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IJP 14902 1–10 International Journal of Pharmaceutics xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Doxorubicin loaded magnetic gold nanoparticles for in vivo targeted drug delivery

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Nihal Elbialy a,b,1, Mohamed Mahmoud Fathy a, * ,1, Wafaa Mohamed Khalil a,1

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Biophysics Department, Faculty of Science—Cairo University, 12613 Giza, Egypt Physics Department (Medical Physics Program), Faculty of Science—King Abdulaziz University, Saudi Arabia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 13 February 2015 Received in revised form 9 May 2015 Accepted 11 May 2015 Available online xxx

Treatment of approximately 50% of human cancers includes the use of chemotherapy. The major problem associated with chemotherapy is the inability to deliver pharmaceuticals to specific site of the body without inducing normal tissue toxicity. Latterly, magnetic targeted drug delivery (MTD) has been used to improve the therapeutic performance of the chemotherapeutic agents and reduce the severe side effects associated with the conventional chemotherapy for malignant tumors. In this study, we were focused on designing biocompatible magnetic nanoparticles that can be used as a nanocarrier’s candidate for MTD regimen. Magnetic gold nanoparticles (MGNPs) were prepared and functionalized with thiol-terminated polyethylene glycol (PEG), then loaded with anti-cancer drug doxorubicin (DOX). The physical properties of the prepared NPs were characterized using different techniques. Transmission electron microscopy (TEM) revealed the spherical mono-dispersed nature of the prepared MGNPs with size about 22 nm. Energy dispersive X-ray spectroscopy (EDX) assured the existence of both iron and gold elements in the prepared nanoparticles. Fourier transform infrared (FTIR) spectroscopy assessment revealed that PEG and DOX molecules were successfully loaded on the MGNPs surfaces, and the amine group of DOX is the active attachment site to MGNPs. In vivo studies proved that magnetic targeted drug delivery can provide a higher accumulation of drug throughout tumor compared with that delivered by passive targeting. This clearly appeared in tumor growth inhibition assessment, biodistribution of DOX in different body organs in addition to the histopathological examinations of treated and untreated Ehrlich carcinoma. To assess the in vivo toxic effect of the prepared formulations, several biochemical parameters such as aspartate aminotransferase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), urea, uric acid and creatinine were measured. MTD technology not only minimizes the random distribution of the chemotherapeutic agents, but also reduces their side effects to healthy tissues, which are the two primary concerns in conventional cancer therapies. ã 2015 Published by Elsevier B.V.

Keywords: Nanoparticles Doxorubicin Drug delivery Iron oxide nanoparticles Magnetic targeted drug delivery Biodistribution

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1. Introduction

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Chemotherapy has significantly improved the cancer treatment over the past half-century. Unfortunately, conventional chemotherapeutic agents lack selectivity where less than 0.1–1% of the drugs are taken up by tumor cells, with the remaining 99% going into healthy tissue (van der Veldt et al., 2010). As drugs are normally intended for a specific region in the body (for example, tumor), this conventional method for delivery is inefficient and requires a larger amount of drug leading to sever side effects to healthy systems. Hence one of the greatest challenges facing

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* Corresponding author. Tel.: +20 235676830; mobile: +20 1119904332. This manuscript was written with contributions from all.

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chemotherapy today is developing drug delivery systems that are Q3 efficacious and have therapeutic selectivity. Freeman and collaborators established the concept of magnetic targeted drug delivery (MTD) using iron oxide nanoparticles. Since this time, the idea of using magnetic nanoparticles (MNPs) to improve techniques for drug delivery has attracted tremendous interest. In MTD, biocompatible MNPs attached with drugs are injected into the blood stream, where they can be concentrated at specific locations in the body by an external magnetic field gradient at the targeted area (Wegscheid et al., 2014; Alexiou et al., 2006; Mishima et al., 2006; Takeda et al., 2006). In addition to MTD there are several targeting techniques capable of directing therapeutic agents to desired locations. These include the use of ultrasound (Pitt et al., 2004; Gao et al., 2005), electric fields (Denet et al., 2004; Barry, 2001), photodynamic therapy (Dougherty et al., 1998), and antigen recognition (Rudnick et al., 2011; Farokhzad

http://dx.doi.org/10.1016/j.ijpharm.2015.05.032 0378-5173/ ã 2015 Published by Elsevier B.V.

