Physica Medica xxx (2014) 1e6

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Preparation and characterization of magnetic gold nanoparticles to be used as doxorubicin nanocarriers Nihal Saad Elbialy*, 1, Mohammed Mahmoud Fathy 1, Wafaa Mohamed Khalil 1 Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 February 2014 Received in revised form 10 May 2014 Accepted 29 May 2014 Available online xxx

Magnetic targeted drug delivery (MTD), using magnetic gold nanoparticles (Fe3O4@Au NPs) conjugated with an anti-cancer drug is a promise modality for cancer treatment. In this study, Fe3O4@Au NPs were prepared and functionalized with thiol-terminated polyethylene glycol (PEG), then loaded with anticancer drug doxorubicin (DOX). The physical properties of the prepared NPs were characterized using different techniques. Transmission electron microscopy (TEM) revealed the mono dispersed nature of Fe3O4@Au NPs with an average size of 20 nm which was confirmed using Dynamic light scattering (DLS) measurements. Zeta potential measurements along with UVeVIS spectroscopy demonstrated surface DOX loading on Fe3O4@Au NPs. Energy Dispersive X-ray Spectroscopy (EDX) assured the existence of both iron and gold elements in the prepared NPs. The paramagnetic properties of the prepared NPs were assessed by vibrating sample magnetometer (VSM). The maximum DOX-loading capacity was 100 mg DOX/mg of Fe3O4@Au NPs. It was found that DOX released more readily at acidic pH. In vitro studies on MCF-7 cell line elucidated that DOX loaded Fe3O4@Au NPs (Fe3O4@Au-PEG-DOX) have more potent therapeutic effect than free DOX. Knowledge gained in this study may open the door to pursue Fe3O4@Au NPs as a viable nanocarriers for different molecules delivery in many diagnostic and therapeutic applications. © 2014 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

Keywords: Nanoparticles Doxorubicin Drug delivery Iron oxide nanoparticles

Introduction The main goal in cancer therapy is to destroy cancer cells without damaging normal cells. Conventional methods for cancer therapy including surgery, radiation and chemotherapy are either invasive or have many undesirable side effects due to the lack of tumor targeting. Recently, numerous drug nanocarriers have been developed to improve the delivery of anticancer drugs to target sites. Magnetic targeted drug delivery (MTD) has been used to improve the therapeutic performance of some chemotherapeutic agents and to reduce their undesirable side effects [1]. MTD involves the binding of an anticancer drug or any other biomolecules to biocompatible magnetic nanoparticles (MNPs) allowing their injection into blood stream as well as their accumulation into tumor site using a suitable external magnetic field [2]. The use of external magnetic field in MTD offers many advantages: (I) Increase the

* Corresponding author. Tel.: þ20 1001200674 (mobile), þ20 235676830 (office), þ20 222873230; fax: þ20 235676830. E-mail addresses: [email protected] (N.S. Elbialy), mohammed.elasal@ gmail.com (M.M. Fathy). 1 This manuscript was written with contributions from all authors.

accumulation of the chemotherapeutic agent into the targeted region. (II) Cellular up take of MNPs increases upon external magnetic field exposure [3]. (III) Finally, it was found that malignant cells proliferation might be inhibited using a static magnetic field [3]. Over the past decade, a wide range of MNPs with several composition and structures have been synthesized using different chemical methods. The most common of these materials are the iron oxides. Other metals and alloys such as Mn3O4, Co, Ni, FePt and FePd are less commonly employed, because of their rapid oxidation in air or their toxic effects [4]. Iron oxide nanoparticles (Fe3O4 NPs) cannot be directly used for drug delivery since (a) Fe3O4 NPs are unstable under physiological conditions [5]. (b) Fe3O4 NPs induced the formation of harmful free radicals [6]. (c) Inefficient surface binding will results in early release of loaded drug into blood stream leading to failure in delivering drug to tumor site [7]. On the other hand, gold nanoparticles (GNPs) are considered as an attractive system for many biological applications owing to the ease of their preparation, the possibility of their bioconjugation, in addition to their biocompatibility [8]. Further, gold nanoparticles are widely used in the field of drug delivery [9]. Based on the previously mentioned advantages of magnetic and gold nanoparticles, a promise combination of both can develop novel drug nanocarriers providing safety and efficacy of anticancer

http://dx.doi.org/10.1016/j.ejmp.2014.05.012 1120-1797/© 2014 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Elbialy NS, et al., Preparation and characterization of magnetic gold nanoparticles to be used as doxorubicin nanocarriers, Physica Medica (2014), http://dx.doi.org/10.1016/j.ejmp.2014.05.012

