Synthesis of Fe₃O₄-Gold hybrid nanoparticles coated by bovine serum albumin as a contrast agent in MR imaging

Abstract

Despite the over spatial separation and the ability to determine soft tissues, insufficient contrast is the shortcoming of magnetic resonance imaging (MRI) that could be circumvented by the use of contrast agents. The use of MRI contrast agents are widely applied to enhance the vision of internal body structures. Nano-sized contrast materials have unique application advantages compared to other contrast agents due to their size and shape. However, for contrast agents such as bare iron (II, III) oxide (Fe₃O₄) magnetic nanoparticles (NPs), aggregation and accumulation are the main shortcomings. Thus, surface modifications are necessary for their use in biopharmaceutical applications. Gold, Au, nanoparticles are of big interesting for use in biomedical purposes due to their chemical stability and oxidation resistance. In this study, we synthesized magnetic Fe₃O₄–Au hybrid NPs with a facile method and coated them with bovine serum albumin (BSA) to increase their chemical stability and biocompatibility. Afterwards, the hybrid nanosystem was characterized by some methods, and their potential to increase MRI contrast was investigated by the phantom MRI experiments. Our data showed that the signal intensity on MR images was significantly reduced, thus confirming the contrast ability of the formulated Fe₃O₄–Au-BSA NPs.

1. Introduction

With of personalized therapeutic methodologies and precise surgical techniques, the emergence of accurate imaging is an urgent need to assist physicians in accurate diagnosis and monitoring of disease or treatment. This issue is crucial for patient survival in the area of oncology, where seen of pathologic features is of particular importance [1].

MRI uses a non-ionizing radiation and merits from big spatial dissociation and superior soft-tissue contrast [2]. Nevertheless, insufficient contrast is a problem in many MRI applications. Therefore, the use of contrast agents is essential as an advanced route to reliable diagnosis [3].

Iron oxide nanoparticles (IONPs) were the early nanoparticular MRI contrast agent to be introduced as a liver contrast agent two decades ago.

Fe₃O₄ is a prevalent and unique nanoparticular agent currently used for MR imaging [4]. The United States Food and Drug Administration (FDA) has given approval to some IONPs. Unfortunately, some of these particles were withdrawn later, because of economic reasons, safety concern, allergic reactions, as well as unclear reasons. Also, there is some IONPs that is still applied in clinical practice [5]. Due to superparamagnetic behavior of Fe₃O₄ nanoparticles could be used as contrast agents in MRI. Fe₃O₄ nanoparticles towards with other nanoparticles as gold, bismuth, or silver could be used as hybrid contrast agents in XCT and MRI. It is easily to synthesize double structures. Fe₃O₄–Au NPs were supposed to be a suitable contrast agents. This nanoplatform offers excellent superparamagnetic ability as it holds a magnetic (Fe₃O₄) unit [6] and have been extensively studied in vivo and pre-clinically in recent years [[6], [7], [8], [9]]. When Au is coated as a shell around Fe₃O₄, the application of Fe₃O₄ core fails to enhance MRI contrast signal because Fe₃O₄ provides T2-weighted MRI, and the Au shell prevents association with water molecules present in the tissue. Compared to the core/shell structure, Fe₃O₄–Au heterodimer structure is perfectly suited to act as contrast agents [10]. Therefore, using Fe₃O₄–Au heterodimers as a single entity allows us to tune magnetic properties [10,11].

Despite they are attractive candidates, the application of Fe₃O₄–Au hybrid NPs undergoes demanding synthetic procedures such as high temperature, high cost, toxicity, and tedious post-reaction surface modifications. Therefore, the synthesis and modification of Fe₃O₄–Au NPs by simple and cost-effective methods has been particularly focused [12,13]. Availability, low cost, easy of purification, ligand-binding making are advantages of BSA in pharmaceutical industries. Therefore BSA, could be applied to cover for stability of nanoparticles such as gold in biological medium. Nosrati et al. investigated safety and radiation of Fe₃O₄–Au hybrid NPs [14]. To this work goals to prepare Fe₃O₄–Au hybrid NPs with a simple method and interact them with BSA to great chemical stability. Subsequently, the potency of these hybrid NPs to induce MRI contrast was investigated.