Please cite this article in press as: Elbialy, N., et al., Doxorubicin loaded magnetic gold nanoparticles for in vivo targeted drug delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.032

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et al., 2006). Compared with the above mentioned techniques, magnetic fields are desirable for directing therapeutics inside patients because they can penetrate deeply into the body and are Q4 considered to be safe even up to very high strengths (8 T in adults, 4 T in children) (Schenck, 2000). In contrast, light and ultrasound have limited tissue penetration depths (Oberti et al., 2010; Haake and Dual, 2005), while strong electric fields (>60 V/cm) are able to damage nerve and muscle cells (Schaefer et al., 2000). Hence, magnetic nanoparticles with an external magnetic field are very attractive in medical technology, which they can be used to deliver anticancer drug to a targeted region of the body such as a tumor. It is well known that the iron oxide nanoparticles are the most pronounced MNPs for biological applications. But iron oxide nanoparticles alone in physiological media is unstable, resulting in oxidation, aggregation and precipitation (Yu et al., 2008; Jeong et al., 2007; Kayal and Ramanujan, 2010). Moreover inefficient surface binding will results in the early release of loaded drug into blood stream leading to failure in delivering drug to tumor site (Babincova et al., 2008; Kettering et al., 2009; Likhitkar and Bajpai, 2012). Therefore, we need to develop safer, more stable magnetic nanoparticles that can carry drugs efficiently and deliver them to the desired area via external magnetic field. Gold nanoparticles (GNPs) in particular have attracted attention in numerous fields in nanomedicine such as cancer targeting (Brown et al., 2010; Paciotti et al., 2004), colorimetric biosensors (Chen et al., 2009; Medley et al., 2008), imaging (Wang et al., 2005; Copland et al., 2004; Sokolov et al., 2003), delivery of therapeutics (Kim et al., 2001a), gene targeting (Wijaya et al., 2009), as well as thermal ablation of tumors (Glazer et al., 2010; Schwartz et al., 2009; Huang et al., 2008). Special interests in GNPs for in vivo nanomedicine applications can be attributed to their biocompatibility (Eustis and El-Sayed, 2006; Jain et al., 2006; El-Sayed et al., 2005). The diverse functional possibilities of GNPs allow a variety of approaches for drug delivery system design. Hydrophobic drugs can be loaded onto GNPs through noncovalent interactions, requiring no structural modification to release drug. Likewise, covalent conjugation to the GNPs through cleavable linkages can be used to deliver drugs to diseased cells, then the drug can be released by an external or internal stimuli (Nikunj et al., 2012). Accordingly, a promise combination of gold and magnetic nanoparticles in core–shell magnetic nanocarriers, magnetic gold nanoparticles (MGNPs), will provide a reasonable chemistry surface for biological application (Verma et al., 2013), owing to presence of gold shell, in addition to active magnetic targeting of iron oxide core. Interestingly, gold shell did not degrade the magnetic properties of iron oxide core (Elbialy and Fathy, 2014; Kim et al., 2001b). The anthracycline doxorubicin (DOX) is a highly efficient antineoplastic agent commonly used in the treatment of various cancers including leukemia, ovarian cancer and especially late stage breast cancer (Chu and DeVita, 2007). The clinical use of DOX is often limited because of its undesirable serious cardiac toxicity, short half-life and low solubility in aqueous solution (Aryal et al., 2009). Moreover, some tumor cells showed multidrug resistance, which has been attributed to the P glycoprotein (P-gp) efflux pump on cell plasma membrane (Wong et al., 2006; Xiong et al., 2010). The effectiveness of drug depends not only on the properties of drug itself, but also on the way of its delivery. Thus, the current work aims to enhance the therapeutic performance of DOX by solving some of the problems that were offered by free DOX such as undesirable serious cardiac toxicity, short blood circulation time, low solubility in aqueous solution, lack of targeting, and wide biodistribution. As the loading of DOX on stabilized MGNPs may provide a better active tumor-targeting upon the application of an external magnetic field, thereby greatly improve the efficacy of the drug and reduce its side effects.

In this study, DOX loaded magnetic gold nanoparticles (MGNPsDOX) nanoconjugates were prepared and injected into blood stream of tumor-bearing mice and were targeted using external magnetic field. Then, the therapeutic efficacy of the developed formulation was assessed. Furthermore, the DOX biodistribution for different organs were measured and many biochemical analyses were also determined. Importantly, a strong magnet (1.14 T of surface strength of magnetic field) has been applied externally to ensure the deep penetration of the magnetic field line throughout the body.