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drug. Thus, using magnetic gold NPs as drug delivery vehicles will offer a reasonable chemistry surface for biological application (owing to presence of gold shell) in addition to the active magnetic targeting of iron oxide core. Moreover, gold shell did not degrade the magnetic properties of iron oxide core [10]. Doxorubicin (DOX), one of the most effective anticancer drugs, is commonly used to treat diverse cancers including ovarian, bladder, thyroid, lung, breast, liver cancer [11]. In biological applications, DOX encountered many drawbacks such as cardiac toxicity, short half-life, lack of targeting and wide biodistribution [12]. To overcome these problems, the present work aims to develop a simple, fast and efficient route for the synthesis of magnetic gold nanoparticles (Fe3O4@Au NPs). The prepared nanoparticles (NPs) were functionalized with thiol-terminated polyethylene glycol PEG (Fe3O4@Au-PEG NPs) in order to improve NPs stability. Finally, Fe3O4@Au-PEG NPs have been used as DOX nanocarriers (Fe3O4@Au-PEG-DOX). Materials and methods Materials Gold (III) chloride (HAuCl4.3H2O,99.99%), sodium citrate (HOC)(COONa) (CH2COONa)2 (2H2O), FeCl3.6H2O, FeCl2.4H2O, 28% w/v% ammonia solution, doxorubicin hydrochloride, Neodymiumeironeboron magnetic discs 1.14 T, and thiol-terminated polyethylene glycol (PEG, MW5000) were purchased from SigmaeAldrich (St. Louis, MO, USA). Synthesis of Fe3O4 NPs

was drawn off with filter paper. The grid was left 5 min to dry at room temperature prior to the beginning of the examination. Absorbance spectra of GNPs, Fe3O4 NPs, Fe3O4@Au NPs and Fe3O4@Au-PEG-DOX NPs were measured using UVeVIS Spectroscopy (Jenway 6405, Barloworld Scientific, Essex, UK). Surface potential and size distribution of Fe3O4 NPs, Fe3O4@Au NPs and Fe3O4@Au-PEG-DOX NPs were determined using Zeta Potential/Particle Seizer (NICOMP TM 380 ZLS, USA). Elemental analysis of the prepared Fe3O4@Au NPs was carried out using EDX (FEI Tecnai G20, Super twin, Double tilt, LaB6 Gun, USA). The saturation of magnetization (Ms) of Fe3O4 NPs, and Fe3O4@Au NPs were evaluated using vibrating sample magnetometer (VSM) (LakeShore 7410, LakeShore, and Westerville, USA). The effect of PEG capping density on DOX-loading efficiency Different concentrations of PEG (0, 0.015, 0.02, 0.025, 0.03, 0.05, 0.10, 0.15 and 0.2 mg/ml) was added to 2 ml of Fe3O4@Au NPs solution (1 mg/ml) and stirred over 24 h. Then, 2 mg of DOX was added to each sample and stirred continuously for another 4 h thereafter, Samples were centrifuged at 13000 rpm for 30 min, and free DOX (unloaded) content in the supernatant was determined by measuring optical density at 498 nm [14]. The concentrations of DOX were calculated from the DOX calibration curve that was performed at 498 nm by UVevisible spectrophotometer. DOX loading on Fe3O4@Au-PEG NPs

Fe3O4 NPs were synthesized according to the previously described method [13]. FeCl3.6H2O and FeCl2.4H2O with a ratio of 1.622:0.9941 g respectively, were dissolved in 40 ml deionized water then 5 ml of ammonia solution (28% w/v%) was added. Ten minutes later, 4.4 g of sodium citrate was added and the reaction temperature was raised to 90  C with continuous stirring for 30 min. After cooling, the precipitate rinsed with acetone two times to remove extra free citrate. During rinsing, the sample was separated from the supernatant using a permanent magnet. Finally, the sample was dried in vacuum pump without heating.

