Doxorubicin loaded on aminosilane modified superparamagnetic magnetic nanoparticles and its characterization

Transcription

1 American Journal of Oil and Chemical Technologies: Volume 4. Issue 3. May 2016 Petrotex Library Archive American Journal of Oil and Chemical Technologies Journal Website: Doxorubicin loaded on aminosilane modified superparamagnetic magnetic nanoparticles and its characterization Rostamzadeh Mansour, S 1 *, Mousavi, M 1 1Department of Chemistry, Ardabil Branch, Islamic Azad University, Ardabil, Iran Abstract: In the past few decades, Magnetite (Fe 3 O 4 ) nanoparticles have attracted growing research interest that this material has many applications in medicine and drug delivery, Coated magnetic particles, called carriers, are very useful for delivering chemotherapeutic drugs. In this work, Magnetite nanoparticles (NPs) were synthesized by co-precipitating Fe 2+ and Fe 3+ in an ammonia solution. Magnetite NPs coated with 3-aminopropyltriethoxysilane (APTES) were prepared by silanization. Then Doxorubicin (DOX) drug was loaded at modified magnetic nanoparticles. The structural, morphological and magnetic properties of as-prepared sample were characterization by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectra, scanning electron microscopy/energy dispersive x-ray analysis (SEM-EDAX) and magnetic measurements were investigated using vibrating sample magnetometer (VSM). We demonstrate that the drug DOX is attached to the nanoparticles surface, that the binding of DOX to the nanoparticles was confirmed by FT-IR analysis. The potential of doxorubicin against the cancer cells K562 was evaluated using the MTT assay. That, cell culture experiments demonstrated the potential of these nanosystems as an effective dual targeting nanoplatform for the delivery of anticancer drugs. Keyword: loaded, Doxorubicin, magnetic nanoparticles, 3-aminopropyl tri methoxysilan, superparamagnetic 1. Introduction 1. Magnetic NPs have attracted much interest because of its extensive applications in the past decades. It was widely used in magnetic resonance imaging (MRI), drug targeted therapy, bioactive molecule separation such as enzyme and protein [1-4]. Among these magnetic NPs, spinel ferrite nanoparticles have attracted much attention because of their electronic, magnetic, and catalytic properties, all of which are different from those of their bulk counterparts. Hyperthermia property in these nanoparticles can be very efficient in killing cancer cells through hyperthermia produced by magnetic nanoparticles guided to the tumor cells using an external magnetic field. While, a possible approach for increasing of the magnetic nanoparticles circulation time in the blood stream is modifying the surface nanoparticles with silica coating films to disperse them and minimize or eliminate protein adsorption [5]. Thus, coating magnetic nanoparticles with silica is becoming a promising. Silica and its derivatives coated onto the surfaces of magnetic nanoparticles may help to change their surface properties. With the appropriate coating, the magnetic dipolar attraction between magnetic nanoparticles may be screened thus minimizing or even preventing aggregation. The coating film could also provide a chemically inert layer for the nanoparticles, which is particularly useful in biological systems [6]. Also, silica-coated magnetic nanoparticles can be easily allowed to conjugate its surface with various functional groups [7]. In this work, our goal is to conjugate the DOX with APTMS coated iron oxide nanoparticle carriers to evaluate the potential and efficiency of the doxorubicin loaded magnetic nano carriers against resistant leukemia cancer cells, K562 by performing MTT assay. XRD, SEM-EADX, FTIR, and VSM were used to characterize the synthesized nanoparticles. Noting that, doxorubicin (Adriamycin), is a powerful drug in the fight against cancer cells joined the oncologic practice in the late 1960s [8]. It exerts antitumor activity through its inhibition of topoisomerase II and thus prevents chain unfolding and separation in DNA replication as well as DNA repair. The clinical use of DOX is limited by the Industrial And Mining Research Centre Cycle Science & Industry Company, Tehran, Iran cs.isi.pjs@gmail.com phone Number:

2 2.1. Materials Authors /American Journal of Oil and Chemical Technologies 4 (2016) resistance developed by cancer cells and by strong side effects. In this condition, drug targeting approach can help to prevent side effects and increase cytotoxicity of anticancer drugs by directly delivering of the drug to the pathological site, leading to the increased drug concentrations at the tumor site [9]. The results obtained in this work indicate that DOX loaded on APTMS coated iron oxide nanoparticle is promising for magnetically targeted drug delivery system. 2. Materials and Methods All the reagents were of analytical grade and used as received without further purification, including FeCl2.4H2O, FeCl3.6H2O, ethanol, NaOH, ammonia solution (25%), (3-aminopropyl)-trimethoxysilane (APTMS) and Doxorubicin hydrochloride (DOX). The Human Leukemia cancer cells, K562 were provided by Pastor Institute (Iranian Type Culture Collection (NCBI Code; C122) and used as a test model. Deionized water was used as a solvent Preparation of samples Preparation of superparamagnetic magnetite nanoparticles g of FeCl 2.4H 2 O (0.016 mol) and g of FeCl 3.6H 2 O (0.028 mol) were dissolved in 320 ml of deionized water, such that Fe 2+ /Fe 3+ = 1/1.75. The mixed solution was stirred under N 2 at 80 C for 1 h. Then, 40mL of NH 3.H 2 O was injected into the mixture rapidly, stirred under N 2 for another 1 h and then cooled to room temperature. The precipitated particles were washed five times with hot water and separated by magnetic decantation. Finally, magnetic NPs were dried under vacuum at 70 C. It is reasonable to assume that growth and nucleation reaction between FeCl 3 and FeCl 2 would take place following the reaction mechanism below: 2FeCl 3 6H 2 O+FeCl 2 4H 2 O+8NH 3 Fe 3 O 4 +8NH 4 Cl + 6H 2 O Preparation of Magnetite nanoparticles coated by (3-aminopropyl)-trimethoxysilane 4.225g magnetic nanoparticles were sonicated in 150 ml ethanol/water (volume ratio, 1:1) solution for 30 min to get uniform dispersion. Then 3 ml of APTMS was added to solution under N 2 atmosphere at 400C for 2 h. After that the solution was cooled to room temperature. The prepared APTMS-modified Fe 3 O 4 NPs were collected with a magnet, and washed with ethanol, followed by deionized water for three times. Finally, APTMS-modified Fe 3 O 4 NPs were dried under vacuum at 70 0 C Doxorubicin (DOX) drug loading The water-soluble anti-cancer drug DOX was chosen as a model drug. The DOX loading was carried out by dispersing 5 mg of core/shell Magnetite nanoparticles in 5 ml aqueous DOX solution (drug concentration=0.1 mg/ml) following the experimental procedure described by Kuznetsov et al. The mixture of silica coated Magnetite nanoparticles in DOX was shaken in a rotary shaker (200 rpm) at 37 C for 26 h to facilitate DOX uptake Cytotoxicity assays The leukemia cancer cells, K562, provided by Pastor Institute were seeded in a standard 96-well plate at 2 X 10 4 cells per well and grown for 24 h. The culture medium was then discarded and cells were treated for 8 and 24 hours with 4, 8, 16, 24, 48, 96 and 192 mg/ml of nanocariers contained doxorubicin. The effect of DOX and DOX-loaded nanocarriers on the cell viability were assessed using a tetrazolium dye (3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, named MTT assay. The medium containing DOX was discarded and remaining cells were rinsed thrice with phosphate buffered saline (PBS) and incubated during 4 h with a 0.5 mg/ml MTT solution in culture medium. The medium was finally replaced by 200 µl of DMSO to dissolve formazan crystals formed by viable cells and the percentage cell viability (%) compared to vehicle-treated control cells. Arbitrary assigned 100% viability was determined by measuring absorbance at 570 nm [10,11] Characterization The powder XRD was performed at room temperature with a PW 1800 (PHILIPS) X-ray diffractometer equipped with a Cu Ka radiation source (k = nm). The lattice constant and the average crystallite size were 2