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2. Material and methods

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2.1. Materials

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Gold (III) chloride (HAuCl4
3H2O, 99.99%), sodium citrate (HOC) (COONa) (CH2COONa)2 (2H2O), FeCl3
6H2O, FeCl2
4H2O, 28% w/v% ammonia solution, Neodymium–ironkboron magnetic discs 1.14 T, Silver Enhancer Kit SE-100, doxorubicin hydrochloride and thiolated polyethylene glycol(PEG-SH, MW5000) were purchased from Sigma–Aldrich (St. Louis, MO, USA).

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2.2. Synthesis of MNPs

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MNPs were synthesized according to the previously described method (Elbialy and Fathy, 2014). FeCl3
6H2O and FeCl2
4H2O with a ratio of 1:0.62 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.

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2.3. Preparation of MGNPs

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Twenty milliliter (0.5 mM) of HAuCl4 deionized water solution was heated and stirred till boiling. Then, 15 ml of the previously prepared MNPs (1 mg/ml) was rapidly added. The color of the solution gradually changed from brown to red. Stirring continued for 10 min after the color change ceased (Elbialy and Fathy, 2014). The heating source was switched off while the stirring continued until the solution cooled to room temperature. The MGNPs were separated by using a permanent magnet.

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2.4. Preparation of MGNPs-DOX

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Thiolated-polyethylene glycol (0.02 mg/mg MGNPs) was added to MGNPs solution and stirred for 24 h. Then, DOX (1 mg/mg MGNPs) was added with continuous stirring for another 4 h. The drug loaded magnetic nanocarriers (MGNPs-DOX) was separated using centrifugation at 13,000 rpm for 30 min. It has been found that the maximum DOX loading capacity was 100 mg DOX/mg MGNPs (Elbialy et al., 2014). The reaction was performed in Tris buffer at pH 7.4.

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2.5. Nanoparticles characterization

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MGNPs and MNPs were visualized by TEM (JEM 1230 electron microscope Jeol, Tokyo, Japan). A drop of solution was applied to TEM grid. The grid was left for 5 min to dry at room temperature prior to the beginning of the examination. Energy-dispersive X-ray spectroscopy (EDX) is an analytical technique used for the elemental analysis or chemical characterization of a sample. So

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the formation of MGNPs was confirmed using EDX (FEI Tecnai G20, Super twin, Double tilt, LaB6 Gun, USA). Zeta potential/particle sizer (zeta potential/particle sizer NICOMP TM 380 ZLS, USA) was used to measure the zeta-potential for the prepared MNPs, MGNPs and MGNPs-DOX nanoparticles.

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2.6. Fourier transform infrared (FTIR) spectroscopy

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The binding of PEG and DOX with MGNPs was studied by FTIR spectroscopy. The lyophilized samples of PEG, DOX, MGNPs and MGNPs-DOX were deposited in KBr disks and were recorded on a NICOLET 6700 FTIR Thermo scientific spectrometer, England. The scanning was done in the range 400–4000 cm1 at room temperature.

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2.7. Cell culture and tumor Inoculation.

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As described previously (Elbialy et al., 2013), Ehrlich ascites carcinoma cells obtained from National Cancer Institute “NCI”— Cairo University, were intraperitoneally injected into female Balb/ c mice. Ascites fluid was collected on the 7th day after injection. Ehrlich cells were washed twice and then resuspended in 5 ml saline. Female Balb/c mice of 22–25 g body weight and 6–8 weeks old (obtained from the animal house of NCI) were then injected subcutaneously in their right flanks where the tumors were developed in a single and solid form. Tumor growth was monitored post-inoculation until the desired volume was about 0.3–0.6 cm3. Then, mice were divided into four groups (20 mice each): (contgroup) negative control injected with saline, (DOX-group) treated with free DOX (10 mg/kg), (MGNPs-DOX-group) treated with MGNPs-DOX (10 mg/kg free DOX equivalent) in the absence of external magnetic field and (MGNPs-DOX-M-group) mice treated with MGNPs-DOX (10 mg/kg free DOX equivalent) followed by immediate external application of neodymium–iron–boron magnetic disc (1.14 T) at tumor site for 3 h (Table 1). Interestingly, the administrated dose of the MGNPs was extremely safe for in vivo application (Li et al., 2011). All drugs were injected intravenously via mice tail. All animal procedures and care were performed using guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee at Cairo University (National Research Council, 1996).