DOX loading was carried out by dispersing 2 mg of Fe3O4@AuPEG NPs (carriers) in 2 ml aqueous DOX solution (1 mg/ml). At fixed time intervals 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4.5, 5.5 and 6.5 h(9 samples were prepared each sample represents one time interval) the carriers were separated from the liquid by centrifugation at 13000 rpm for 30 min and the optical density of residual DOX in the supernatant was measured at 498 nm by UVevisible spectrophotometer. The drug loading was determined as the difference between the initial DOX concentration and the DOX concentration in the supernatant (unloaded). As mentioned previously, the concentrations of DOX were calculated from the DOX calibration curve that was performed at 498 nm by UVevisible spectrophotometer.

Preparation of Fe3O4@Au NPs

DOX release from Fe3O4@Au-PEG-DOX NPs surface

Twenty ml of 0.5 mM HAuCl4 solution was heated and stirred till boiling. 15 ml of Fe3O4 NPs solution (1 mg/ml) was rapidly added and the color of the solution gradually changed from brown to red. Stirring continued for 10 min after the color change ceased. The heating source was switched off while the stirring continued until the solution cooled to room temperature. The Fe3O4@Au NPs were separated using a permanent magnet.

Fe3O4@Au-PEG-DOX NPs were diluted into phosphate buffer saline at pH 7.5 and acetate buffer at pH 5.5 at a concentration equivalent to 5 mg/ml of free DOX. Then, the samples were incubated at a shaking incubator at 37  C and 100 rpm for 0.5, 1, 1.5, 2.5, 4, 8, 18, 24 and 48 h. Similarly, free DOX was diluted in the same buffers to 5 mg/ml and incubated under the same conditions to be served as a control. After centrifugation, the amount of the released DOX was determined by measuring optical density of the supernatant at 498 nm.

Preparation of Fe3O4@Au-PEG-DOX Thiol-terminated polyethylene glycol (0.02 mg/mg Fe3O4@Au NPs) was added to Fe3O4@Au NPs solution and stirred for 24 h. Then, DOX (1 mg/mg Fe3O4@Au NPs) was added with continuous stirring for another 4 h. The drug loaded magnetic carriers was separated using centrifugation at 13000 rpm for 30 min. Nanoparticles characterization Fe3O4@Au and Fe3O4 NPs were visualized by TEM (JEM 1230 electron microscope. Jeol, Tokyo, Japan). A drop of solution was applied to a carbon grid coated with copper and the excess sample

In vitro cytotoxicity According to Gupta et al. [15], Human breast adenocarcinoma (MCF-7) cells were seeded in 96-well plates at a concentration of 5000 cell/well in a fresh medium and left for 24 h. Cells were incubated for 24 and 48 h with the Fe3O4@Au-PEG, Fe3O4@Au-PEGDOX and free DOX at concentrations equivalent to 9, 11, 13, 14 and16.3 mM of free DOX. After washing, each well was filled with 100 ml fresh media. Inverted microscopy (Leica DM IL LED, Germany) was used to examine the morphological changes of cancer cells. 10 mL of MTT reagent was added and the plates were

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incubated for 4 h till purple precipitate appear. Then, detergent reagent was added and the plates were left at room temperature in the dark for 3 h. Finally, the absorbance was recorded at 570 nm using ELIZA microplate reader (Meter tech. S 960, U.S.A.). The mean background absorbance was automatically subtracted. The experiment was repeated 3 times for each concentration. SPSS version 17 was used for statistical analysis. Results

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The average zeta potentials of the prepared Fe3O4 NPs, Fe3O4@Au NPs, and Fe3O4@Au-PEG-DOX NPs were found to be 45.1 ± 5, 31.1 ± 2.5 mV and 25.9 ± 2.4 mV respectively (Table 1). The average diameter measured by DLS was 11 ± 3.2 nm, 24.06 ± 1.5 nm, and 35.73 ± 2.5 nm for Fe3O4 NPs, Fe3O4@Au NPs, and Fe3O4@Au-PEG-DOX NPs respectively (Table 1). According to DLS measurements and TEM micrographs of Fe3O4 NPs and Fe3O4@Au NPs, the thickness of gold shell of the prepared magnetic gold nanoparticles was estimated to be 10e15 nm.