3 determined by the positions and full width at half maximum (FWHM) of the (311) peaks by using Scherrer formula. The element analysis was carried out by an energy dispersive X-ray analysis (EDAX) on a Field emission scanning electron microscopy (FESEM, Hitachi F4160 oxford instrument). The Fourier transform infrared spectra (FT-IR) (NICOLET 5700) were also measured. The magnetic measurements were performed by using a vibrating sample magnetometer (VSM) on a physical property measure system (VSM Lecshore). The DOX concentration was measured by a UV VIS (Shimadzu UV 1700) spectrophotometer equipped with quartz 1 cm optical length cuvettes (Hellma). 3. Results and discussion 3.1. XRD Characterization The structures of the Fe 3 O 4 /APTMS nanoparticles were investigated by X-ray diffraction and FT-IR spectroscopy. Figure 1 (a, b) shows the X-ray diffraction patterns of Fe 3 O 4 and Fe 3 O 4 /APTMS nanoparticles. The diffraction peaks assigned to the scattering from (220), (311), (400), (422), (511) and (440) planes of the spinel Fe 3 O 4 are in agreement with the reported data (JCPDS card NO ) and confirm maintaining ferrite spinel structure in both the case. The average of crystalline size of Fe 3 O 4 and Fe 3 O 4 /APTMS nanoparticles at the characteristic peak (311) were calculated using Scherrer s formula and were nm and nm respectively. (b) (a) 3.2. FT-IR spectra Figure1. XRD patterns of Fe 3 O 4 (a) and Fe 3 O 4 /APTMS (b) nanoparticles. Figure 2 shows the FT-IR spectra of Fe 3 O 4 (a) and Fe 3 O 4 /APTMS (b) nanoparticles. In the spectra, the strong peaks near 582 cm -1 and 465 cm -1 are associated with the stretching vibrations of the Fe-O bonds, which represents the presence of magnetic NPs. The peaks around cm -1 and 1624 cm -1 are related to the stretching and bending vibrations of the H O H bond, respectively, showing the physical absorption of H 2 O molecules on the surfaces. As shown in Fig. 2(b), the characteristic peaks of spinel Fe 3 O 4 appear in the infrared spectrum of Fe 3 O 4 /APTMS nanoparticles. The band at 1062 cm 1 can be attributed to the Si O Si asymmetric stretching vibrations; these vibrations are expected to have strong IR absorption consistent with how they appear in the spectrum. The weak band at 806 cm 1 is due to Si OH deformation. Fig. 3 shows the FTIR spectra of pure DOX and 3

4 DOX-conjugated APTMS coated iron oxide nanoparticles. This FTIR result, it can be interpreted that attachment of DOX to the APTMS coated iron oxide nanoparticles occurs via the interaction of NH 2 and OH groups of DOX with NH 2 groups of APTMS through hydrogen bonding which is consistent with previous report [12]. Figure2. FT-IR spectrum of Fe 3 O 4 (a) and Fe 3 O 4 /APTMS(b) nanoparticles Figure3. FTIR spectra of (a) pure DOX and (b) DOX-conjugated APTMS coated iron oxide nanoparticles SEM and EDAX The surface morphology of the magnetic nanoparticles have been studied by scanning electron microscopy (SEM) method. Fig. 4(a,b) represents SEM images of Fe 3 O 4 and Fe 3 O 4 /APTMS nanoparticles. The X-ray energy dispersive data (EDAX) of these two materials are given in Fig. 5 (a, b. The elemental analysis shows the presence of Fe, O (see Fig.5.a) indicates the formation of ferrite. Also elemental analysis shows the presence of Fe, O, C and Si (see Fig.5.b) indicates the formation of Fe 3 O 4 /APTMS nanoparticles. 4

5 Figure4. SEM images of the Fe 3 O 4 (a) and Fe 3 O 4 /APTMS nanoparticles (b) Figure5. Dispersive analysis of X-ray (EDAX) of Fe 3 O 4 (a) and Fe 3 O 4 /APTMS nanoparticles (b) 3.4. Magnetic properties Figure 6 shows the magnetic hysteresis loops of the as-prepared Fe 3 O 4 and Fe 3 O 4 /APTMS nanoparticles at room temperature. The absence of remanence in the hysteresis curves indicates that magnetic particles are superparamagnetic. The magnetic parameters such as saturation magnetization (Ms) and coercivity (Hc) of Fe 3 O 4 determined by hysteresis loops (Table 1) clearly decrease upon coating. Figure 6(b) shows the saturation magnetization and coercivity of Fe 3 O 4 decreased after APTMS coating. Core/shell ferrite nanoparticles show lower magnetization saturation than the uncoated ferrite nanoparticles, this is due to the effect of silica shell coating where 5

6 each particle was separated from its neighbors by the shell layer leading to decrease the magnetostatic coupling between the particles. Figure6. Hysteresis loops of the as-prepared Fe 3 O 4 (a) and Fe 3 O 4 /APTMS (b) nanoparticles with a magnetic field of 15kOe Table 1. Magnetic parameters of Fe 3 O 4 and Fe 3 O 4 /APTMS nanoparticles Samples Ms (emu/g) Hc (Oe) Fe 3 O Fe 3 O 4 /APTMS Cytotoxicity evaluation of DOX-conjugated APTMS coated iron oxide nanoparticles Cell mortality experiments The effect of cytotoxicity of DOX and Fe 3 O 4 / APTMS/DOX suspensions on K562 cells viability within 8 and 24 h, studied in this work is presented in fig. 7. The results showed that DOX loaded on Fe 3 O 4 /APTMS was significantly more active against K562 cells than pure DOX solution: Thus Fe 3 O 4 alone has no cytotoxic effect on K562 cells, while DOX loaded Fe 3 O 4 /APTMS induced higher cell mortality than conventional drug. These data indicate that the 6