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2.8. Intratumoral accumulation of MGNPs-DOX

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Prior to in vivo application, both passive and magnetic targeting effects were qualitatively examined using silver enhancement staining experiment. One day post intravenous injection of MGNPs-DOX, one mouse from each treated group MGNPs-DOXgroup and MGNPs-DOX-M-group were sacrificed. Tumor tissues were excised and fixed in 10% formalin for 24 h then they were sectioned, with thickness 5 mm, and stained with silver (according to the manufacturer’s instructions of Silver Enhancer Kit SE-100) to visualize the accumulation of MGNPs in tumor tissues (Dickerson et al., 2008).

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2.9. Histopathological examination

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Mice of the treatment groups DOX-group, MGNPs-DOX-group and MGNPs-DOX-M-group were sacrificed 3 days post drug injection. The tumors were excised, fixed in 10% neutral formalin, embedded in paraffin blocks and sectioned. Tissues sections were stained with hematoxylin and eosin (H&E). Previous procedures were repeated for the control group. All tissue sections were examined using light microscope (CX31 Olympus microscope) connected with a digital camera (Canon).

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2.10. Tumor size measurements

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Due to the high growth rate in Ehrlich tumor model, change in tumor volume (DV) was monitored over 18-day period for the four treated groups: cont-group, DOX-group, MGNPs-DOX-group and MGNPs-DOX-M-group. Ellipsoidal tumor volume (V) was assessed every three days and calculated using the formula   P ðd 2ÞðDÞ V¼ 6

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where D and d are the long and short axes respectively measured with a digital caliper (accuracy 0.01 mm). Fisher’s LSD (least significance difference) multiple-comparison test was conducted to check the significance between group pairs. SPSS version 17 was used for statistical analysis.

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2.11. Quantitative determination of doxorubicin amount in different organs

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In vivo DOX biodistribution was assessed in different organs for the three treated mice groups DOX-group, MGNPs-DOX-group, and MGNPs-DOX-M-group. Heart, liver, spleen, lung, kidney, brain and tumor were collected at 1, 3, and 24 hr post single intravenous injection of different formulations. Then, the collected tissues were washed, weighted and homogenized in 5 volumes of acidic ethanol (0.3 M HCl:EtOH, 3:7, v/v) (Wei et al., 2012; Huang et al., 2011). Tissue homogenates were centrifuged at 13,000 rpm for 10 min. The supernatant were then isolated and quantitative analysis of DOX was measured using spectrofluorometer (Shimadzu, RF- Q6 5301PC, Japan). The concentrations of DOX were calculated from the calibration curve at an excitation wave length of 480 nm and emission wave length of 585 nm. The final doxorubicin concentrations were expressed as the microgram DOX per gram of tissue

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2.12. Serum biochemical analysis

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For mice groups (cont-group, DOX-group, MGNPs-DOX-group, and MGNPs-DOX-M-group), blood samples were collected 2 weeks post single intravenous injection of different drug formulations. Mice were sacrificed and terminally bled by cardiac puncture. Blood samples were incubated on ice for 30 min to coagulate and were centrifuged for 10 min at 5000 rpm to separate serum (Lenaerts et al., 2005). Using a Konilab PRIME 30 fully automated clinical chemistry analyzer, serum biochemical analysis was

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Table 1 The administrated dose of DOX and MGNPs for each treated group. Group name

Treatment regimen

Equivalent dose of DOX

Equivalent dose of MGNPs

Cont-group DOX-group MGNPs-DOX-group MGNPs-DOX-M-group

Saline Free DOX MGNPs-DOX MGNPs-DOX + external magnet (1.14 T) was applied on tumor for 3 h

No 10 mg/kg 10 mg/kg 10 mg/kg

No No 100 mg/kg 100 mg/kg

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carried out. The levels of aspartate aminotransferase (AST) and alanine transaminase (ALT) were measured to evaluate liver functions. Serum LDH and CK-MB levels were used as markers for the diagnosis of cardiac toxicity. Moreover, levels of urea, uric acid and creatinine were measured to assess the kidney functions. All the chemicals used for biochemical measurements were purchased from ELI Tech (Paris, France).