2. Materials and methods

2.1. Materials

All of the materials used in the this study were of analytical grade, and were prepared from the Sigma-Aldrich (St. Louis, USA) and Merck (Kenilworth, USA).

2.2. Methods

2.2.1. Synthesis of Fe₃O₄

For the synthesis of Fe₃O₄, our previous protocol was used [14]: First, under vigorous stirring and N₂ atmosphere, 7.0 mL NH₄OH (25%) were added dropwise to hot (60 °C) aqueous solution of 110.0 mg FeCl₃·6H₂O, and 40.0 mg FeCl₂·4H₂O. When the black color became apparent, the solution was placed on a stirrer for 2 h and the Fe₃O₄ particles were separated using a high power magnet and washed three times with distilled water. Subsequently, the colloidal stability of bare Fe₃O₄ was increased with ultrasonic force. This step is the key to regulate the dispersion of bare Fe₃O₄. Finally, the fabricated NPs were dispersed in deionized water.

2.2.2. Synthesis of Fe₃O₄–Au heterodimers

Fe₃O₄–Au heterodimers were synthesized as follows [14]: 152.0 μL HAuCl₄ (2.0 M) and 12.0 mL sodium citrate (1% W/V) were added to a suspension of 30.0 mg Fe₃O₄ in 90.0 mL H₂O. Then, the reaction mixture was stirred for 15 min, and 15.0 mL of freshly prepared NaBH₄ (0.6 mg/mL) was added dropwise. The appearance of cherry color at this stage is the confirmation of Au nanoparticle synthesis. Finally, the fabricated Fe₃O₄–Au heterodimers were magnetically separated from the reaction medium and washed with deionized H₂O.

2.2.3. Preparation of protein coated Fe₃O₄–Au

The synthesized Fe₃O₄–Au heterodimers were modified with BSA protein as stabilizer and stealth agent. Under magnetic stirring at room temperature, 100.0 mg of aqueous Fe₃O₄–Au solution was added to 300.0 mg BSA solution (50.0 mg BSA/mL). The reaction continued for 24 to allow BSA to attach to the surface of Fe₃O₄–Au heterodimers. The resulting Fe₃O₄–Au-BSA hybrids were collected by centrifugation at 18,000 rpm and washed with deionized water.

2.3. Characterization

The size and morphology of the synthesized nanoparticles were analyzed using transmission electron microscopy (TEM, FEI 120 kV), field emission scanning electron microscopy (FESEM, FEI Quanta 200FEG), as well as atomic force microscopy (AFM, XE‐ 100E PSIA). Energy dispersive X-ray spectrometry (EDX) was used to determine the elemental composition of nanoformulations. EDX spectra were analyzed using a vector-based algorithm to determine the relative abundance of carbon (C), iron (Fe), oxygen (O), nitrogen (N) and gold (Au) elements. FTIR spectra were recorded with an infrared microscope (Bruker, Tensor 27, USA). UV–Vis absorption spectra were recorded using a UV–Vis spectrophotometer (T80 double beam spectrophotometer, PG Instruments Limited). In addition, X-ray diffraction (XRD) analysis was performed using a powder X-ray diffractometer system (Malvern, PANalytical) to study the crystallographic structure of nanoparticles. Dynamic light scattering (DLS) (Malvern Instruments, Worcestershire, UK, ZEN 3600 model Nano ZS) was used for the size distribution and surface charge. Monitoring the particle size for 30 was used of physical stability.

2.4. MR imaging

In order to measure the transverse (R2) relaxation rates, a clinical 1.5 T whole body magnetic resonance scanner (Siemens Avanto Medical Systems, Berlin, Germany) was used. T2‐weighted phantom MRI images of the final nanoparticle formulations were obtained in a series of colloidal suspensions of various concentration. A series of pulse sequences with repetition times (TR) of 1600 ms and variable echo times (TE) of 47.0, 70.5, 105.8 and 141.1 ms (slice thickness: 4.5 mm) were applied to acquire T2-weighted images.