7 intracellular action of internalized Fe 3 O 4 /APTMS /DOX plays an important role in the gain of cytotoxicity. These complex mechanisms may become the subject of more extensive studies in the future. Figure7.The percentage K562 cells viability (%) for DOX and DOX loaded Fe 3 O 4 /APTMS within 8h (a) and 24 h (b) compared to the vehicle-treated control cells, arbitrary assigned 100% viability K562 cells morphology The morphology and behavior of DOX loaded Fe 3 O 4 /APTMS against leukemia cancer cells, K562 cultured in vitro was observed under phase-contrast microscope and evaluated by MTT assays. The phase-contrast micrographs obtained in figure 8show cell attachments on the DOX loaded Fe 3 O 4 /APTMS after culture for 24h at concentration 1 and 4 μm. Figure8. Phase contrast micrographs of K562 cell before and after treatment with DOX loaded Fe 3 O 4 /APTMS nanocarriers for 24h at concentration 1 and 4 μm 7

8 4. Conclusions Authors /American Journal of Oil and Chemical Technologies 4 (2016) In this work, Magnetite nanoparticles (NPs) were synthesized by co-precipitating Fe 2+ and Fe 3+ in an ammonia solution. Magnetite NPs coated with 3-aminopropyltriethoxysilane (APTES) were prepared by silanization. The structural, morphological and magnetic properties of as-prepared sample were characterization by X- ray diffraction (XRD), Fourier transform infrared (FT-IR) spectra, scanning electron microscopy/energy dispersive x-ray analysis (SEM-EDAX) and magnetic measurements were investigated using vibrating sample magnetometer (VSM). That the binding of DOX to the nanoparticles was confirmed by FT-IR analysis. The potential of doxorubicin against the cancer cells K562 was evaluated using the MTT assay. That, cell culture experiments demonstrated the potential of these nanosystems as an effective dual targeting nanoplatform for the delivery of anticancer drugs. 5. References: [1] M. Faraji, Y. Yamini, M. Rezaee, Magnetic Nanoparticles: Synthesis, Stabilization, Functionalization, Characterization, and Applications, J. Iran. Chem. Soc, 7,1-37,2010. [2] C. Zhang, T. Liu and JN. Gao, Recent Development and Application of Magnetic Nanoparticles for cell Labeling and Imaging, J. Mini-Rev. Med. Chem, 10, ,2010. [3] P. Tartaj and MD. Morales, Veintemillas-Verdaguer S., (2003), The Preparation of Magnetic Nanoparticles for Applications in Biomedicine, J. Phys. D: Appl. Phys, 36, ,2003. [4] J.Giri, P. Pradhan and V. Somani, Synthesis and Characterizations of Water-based Ferrofluids of Substituted Ferrites [Fe 1-x BxFe 2 O 4, B=Mn, Co (x=0-1)] for Biomedical Applications, J. Magn. Magn. Mater, 320, ,2008. [5] Y.H. Deng, C.C. Wang, J.H. Hu, W.L. Yang and S.K. Fu, Investigation of formation of silica-coated magnetite nanoparticles via sol-gel approach, Collid and surf physic and engine, 262, 87-93, [6] N. Sounderya and Y. Zhang, Use of core/shell structured nanoparticles for biomedical applications, Recent Patents on Biome Engine,1,34-42, [7] A. K. Gupta and M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomaterials, 26, , [8] P.K. Singal and N. Iliskovic Doxorubi, cin-induced cardiomyopathy, N Engl J Med, 339, , [9] O. Vieseh, J. Gunn and M. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv drug deliv rev, 11, 002, [10] C. Sun, J.S.H. Lee and M.Q. Zhang, Magnetic nanoparticles in MR imaging and drug delivery, Advanced Drug Delivery Reviews, 60, , [11] T. Mosmann, Evaluation of Tetrazolium-based Semiautomatic Colorimetric Assay for Measurement of Human Antitumor Cytotoxicity, J Immunol Methods, 56, 55-63, [12] S. Kayal and R. V. Ramanujan, Doxorubicin loaded PVA coated iron oxide nanoparticles for targeted drug delivery, 30, ,