In this study, Fe3O4@Au NPs were prepared and stabilized using thiol-terminated polyethylene glycol (PEG).The prepared NPs were loaded with anti-cancer drug doxorubicin (DOX). The prepared nanoparticles were characterized by different techniques.

The influence of PEG capping density on DOX loading capacity

Physical characterization of the prepared nanoparticles

Efficiency of DOX loading on the surface of Fe3O4@Au-PEG NPs

The prepared Fe3O4@Au NPs were approximately spherical, well dispersed and uniform in size as observed by TEM (Fig. 1A). Further, EDX confirmed that the prepared nanoparticles contained the elements of Fe, Au, and O (Fig. 1C). The detected signals of copper and carbon arise from the TEM grid. Fig. 1B, showed the spherical Fe3O4 NPs with a diameter of about 10 nm. The Measured magnetic properties of Fe3O4@Au NPs and Fe3O4 NPs using VSM at room temperature showed that both Fe3O4 and Fe3O4@Au NPs exhibited a superparamagnetic behavior as no remanence in their hysteresis loops was observed (Fig. 1D). The saturation magnetization (Ms) of Fe3O4 NPs and Fe3O4@Au NPs was 58 emu/g and 19 emu/g respectively. The absorption spectrum of Fe3O4@Au NPs solution showed a characteristic Surface Plasmon Resonance (SPR) band at 525 nm indicating the formation of gold shell, while Fe3O4 NPs had no significant peak in this region (Fig. 2A). Also, surface modification of Fe3O4@Au NPs induced by DOX and PEG caused a red shifted of the SPR band with about 10 nm.

DOX loading profile of Fe3O4@Au-PEG NPs initially showed a rapid adsorption of DOX at the first 1.5 h. Then, the adsorption rate slowed down and finally reached the saturation value after about 4 h (Fig. 3A). It has been found that the maximum DOX loading capacity was 100 mg DOX/mg Fe3O4@Au NPs.

Fig. 2B showed that PEG concentration at 0.02 mg PEG/mg of Fe3O4@Au NPs resulted in the highest DOX loading on NPs surface.

DOX release from Fe3O4@Au-PEG-DOX nanoconjugates in vitro It is apparent from DOX release profile (Fig. 3B) that there was a continuous release of drug up to 4 h beyond which it slows down. As shown in Fig. 3B, the DOX release rate from the Fe3O4@Au-PEGDOX NPs in acidic medium (i.e., pH 5.5) was dramatically higher than that in physiological condition (i.e., pH 7.5). The maximum DOX release from the surface of Fe3O4@Au-PEG-DOX NPs at pH 5.5, and pH 7.5, after 48 h, was about 55%, and 18% respectively. The pH levels at 7.5 and 5.5 were chosen to simulate the physiological body fluid and the characteristic acidity of the tumor microenvironment respectively.

Figure 1. TEM images of (A) Fe3O4@Au NPs and (B) Fe3O4 NPs, (C) EDX spectrum of Fe3O4@Au NPs. (D) VSM plot of magnetization versus magnetic field at room temperature of Fe3O4 NPs, and Fe3O4@Au NPs.

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DOX Loading( μg/ mg Fe3O4@Au NPs)

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Figure 2. (A) The effect of PEG capping density on DOX loading efficiency. (B) The absorption spectra of GNPs, Fe3O4 NPs, Fe3O4@Au-PEG-DOX NPs, and Fe3O4@Au NPs. (C) Random coil conformation of PEG on nanoparticles surfaces.