3. Result and discussion

3.1. Characterization

Nanoparticle characterizations were carried out using several techniques. As shown by the TEM image in Fig. 1a, it has been confirmed that Fe₃O₄–Au heterodimers have been successfully formed, since both Fe₃O₄ and Au nanoparticles are recognized separately by bright and dark contrasts. Due to the higher electron density, Au nanoparticles appeared as darker regions. In addition, spherical and monodisperse Fe₃O₄–Au heterodimers are also evident in the 500 nm and 1 μm scale SEM images shown in Fig. 1b and c, respectively. These findings are further supported by the AFM images shown in Fig. 1d. The results obtained by SEM and AFM show good uniformity in the distribution of nanoparticle size. The size of Fe₃O₄–Au nanoparticles was 78 nm. The zeta potential of the nanoparticles was −22.53 mV. For monitoring of the stability of Fe3O4–Au nanoparticles was evaluated by their size for one month using DLS. No great size changes during the 30-day monitoring Fe₃O₄–Au nanoparticles.

Fig. 1.

(a) TEM images of Fe₃O₄–Au-BSA NPs; (b and c) SEM images of Fe₃O₄–Au NPs; (d) AFM images of Fe₃O₄–Au-BSA NPs.

SEM images of different particle classes and their EDX spectra are shown in Fig. 2a Fe3O4 NPs, Fig. 2 (b) Fe3O4–Au NPs, and Fig. 2 (c) Fe3O4–Au-BSA NPs (column 1). Columns 2–6 show EDX elemental maps of Fe, O, C, Au and N to identify the distribution of these elements within representative NPs. Characteristic peaks of all elements expected to be present in structures have been observed in EDX spectra (column 1), and the maps of these elements indicate that they show a homogeneous distribution in the nanoformulations.

Fig. 2.

SEM images and EDX spectra and elemental maps of representative nanoparticles: (a) Fe₃O₄ NPs, (b) Fe₃O₄–Au NPs, and (c) Fe₃O₄–Au-BSA NPs. Elemental maps of Fe (orange), O (purple), C (blue), Au (red), and nitrogen (green) are shown for each class. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

XRD analysis was used to determine the crystal structure and composition of the nanoparticles produced. Fig. 3a shows the characteristic Fe₃O₄ peaks marked by # and correspond to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 40) planes, which are in good agreement with the standard XRD card of Fe₃O₄ nanoparticles (JCPDS file no. 019–0629). In addition, peaks marked by * at 2 theta degrees of 38.26, 44.50, 64.89, and 77.84 are associated with (111), (200), (220), and (311) planes of Au nanoparticles and well-suited with the standard XRD card of Au NPs (JCPDS 65–2870) [15]. These results confirm the presence of Fe₃O₄–Au heterodimers.

Fig. 3.

(a) XRD pattern of Fe₃O₄–Au NPs; (b) FTIR spectra of Fe₃O₄–Au NPs, pure BSA, and Fe₃O₄–Au-BSA NPs; (c) UV–vis spectra of bare Fe₃O₄, Fe₃O₄–Au, and Fe₃O₄–Au-BSA NPs.

The presence of the components of the nanoformulation was determined by FTIR analysis. The FTIR spectra of the Fe₃O₄–Au heterodimer, BSA, and Fe₃O₄–Au-BSA are shown in Fig. 3b. The Fe₃O₄–Au spectrum shows a sharp band at 540 cm⁻¹ corresponding to iron oxide in the Fe₃O₄–Au formulation [16]. In the next formulation, the amide I peak was seen for a protein with a high α-helix structure was observed at 1661 cm⁻¹ [17], which reveals the presence of BSA added to form a protein corona around heterodimer to active biocompatibility and colloidal stability [18]. Also, the characteristic primary amine bond can be seen at 1535 cm⁻¹. The broad band located at 3435 cm⁻¹ is also indicative of the primary amine in BSA. After the BSA unit was conjugated to the Fe₃O₄–Au heterodimer, the characteristic peaks of BSA shifted from 1661 cm⁻¹, 1535 cm⁻¹ and 3435 cm⁻¹ to 1700 cm⁻¹, 1800 cm⁻¹ and 3200 cm⁻¹, respectively. All the absorption features of the components on the final nanostructure are apparent in the FTIR spectrum of Fe₃O₄–Au-BSA, which confirms the successful fabrication of Fe₃O₄–Au-BSA.

