered changes in orientation, conformation and formation of two-dimensional structures to explain observed adsorption phenomena. Most proteins have a tertiary structure that is spheroidal with a major and minor axis, rather than spherical ^[36]. Hence, initial adsorption to a surface can occur in more than one orientation with respect to the surface. Subsequent transition in time from one orientation to the other is likely if a favorable higher contact area is created. When the concentration of bulk protein is high, overshoots in the adsorbed amount, have been reported in the literature ^[37].

The activity of the free XO enzyme and the enzyme immobilized on different types of magnetic nanoparticles was measured by using nine different concentrations of xanthine as a substrate and at human body temperature (37.5°C). The Lineweaver-Burk plots of the three experiments were shown in Figure 7.

Kinetic parameters Vmax and Km were calculated from the regression equation of each line and presented in Table 2. The activity of the enzyme at the highest concentration of xanthine used in the experiments is also cited in the Table 2.

Table 2. Vmax and Km of the free XO enzyme, immobilized on MNPs, MNPs@Cholesterol, and MNPs@Sulphadiazine

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The changes in the V_maxand K_m indicated a mixed inhibition of the activity of the XO enzyme after interaction with magnetic NPs. These changes may be due to the changes in the secondary and tertiary structures of the enzyme as a result of the interaction between the XO molecule with the active sites on the NPS. Proteins are flexible molecules that undergo conformational changes as part of their interactions with other proteins or drug molecules ^[38]. Many proteins undergo changes in their structure during biochemical processes such as binding and transport. Such conformational changes are crucial to the functioning of proteins, and by extension, to most biological processes.

Figure 7. Lineweaver-Burke lines of the Free XO and XO immobilized on MNPs, MNP@Cholesterol, and MNP@Sulphadiazine

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Binding of proteins on planar surfaces often induces significant changes in the secondary structure, but the high curvature of NPs aids proteins in retaining their original structure. However, a study of a variety of nanoparticle surfaces and proteins indicates that perturbation of protein structure still occurs to varying extents. The proteins show a rapid conformational change at both secondary and tertiary structure levels ^{[39, 40]}. The structure and composition of the protein corona depends on the shape, composition, size, surface charges, and surface functional groups of the nanomaterial. Furthermore, the nature of the physiological compartment e.g., blood, interstitial fluid, etc., and the duration of exposure affect the protein corona structure and composition which alters the biological identity of the original biomolecules ^[41].

4. Conclusions

Two new magnetic nanoparticles were prepared and identified; MNP@Cholesterol and MNP@Sulfadiazine. These new particles in addition to magnetic Fe₃O₄ have the ability to adsorb and immobilize xanthine oxidase efficiently for future medical applications. Desorption quantities of the XO`s from the surface of NPs were low in general and the adsorption processes were exothermic.

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