Therapeutic efficacy of Fe3O4@Au-PEG-DOX NPs in vitro Results showed that the Fe3O4@Au-PEG-DOX NPs have more potent therapeutic effect than free DOX on MCF-7 cancer cells (Fig. 4A & B). Cell inhibition rate of cancer cells treated with Fe3O4@Au-PEG-DOX NPs was higher than that of cells treated with free DOX. Moreover, cell inhibition rate increased with increasing drug concentration and drug exposure time. The morphology of untreated and treated MCF-7 cells is in agreement with MTT assay toxicity results (Fig. 5). Post cells incubation with Fe3O4@Au-PEG-DOX NPs the morphology of the cells was completely changed and became spherical rather than their normal spindle shape indicating apoptosis. The observed dark aggregates in the Fe3O4@Au-PEG-DOX NPs treated cells revealed the accumulation of NPs inside the cells.

Table 1 The measured particle size and zeta potential of Fe3O4 NPs, Fe3O4@Au NPs and Fe3O4@Au-PEG-DOX NPs. Data represent mean ± standard deviation of triplicate experiments.

Size (nm) Zeta pot. (mV)

Fe3O4 NPs

Fe3O4@Au NPs

Fe3O4@Au-PEG-DOX NPs

11 ± 3.2 45.1 ± 5

24.06 ± 1.5 31.1 ± 2.5

35.73 ± 2.5 25.9 ± 2.4

Figure 3. (A) DOX loading profile of Fe3O4@Au NPs, (B) pH dependent release profile of DOX from Fe3O4@Au-PEG-DOX NPs.

Discussion Recently, the unique structure and magnetic-optical properties of Fe3O4@Au NPs have made them important nanomaterials for a wide range of biological applications such as cell labeling, tracking, imaging, drug delivering, and sensing. Our results indicated that Fe3O4@Au NPs is a promising drug delivery system capable of loading and releasing the anticancer drug DOX as well as its targeting to tumor site. Firstly, the study revealed that the superparamagnetic, spherical, well dispersed Fe3O4@Au NPs with an average size of 24 nm could be synthesized by simplified method. The magnetic measurements confirmed that the Ms of Fe3O4 NPs drops when the surface was coated with gold, and the resulting Fe3O4@Au NPs still show good superparamagnetic behaviors, which is consistent with previous study [13]. This reduction was expected as the surface magnetic order can be affected by structural distortions that cause spin canting [16]. The spectrophotometry assessment showed that Fe3O4 NPs do not exhibit any surface plasmon resonance, while Fe3O4@Au and GNPs have surface plasmon resonance at wavelengths 525 nm and 525 nm respectively. The red shift of SPR band induced by loading of PEG and DOX may be attributed to the interaction of both (eNH2) groups of DOX and (eSH) groups of PEG with the gold shell affecting their SPR gold peak position (Fig. 2A) [17]. The high negative zeta potential imparted by Fe3O4@Au NPs compared with Fe3O4@Au-PEG-DOX NPs confirmed that a fraction of negative charges was neutralized due to the electrostatic

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Fe3o4@Au-PEG Free DOX Fe3o4@Au-PEG-DOX

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Figure 4. Cells survival % as a function of DOX concentration (A) after 24 h and (B) after 48 h. The Viability of cells was determined using the MTT assay. Data represent mean ± standard deviation of triplicate experiments.