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3. Results and discussion

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The physicochemical properties of nanomaterials are highly dependent on particle parameters such as size, shape and surface coating. Hence, we need to characterize the prepared nanoparticles to confirm that they have the desired properties allowing them to be used in drug delivery applications. In this study, we have used various characterization techniques to study morphology, size, composition, and surface properties of the prepared NPs. The morphologies and the size of the prepared MNPs and MGNPs were investigated by TEM (Fig 1A,B). TEM micrograph clearly shows that MNPs are almost spherical in shape with a size of about 10 nm (Fig. 1A). TEM image of MGNPs revealed that most prepared nanoparticles were spherical shapes and they have less aggregation with an average diameter about 22 nm (Fig. 1B). In general, TEM images cannot show the core–shell structure of MGNPs, since the electronic density of gold is much higher than that of iron oxide. For this reason, energy dispersive X-ray spectroscopy (EDX) is a complementary evidence needed to characterize the composite nanoparticles of this kind (Brown et al., 2000). Fig. 1C, shows the EDX spectrum of the prepared MGNPs. The spectrum validates that the prepared nanoparticles contained the elements of Fe, Au, and O. The detected signals of copper and carbon arise from the TEM grid. The magnitude of the zeta potential gives an indication of the potential stability of the colloidal systems. The average zeta potential of the prepared MNPs and MGNPs was found to be 45.1  5 mV and 31.1  2.5 mV respectively (Fig. 2). These high negative values of zeta-potential confirmed the presence of negatively charged carboxylate groups on the surface of the prepared MGNPs due to the absorption of citrate onto their surfaces. When MGNPs loaded with DOX the average zeta potential of the MGNPs-DOX was reduced to 25.9  2.4 (Fig. 2). It was reported that the majority of DOX molecules carry positive charges at pH 7.4 (Anderson et al., 2002; Raghunand et al., 2003; Kataoka et al.,

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Fig. 1. TEM images of (A) MNPs and (B) MGNPs. (C) EDX spectrum of MGNPs.

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Zeta Potential

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MGNPs-DOX

Fig. 2. Zeta potential of the prepared MNPs, MGNPs, and MGNPs-DOX with average zeta potential of about 45.1  5 mV, 31.1  2.5 mV, and 25.9  2.4 mV respectively (n = 5). All zeta potential measurements were performed at 25  C and at pH 7.4.

2000). Consequently, the reduction in zeta potential value indicated that a fraction of negative charges was neutralized due to the electrostatic interaction between (NH3+) of DOX molecules and (COO) groups of NPs surfaces (Kayal and Ramanujan, 2010). Also it was found that coating the surfaces of nanoparticles with PEG reduces the zeta-potential value (England et al., 2013). Fourier transform infrared (FTIR) spectroscopy was used to monitor different types of interactions. So, the binding of DOX and PEG with MGNPs surfaces was investigated by FTIR analysis. FTIR spectra of PEG, DOX, MGNPs and MGNPs-DOX were shown in Fig. 3a–d respectively. FTIR spectrum of PEG revealed various bands at, 2885 cm1 (asymmetric stretching vibrations of the  CH2), 1347 cm1(C H 1 bending of  CH2 and  CH3), 1108 cm (C O C stretching) and 600–900 cm1 (N H wagging) (Fig. 3a) (Manson et al., 2011). Fig. 3b shows FTIR spectrum of pure DOX. The characteristic bands at 1620 cm1, 1734 cm1 and 2917 cm1 were attributed to N H bending, C¼O stretching vibration and C–H stretching vibration respectively. The bands at 1000–1620 cm1 are corresponding to the quinine and ketone groups of the DOX (Rana et al., 2007). Importantly, for pure DOX, the characteristic band at 3430 cm1 was due to NH stretching vibrations for primary amine structure. For MGNPs FTIR spectrum, the IR band observed at 578 cm1 in MGNPs spectrum can be attributed to the Fe O stretching vibrational mode of Fe3O4 (Zhou et al., 2012) (Fig. 3c). Also the observed peaks at 3465 cm1 (HO stretching) and 1626 cm1 (HO H bending) are due to adsorbed water molecules on the NPs surfaces (Kayal and Ramanujan, 2010). In case of MGNPs-DOX complex, the amine peak of pure DOX at 3430 cm1 was broadened and shifted to 3440 cm1, indicating the formation of electrostatic interaction between protonated amine groups of the doxorubicin molecule with the surface of MGNPs (Fig. 3d) (Mirza and Shamshad, 2011). Also surface modification of MGNPs with PEG and DOX resulted in the appearance of newly characteristic peaks at (621 cm1, 1070 cm1, 1270 cm1, and 1400 cm1). According to FTIR analysis, it could be suggested that: (1)PEG and DOX molecules were successfully attached to NPs surfaces. (2)  NH2 group of DOX is the active site for the attachment to the MGNPs. To confirm the accumulation of MGNPs-DOX throughout tumor, histological examination of tumor tissues was performed using

