br conjugation with antibodies and or other bio functionaliz
conjugation with Glycoursodeoxycholic acid and/or other bio-functionalized substances more feasible. The silane-coated MNPs produced for this study are biocompatible and suitable for use in bioseparation [39,40].
Next, Herceptin was used as a humanized monoclonal antibody against epidermal growth factor receptor 2 (EGFR2, HER2, i.e. trastu-zumab, approved by the FDA in 1998)  as the targeting moiety to selectively target HER2-positive tumors. HER2 is a receptor protein over expressed on the surface of diﬀerent tumor cells, including 20–30% of human invasive breast, ovarian, lung and gastric carci-nomas, and which correlates strongly with the pathogenesis and prog-nosis of BC . Because it is an easily accessible cell surface receptor, it is easy for the nanoparticle conjugates to target. In addition, the ef-ficient internalization of HER2 following interaction with Herceptin Journal of Magnetism and Magnetic Materials 490 (2019) 165479
provides eﬃcient uptake of the antibody alone or when linked to drugs or drug carrier systems [42,43]. The Herceptin-conjugated MNP-Si were used in present study to bioseparate a HER2-expressing BC cell line, SK-BR-3, under a low magnetic field gradient.
2. Materials and methods
MNPs in the form of nano-powder (nano-Fe3O4) with 98% purity which were coated with 1% polyvinylpyrrolidone (PVP), ranging in diameter between 20 and 30 nm, were purchased from US Research Nanomaterials (USA). Herceptin was purchased from Roche (Switzerland). (3-aminopropyl) trimethoxysilaneas well as materials for Herceptin conjugation including N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was purchased from Sigma Aldrich (USA). Antibodies for flow cyto-metry including PE Mouse Anti-Human EpCAM (EpCAM-PE) and fluorescein isothiocyanate (FITC) Mouse Anti-Human IgG (IgG) were provided by BD Biosciences (USA). FITC anti-human CD45 antibody (CD45-FITC) and 7-amino-actinomycin D (7-AAD) were obtained from Bio-Legend (USA). Materials for cellular studies including fetal bovine serum (FBS), Trypsin-EDTA, penicillin-streptomycin and RPMI-1640 were purchased from Biosera (France). Human BCcell line SK-BR-3 was purchased from the Pasteur Institute of Iran (Iran). All chemicals were used as received without further purification.
Fourier-transform infrared (FT-IR) spectra were recorded with Bruker AXS (USA) Vertex 70 spectrometer to characterize the chemical structures of MNPs and MNP-Si. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) were performed on a TescanVEGA3 instrument (Czech); to prevent unfavorable electron charging, the sample was gold sputter-coated before the test. Vibrating sample magnetometer (VSM) testing was done with a VSM/AGFM instrument (Meghnatis Danesh Pajouh, Iran) at ambient temperature under a magnetic field. X-ray diﬀraction (XRD) was used to obtain the crystal-lography pattern of the MNPs and MNP-Si, and average size of the MNPs was calculated with Scherrer’s formula. The data were taken from a Bruker AXS D8 ADVANCE X-ray diﬀractometer (Cu Kα radiation with λ = 1.540 Å) at 25 °C. Thermogravimetric analysis (TGA) was used to compare MNP and MNP-Si with a TGA/DSC1 instrument (Mettler Toledo, USA) under environmental atmosphere with a heating rate of 10 °C/min up to 800 °C. A BD FACS Calibur flow cytometer (USA) was used to detect Herceptin on the MNP-Si and to couple HER2+ cells to Ab/MNP-Si. Attachment of the HER2+ cells to Ab/ MNP-Si was also verified by fluorescence microscopy (BX61 Olympus, Japan).
2.2. Coating MNPs with aminopropyltrimethoxysilane
MNPs were coated with silane by adding APTES as previously de-scribed [44,45]. Accordingly, in the first step, 1 g MNPs was sonicated in 50 mL dried toluene, followed by adding APTES (total amount of 4 mL), and the components were left to react with each other for 4 days at 80 °C. Then MNPs were collected with a magnet and washed three times with acetone and ethanol. Finally, coated MNPs (MNP-Si) were dried at 50 °C in a vacuum for 24 h. Further evaluations were done to verify silane coating on the MNP surface.
2.3. Bio-conjugation of MNP-Si with anti-HER2 antibody
To covalently bond Herceptin (anti-HER2) with MNP-Si, 50 µL Herceptin (22 mg/mL) was first activated for 30 min in the presence of 12 mg (0.1 mol) NHS and 8 mg (0.04 mol) EDC, which were dissolved in 50 µL deionized water. Next, 0.350 mLMNPs (2.5 mg/mL) was added to activated Herceptin and stirred for 4–5 h at room temperature. To re-move EDC/NHS and unreacted antibodies, the Ab/MNP-Si mixture was
Fig. 5. EDX spectra from sample of MNPs with (MNP-Si) and without APTES coating (MNP), with quantitative results.