interaction between DOX molecules and NPs surfaces leading to reduction in zeta potential value [6]. The average diameter of Fe3O4@Au NPs and Fe3O4@Au-PEG-DOX NPs measured by DLS showed 11 nm increases in size of the Fe3O4@Au NPs indicated the attachment of both PEG and DOX to the NPs surfaces (Table 1). The end to end length of 5000 Mw PEG molecule based on a Gaussian random coil model is about 8 nm compared to a zigzag model which about 40 nm. Accordingly, we suggested that PEG chains have been extended in a random coil conformation (Fig. 2C). Polyethylene glycol (PEG) can improve the biocompatibility and blood circulation time of the drug-nanocarriers. Consequently, the current study investigated the influence of PEG capping density on the DOX loading capacity. It was obvious that coating NPs with PEG might enhance the colloidal stability which in turn minimize the aggregation of NPs and maximize DOX loading capacity on their surface (Fig. 2B). On the other hand, increasing the PEG-coating density lowering the interaction between Fe3O4@Au NPs and DOX molecules as the steric hindrance of PEG prevent DOX molecules from being attached to NPs surfaces. Consequently, up to certain level of PEG coating density, the loading efficiency of DOX decreased with increasing PEG coating. It was previously reported that the saturation of the NPs surface with PEG occurs at a PEG density ~1.13 PEG chains/nm2 of the NPs surface [18]. In the present work, we observed that PEG-capping density ~0.2 chains/nm2 of the NPs surface (i.e. lower than saturation by 82%) achieved maximum DOX loading on the nanocarriers surfaces.

Figure 5. MCF-7 cells morphology of untreated (control) and treated with free DOX and Fe3O4@Au-PEG-DOX NPs (DOX conc. 16.3 mM) using inverted microscopy at 10.

The loading and release patterns of DOX from Fe3O4@Au NPs showed that DOX could be efficiently loaded onto and released from Fe3O4@Au NPs. The observed higher drug release at lower pH is attributed to the increased protonation of the negatively charged groups on the surface of gold shell. As a result, the electrostatic interaction between DOX amine group and gold surface was reduced leading to higher drug release [19]. These results indicated that a fast DOX release occurs once the Fe3O4@Au-PEG-DOX NPs step inside the tumor owing to the acidity of tumor extracellular microenvironment. It was also proposed that electrostatic interactions between gold surface and amine group of doxorubicin might be the main factor in the process of doxorubicin adsorption on Fe3O4@Au NPs surfaces [3]. MTT assay was used to explore the synergistic enhancement effect of DOX conjugated Fe3O4@Au NPs on the growth of the relevant breast cancer cells. It was observed that loading of DOX on Fe3O4@Au NPs could enhance therapeutic efficacy of the drug. This might be attributed to many factors (a) NPs loaded DOX was more rapidly taken into the cancer cells, via an endocytic process, than free DOX [20], (b) it was found that the DOX conjugated NPs and the DOX released from them were localized within the cells at the perinuclear regions as well as the nuclei, which can enhance the cytotoxicity of DOX [12]. (c) PEG-modification facilitates the intracellular accumulation that disrupts mitochondrial DNA synthesis in the cytoplasm, resulting in cell death [20]. Cytotoxicity assessments showed that Fe3O4@Au-PEG-DOX treatment resulted in relatively higher therapeutic activity than that of the free DOX, but only for concentrations below the IC50 values [21]. However, as the incubation time and DOX concentration increase the killing effect of DOX masked the synergistic enhancement effect of the DOX conjugated Fe3O4@Au NPs [21]

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Interestingly, Fe3O4@Au-PEG NPs toxicity is very crucial [3] because gold shell [15] and PEG sheath improve the NPs properties and decrease their toxicity. Conclusion This study reports a simple method for the preparation of doxorubicin-loaded superparamagnetic gold coated iron oxide Fe3O4@Au NPs. Maximum loading of DOX was achieved at capping density of the PEG molecules ~0.2 chains/nm2 of NPs surface. DOX loaded NPs showed high drug release at pH 5.5 similar to the acidic microenvironment of tumor. Fe3O4@Au NPs showed great potential as promise magnetic drug nanocarriers through their binding with anti-cancer drug DOX. These multifunctional DOX conjugated Fe3O4@Au NPs may also be used for magneto-thermal cancer therapy. Conflict of interests The authors report no conflict of interests. The authors alone are responsible for the content and the writing of the paper. References [1] Jon D. Magnetic nanoparticles for drug delivery. Drug Dev Res 2006;67: 55e60. [2] Alexiou C, Schmid RJ, Jurgons R, Kremer M, Wanner G, Bergemann C, et al. Targeting cancer cells: magnetic nanoparticles as drug carriers. Eur Biophys J 2006;35:446e50. [3] Chao X, Shi F, Zhao Y, Li K, Peng ML, Chen C, et al. Cytotoxicity of Fe3O4/Au composite nanoparticles loaded with doxorubicin combined with magnetic field. Pharmazie 2010;65:500e4. [4] Latham AH, Williams ME. Controlling transport and chemical functionality of magnetic nanoparticles. Acc Chem Res 2008;41:411e20. [5] Yu MK, Jeong YY, Park J, Park S, Kim JW, Min JJ, et al. Drug-loaded superparamagnetic iron oxide nanoparticles for combined cancer imaging and therapy in vivo. Angew Chem Int Ed 2008;47:5362e5.