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Fig. 4. Silver enhancement staining of Ehrlich tumor tissue for mice administrated with MGNPs-DOX (A) in absence of external magnetic field (passive targeting) and (B) in presence of external magnetic field (active targeting). The nanoparticles appear as dark small dots or aggregates (300).

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silver enhancement staining. The mechanism of nanoparticles visualization was taken place when the silver stain enlarges the accumulated nanoparticles by precipitation of metallic silver on gold surface. This silver coating increases photoemission and gives a high contrast signal visible under a light microscope (Birrel et al., 1986), so the NPs seen as dark small dots or aggregates (Elbialy et al., 2013). Histological examination of tumor tissue, using silver enhancement staining, clearly showed a higher accumulation of magnetically targeted MGNPs-DOX (Fig. 4b) over that of passively targeted MGNPs-DOX (Fig. 4a). These passively targeted NPs to tumor were owing to the by enhanced permeability and retention

(EPR) effect of tumor vasculature regardless of the absence or presence of external magnet. The antitumor activity of the developed nanocarrier (MGNPsDOX) was assessed by following up the average change in tumor volume (DV) over 18 days for the four experimental animal groups (cont-group, DOX-group, MGNPs-DOX-group, and MGNPs-DOXM-group). Fig. 5, revealed a pronounced inhibition in tumor growth for MGNPs-DOX-M-group compared with MGNPs-DOX-group and Cont-group. This marked decrease in Ehrlich tumor volume, for MGNPs-DOX-M-group, was attributed to the implementation of MTD protocol that achieved maximum attraction of MGNPs-DOX into tumor site. Consequently, the therapeutic index of the drug was improved by increasing DOX concentration in the targeted region (tumor) (Bajaj and Yeo, 2010; Lencioni, 2010). The obvious tumor regression in MGNPs-DOX-group, compared with that of Cont-group and DOX-group, corroborated that the MGNPs-DOX could be passively targeted to tumor by EPR effect leading to the release of DOX at tumor site. Hence, MGNPs-DOX-group showed a delay in the tumor growth rate compared with the cont-group. One way analysis of variance (ANOVA) was used to test the effect of time on the tumor growth. Additionally, the least significant difference was performed to compeer between different treatment regimens and controls. Histopathological examination was performed for the treated and control experimental groups in order to assess the degree of cell necrosis (Fig. 6a–d). Examination of the entire tumor sections for the various groups (Cont-group, DOX-group, MGNPs-DOXgroup, and MGNPs-DOX-M-group) revealed marked differences in the cellular features with varying degrees in tumor cell necrosis. Tumor section of cont-group showed a neoplastic feature with normal necrosis percentage of focal and diffuse necrosis (Fig. 6a).

Fig. 6. Section of Ehrlich tumor tissues, stained with H&E for (A) cont-group, (B) DOX-group, (C) MGNPs-DOX-group, and (D) MGNPs-DOX-M-group. At magnefication 150.

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Fig. 7. Biodistribut ion of DOX for the treatment groups DOX-group, MGNPs-DOXgroup, and MGNPs-DOX-M-group. (A) one hour post drugs injection, (B) Three hours post drugs injection and (C) 24 h post drugs injection. Data represent mean  standard deviation (n = 4). 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

Tumor sections of the DOX-group and MGNPs-DOX-group of treated mice revealed mild and moderate cell coagulative necrotic regions respectively (Fig. 6b,c). The mild tumor cell necrosis observed in DOX-group is due to the normal distribution of DOX by blood circulation. While, the moderate tumor cell necrosis of MGNPs-DOX-group was attributed to the passively targeted MGNPs-DOX at tumor site induced by EPR effect. Ehrlich tumor section of MGNPs-DOX-M-group, treated with MGNPs-DOX in presence of external magnetic field, showed an extensive necrosis, complete loss of cellular details “ghosts”, with scattered residual of viable tumor cells (Fig. 6d). Biodistribution studies will be at the core of any safety evaluation of products containing nanomaterials. Biodistribution is an imperative study to know where in the body the nanotherapeutics are distributed, how long they remain at different organs, and how it is cleared from these organs. This information would allow researchers to more accurately interpret any toxicological finding that might be observed in the preclinical studies.