UV–Vis analysis was also applied as a valuable tool to identify the synthesized hybrid nanostructures and to study the interactions between the components. This analysis helped us verify the step-by-step synthesis of the nanoparticles. In this regard, Fig. 3c clearly showed that the Fe₃O₄ nanoparticles did not represent any characteristic peak in the 200–510 nm region of the UV–Vis spectrum. Au nanoparticles are a characteristic plasmon resonance absorption peak at approximately 520 nm, which undergoes a red shift towards the upper wavelength due to interactions [19,20]. These results are utterly consistent with our data in Fig. 3c showing the characteristic Au peak at 529 nm in the spectrum of Fe₃O₄–Au. In addition, the characteristic BSA peak at 280 nm in the Fe₃O₄–Au-BSA spectrum is indicative of BSA conjugation to Fe₃O₄–Au. Thus, the final formulation of Fe₃O₄–Au-BSA was also confirmed by of UV–Vis spectroscopic data.

3.2. Evaluation of Fe₃O₄–Au-BSA NPs as MRI contrast agent

Magnetic resonance imaging (MRI) is an advanced technology used to detect, diagnose and monitor the disease by producing detailed three-dimensional anatomical images. It excites and detects the changes in the direction of the rotational axis of the protons in the water that make up living tissues. By changing the order of applied and collected external radio frequency (RF) energy pulses, different types of images are created. T2-weighted images are produced using longer echo time (TE) and repetition time (TR). In these images, contrast and brightness are predominantly determined by the transverse relaxation time (T2) properties of tissue. The contrast ability of the Fe₃O₄–Au-BSA nanosystem was studied in MR imaging on phantom agar gel. Phantom MRI experiments were performed to assess the detectability of these nanoparticles. Our data in Fig. 4 suggests that at a constant concentration, the signal intensity decreases with the increase in TE, which is the time between sending the RF pulse and receiving the echo signal. Also, at a constant TE, the signal intensity decreases with the increase in concentration of Fe. Altogether, with the increase in both Fe concentration and TE, the signal intensity decreases dramatically. These findings confirm that the contrast in T2-weighted images is enhanced using Fe₃O₄–Au-BSA nanoparticles.

Fig. 4.

Phantom MRI scans: Signal intensity weighted images and T2 relaxation analysis curves of Fe₃O₄–Au-BSA NPs in phantom agar gel at different concentrations (ppm). Data as mean intensity within the region of interest (ROI).

4. Conclusion

Early and accurate detection of pathologic features is crucial, especially in oncology. Therefore, a big deal of attention has been paid to the evaluated of contrast agents to improve MR imaging. In the current study, Fe3O4–Au hybrid NPs have been produced by a convenient method and coated with BSA to increase the chemical stability and biocompatible nature of magnetic NPs. As a result of the characterizations carried out, it has been shown that the nanoparticles exhibit good size and morphological properties. Impressively, the phantom MRI experiment revealed the potential of Fe3O4–Au-BSA formulation as a contrast agent capable of reducing transverse relaxation time, T2. This study provides important data on the way to explore an appropriate contrast agent for evaluation of tumors through MR imaging.

Author contribution statement

Hossein Danafar: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.

Yasamin Baghdadchi: Analyzed and interpreted the data; Wrote the paper.

Murat Barsbay, Mohammadreza Ghaffarlou, Navid Mousazadeh, Ali Mohammadi: Performed the experiments.

Funding statement

This work was supported by the Deputy of Research of Zanjan University of Medical Sciences [A-12-430-60, ethical code:IR. ZUMS.REC.1400.444], ].

Data availability statement

The authors do not have permission to share data.

Declaration of interest’s statement

The authors declare no conflict of interest.