[6] Kayal S, Ramanujan RV. Anti-Cancer drug loaded IroneGold CoreeShell nanoparticles (Fe@Au) for magnetic drug targeting. J Nanosci Nanotechnol 2010;10:1e13. [7] Likhitkar S, Bajpai AK. Magnetically controlled release of cisplatin from superparamagnetic starch nanoparticles. Carbohydr Polym 2012;87: 300e8. [8] El-Sayed IH, Huang X, El-Sayed MA. Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano Lett 2005;5:829e34. [9] Mady MM, Fathy MM, Youssef T, Khalil WM. Biophysical characterization of gold nanoparticles-loaded liposomes. Phys Medica 2012;28:288e95. [10] Lin J, Zhou W, Kumbhar A, Wiemann J, Fang J, Carpenter EE, et al. Gold-coated iron (Fe@Au) nanoparticles: synthesis, characterization, and magnetic fieldinduced self-assembly. J Solid State Chem 2001;159:26e31. [11] Chu E, DeVita VT. Physician’s cancer chemotherapy drug manual. Sudbury: Jones and Barlett publisher; 2007. [12] Aryal S, Grailer JJ, Pilla S, Steeber DA, Gong S. Doxorubicin conjugated gold nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers. J Mater Chem 2009;19:7879e84. [13] Zhoua H, Leea J, Parkb TJ, Leec SJ, Parkd JY, Lee J. Ultrasensitive DNA monitoring by AueFe3O4 nanocomplex. Sens Actuators B Chem 2012;163: 224e32. [14] Arruebo M, Pacheco RF, Irusta S, Arbiol J, Ibarra MR, Santamara J. Sustained release of doxorubicin from zeoliteemagnetite nanocomposites prepared by mechanical activation. Nanotechnol 2006;17:4057e64. [15] Gupta AK, Gupta M. Cytotoxicity suppression and cellular uptake enhancement of surface modified magnetic nanoparticles. Biomaterials 2005;26: 1565e73. [16] Pal S, Morales M, Mukherjee P, Srikanth H. Synthesis and magnetic properties of gold coated iron oxide nanoparticles. J Appl Phys 2009;7:504e7. [17] Yokoyama K, Welchons DR. The conjugation of amyloid beta protein on the gold colloidal nanoparticles' surfaces. Nanotechnol 2007;18:105101e7. [18] Manson J, Kumar D, Meenan BJ, Dixon D. Polyethylene glycol functionalized gold nanoparticles: the influence of capping density on stability in various media. Gold Bull 2011;44:99e105. [19] You J, Zhang G, Li C. Exceptionally high payload of doxorubicin in hollow gold nanospheres for near-infrared light-triggered drug release. ACS Nano 2010;4: 1033e41. [20] Gu YJ, Cheng J, Man CW, Wong WT, Cheng SH. Gold-doxorubicin nanoconjugates for overcoming multidrug resistance. Nanomedicine Nanotechnol Biol Med 2012;8:204e11. [21] Munnier E, Cohen-Jonathan S, Linassier C, Douziech-Eyrolles L, Marchais H,  M, et al. Novel method of doxorubicindSPION reversible association for Souce magnetic drug targeting. Int J Pharm 2008;363:170e6.

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Preparation and characterization of magnetic gold nanoparticles to be used as doxorubicin nanocarriers.

Magnetic targeted drug delivery (MTD), using magnetic gold nanoparticles (Fe3O4@Au NPs) conjugated with an anti-cancer drug is a promise modality for ...
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