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Studies have proven that the most important factors for magnetic drug targeting are magnetic flux density and magnetic field exposure time (Alexiou et al., 2002). It was found that Application of 0.5 T permanent magnet on liver cause an enrichment of drugs in the targeted region of liver. Thus the concentration of drug was reduced in other organs (Chao et al., 2011). Also previous results shown that the drug concentrations in tumors of mice in presence of 0.5 T permanent magnet were higher than those from the passively targeted group (Chao et al., 2012). In this study stronger magnet (1.14 T) was used to enhance the targeting of drugs and reducing their side effects. Hence, in vivo studies were carried out to investigate the time dependent magnetic targeting of DOX for three experimental treated groups of mice DOX-group, MGNPs-DOX-group, and MGNPs-DOX-Mgroup. DOX concentrations in different organs were measured at different time intervals; 1 h, 3 h, and 24 h post intravenous injection of drug (Fig. 7a–c). For DOX-group, 1 h post drug administration, the DOX had a wide distribution in tissues, and was mainly concentrated in the liver (10. 56 mg DOX/g liver), which is the major site for the metabolism of this drug (Bachur et al., 1974), followed by the heart (7.9 mg DOX/g heart), and the kidney (5.15 mg DOX/g kidney) with a comparatively low accumulation in the tumor tissues (1.2 mg DOX/ g tumor) (Fig. 7a). No significant difference was observed between DOX levels, in almost all organs, at 1 h and 3 h post free DOX administration (Fig 7a,b). Twenty four hours post free DOX administration, the concentrations of DOX were (8.025 mg DOX/ g liver), (6.6 mg DOX/g kidney), (5.2 mg DOX/g heart), and (2.0 mg DOX/g tumor) for liver, kidney, heart, and tumor respectively (Fig. 7c). This indicated that the concentration of DOX decreased in almost all organs which attributed to the clearance of drug from the body (mainly by liver). In case of MGNPs-DOX-group, 1 h post drug administration, the DOX concentrations in liver, spleen, lung, and heart were (9.1 mg DOX/g liver), (4.6 mg DOX/g spleen), (3.1 mg DOX/g lung), and (5.1 mg DOX/g heart) respectively (Fig. 7a). These results suggested that the conventional i.v. administration of MGNPs-DOX dramatically captures the nanocarriers in liver, heart, lung, and spleen and some of the nanocarriers could be passively accumulated in the tumor (Tietze et al., 2013). Interestingly, for MGNPs-DOX-group, the DOX concentrations in tumor tissues were (5.0 mg DOX/g tumor) and (5.7 mg DOX/g tumor), 3 h and 24 h post drug administration respectively. This relatively higher accumulation, compared with DOX-group, assured the passive accumulation of NPs throughout tumor tissues which induced by the leaky nature of tumor vasculature. Obviously, administration of MGNPs-DOX in presence of an external magnetic field (active targeting) dramatically changed the DOX concentrations in the different organs (MGNPs-DOX-Mgroup) (Fig 7a–c). In case of MGNPs-DOX-M-group, DOX concentrations in tumor tissues were (5.2 mg DOX/g tumor) and (12.2 mg DOX/g tumor) at 1 h and 3 h post drug administration respectively. These results indicated that the application of magnetic field on tumor region leads to a selective biodistribution of DOX at tumor site, while minimizing its concentration at other healthy tissues. For MGNPs-DOX-M-group, 24 h post drug administration, the measured DOX concentration in tumor was (10.3 mg DOX/g tumor). This marked retention of DOX in tumor region, even though the magnetic field was removed, is due to the lake of lymphatic drainage in tumor and the ability of tumor tissue to retain the accumulated MGNPs-DOX according to their size. Intriguingly, 24 h post injection, our results demonstrated that the measured DOX concentrations in tumor of MGNPs-DOX-Mgroup showed 2-folds and 5-folds increase over that of MGNPsDOX-group and DOX-group respectively. This indicated the

Please cite this article in press as: Elbialy, N., et al., Doxorubicin loaded magnetic gold nanoparticles for in vivo targeted drug delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.032

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Fig. 8. Various biochemical parameters for the treatment groups DOX-group, MGNPs-DOX-group, and MGNPs-DOX-M-group as well as the control group. (A) The level of lactate dehydrogenase (LDH) (U/L), (B) The level of CK-MB (U/L), (C) The level of urea (mg/dl), (D) The level of creatinine(mg/dl), (E) The level of uric acid (mg/dl) and (F) The level of enzymes (U/L). Data represent mean  standard deviation (n = 4).

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effectiveness of the active targeting using an external magnetic field. In summary we can conclude that,  DOX-group showed the highest DOX accumulation in heart and liver suggesting its toxicity to these organs are more than MGNPs-DOX-group and MGNPs-DOX-M-group.  Utilizing MGNPs as DOX carriers that interact with an external magnetic field (MTD) offers the chance to selective targeting of the chemotherapeutic agent in tumor tissues in comparison to other modalities of treatment. In vivo toxicity of the prepared formulations was assessed by measuring various biochemical parameters for heart, liver and kidney. Serum LDH and CK-MB levels were extensively used in clinical practice as markers for the diagnosis of cardiac necrosis and toxicity (Andreadou et al., 2007). Likewise, the level of ALT and AST (liver enzymes) is an indicator for the proper performance of liver. Any damage in liver tissue may lead to a disturbance in the secreted amount of these enzymes in blood stream (Shrestha et al., 2007). Furthermore, the levels of urea, creatinine and uric acid in blood are associated with the functionality of the kidney. For DOX-group, the results showed that the LDH, CK-MB, AST, and ALT levels were significantly elevated after a single dose administration of free DOX compared with their levels in the control group (p < 0.01, p < 0.05, p < 0.05and p < 0.01 respectively) (Fig. 8a,b and f). This elevation in enzymes level was attributed to the hepatic and cardiac toxicity of free DOX (Injac and Strukelj, 2008; Iqbal et al., 2008). Such DOX toxicity is generally mediated through the generation of free radicals (Bulucu et al., 2009). In addition to such oxidative damage, it was found that DOX toxicity has been extended to induce inflammation in heart and liver

tissues upon DOX administration in rats (Deepa and Varalakshmi, 2005). In the current study, mice administrated with MGNPs-DOXgroup showed a significant decrease in LDH and CK-MB levels as compared to that administrated with free DOX (p < 0.01and p < 0.05 respectively). Hence, the loading of DOX on magnetic gold nanocarriers resulted in lower cardiac toxicity indicated the reduction of the accumulated MGNPs-DOX in heart. Importantly, for MGNPs-DOX-M-group, no significant changes were observed in LDH, CK-MB, AST and ALT serum levels compared to their normal levels in control mice group. This indicated that the active targeting of drug nanocarriers increases the selective accumulation and maintenance of the chemotherapeutic agent into the target region. Providing vital organs (heart and liver) protection from excessive dose of drug. The levels of urea, creatinine and uric acid values were changed in a non significant manner for groups DOX-group, MGNPs-DOX-group and MGNPs-DOX-M-group compared with the cont-group. However, these observed changes in the kidney of urea, creatinine and uric acid might be due to the highest clearance of different drug formulations by the kidney. Interestingly, previous studies reported that renal destruction and apoptosis were observed in animals administrated with DOX nevertheless, serum creatinine level was in normal range (Bertani et al., 1982; Weening and Rennke, 1983). Also, it was reported that the accumulation of GNPs in different organs after repeated administration did not produce any mortality or any indication of toxicity as assessed by animal behavior, tissue morphology, serum biochemistry, hematological analysis, and histopathological examination (Lasagna-Reeves et al., 2010). These results were similar to tumor treatment with liposomal DOX where drug toxicity to heart, liver and kidney is minimized

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owing to lower cumulative dose of DOX in these tissues, compared to that of free drug (Goyal et al., 2005; van Hoesel et al., 1984).

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This study reports a simple method for the preparation and characterization of doxorubicin-loaded paramagnetic gold coated iron oxide nanoparticles. Loading of DOX was confirmed by FTIR measurements. Biodistribution studies revealed that the MGNPsDOX could be successfully retained throughout tumor in the presence of suitable external magnetic field. Administration of MGNPs-DOX, in presence of external magnetic field, showed the best therapeutic anticancer activity and lowest systemic toxicity compared to that of free DOX.

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Please cite this article in press as: Elbialy, N., et al., Doxorubicin loaded magnetic gold nanoparticles for in vivo targeted drug delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.032

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