International Journal of Pharmaceutics 490 (2015) 190–199

Pharmaceutical nanotechnology

Doxorubicin loaded magnetic gold nanoparticles for in vivo targeteddrug delivery

Nihal Saad Elbialya,b,1, Mohamed Mahmoud Fathyb,*,1, Wafaa Mohamed Khalilb,1

a Physics Department, Faculty of Science, King Abdulaziz University, Saudi ArabiabBiophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

A R T I C L E I N F O

Article history:Received 13 February 2015Received in revised form 9 May 2015Accepted 11 May 2015Available online 18 May 2015

Keywords:NanoparticlesDoxorubicinDrug deliveryIron oxide nanoparticlesMagnetic targeted drug deliveryBiodistribution

A B S T R A C T

Treatment of approximately 50% of human cancers includes the use of chemotherapy. The major problemassociated with chemotherapy is the inability to deliver pharmaceuticals to specific site of the bodywithout inducing normal tissue toxicity. Latterly, magnetic targeted drug delivery (MTD) has been used toimprove the therapeutic performance of the chemotherapeutic agents and reduce the severe side effectsassociated with the conventional chemotherapy for malignant tumors. In this study, we were focused ondesigning biocompatible magnetic nanoparticles that can be used as a nanocarrier’s candidate for MTDregimen. Magnetic gold nanoparticles (MGNPs) were prepared and functionalized with thiol-terminatedpolyethylene glycol (PEG), then loaded with anti-cancer drug doxorubicin (DOX). The physical propertiesof 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 theprepared nanoparticles. Fourier transform infrared (FTIR) spectroscopy assessment revealed that PEGand DOX molecules were successfully loaded on the MGNPs surfaces, and the amine group of DOX is theactive attachment site to MGNPs. In vivo studies proved that magnetic targeted drug delivery can providea higher accumulation of drug throughout tumor compared with that delivered by passive targeting. Thisclearly appeared in tumor growth inhibition assessment, biodistribution of DOX in different body organsin addition to the histopathological examinations of treated and untreated Ehrlich carcinoma. To assessthe in vivo toxic effect of the prepared formulations, several biochemical parameters such as aspartateaminotransferase (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 randomdistribution of the chemotherapeutic agents, but also reduces their side effects to healthy tissues, whichare the two primary concerns in conventional cancer therapies.

ã2015 Elsevier B.V. All rights reserved.

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International Journal of Pharmaceutics

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1. Introduction

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

* Corresponding author. Tel.: +20 235676830; mobile: +20 1119904332.E-mail addresses: n_elbialy@hotmail.com (N.S. Elbialy), mfathy@sci.cu.edu.eg

(M.M. Fathy).1 This manuscript was written with contributions from all.

http://dx.doi.org/10.1016/j.ijpharm.2015.05.0320378-5173/ã 2015 Elsevier B.V. All rights reserved.

chemotherapy today is developing drug delivery systems that areefficacious and have therapeutic selectivity.

Freeman and collaborators established the concept of magnetictargeted drug delivery (MTD) using iron oxide nanoparticles. Sincethis time, the idea of using magnetic nanoparticles (MNPs) toimprove techniques for drug delivery has attracted tremendousinterest. In MTD, biocompatible MNPs attached with drugs areinjected into the blood stream, where they can be concentrated atspecific locations in the body by an external magnetic fieldgradient at the targeted area (Wegscheid et al., 2014; Alexiou et al.,2006; Mishima et al., 2006; Takeda et al., 2006). In addition to MTDthere are several targeting techniques capable of directingtherapeutic agents to desired locations. These include the use ofultrasound (Pitt et al., 2004; Gao et al., 2005), electric fields (Denetet al., 2004; Barry, 2001), photodynamic therapy (Dougherty et al.,1998), and antigen recognition (Rudnick et al., 2011; Farokhzad

N.S. Elbialy et al. / International Journal of Pharmaceutics 490 (2015) 190–199 191

et al., 2006). Compared with the above mentioned techniques,magnetic fields are desirable for directing therapeutics insidepatients because they can penetrate deeply into the body and areconsidered to be safe even up to very high strengths (8 T in adults,4 T in children) (Schenck, 2000). In contrast, light and ultrasoundhave limited tissue penetration depths (Oberti et al., 2010; Haakeand Dual, 2005), while strong electric fields (>60 V/cm) are able todamage nerve and muscle cells (Schaefer et al., 2000). Hence,magnetic nanoparticles with an external magnetic field are veryattractive in medical technology, which they can be used to deliveranticancer drug to a targeted region of the body such as a tumor.

It is well known that the iron oxide nanoparticles are the mostpronounced MNPs for biological applications. But iron oxidenanoparticles alone in physiological media is unstable, resulting inoxidation, aggregation and precipitation (Yu et al., 2008; Jeonget al., 2007; Kayal and Ramanujan, 2010). Moreover inefficientsurface binding will results in the early release of loaded drug intoblood 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 magneticnanoparticles that can carry drugs efficiently and deliver them tothe desired area via external magnetic field.

Gold nanoparticles (GNPs) in particular have attracted attentionin 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 asthermal ablation of tumors (Glazer et al., 2010; Schwartz et al.,2009; Huang et al., 2008). Special interests in GNPs for in vivonanomedicine applications can be attributed to their biocompati-bility (Eustis and El-Sayed, 2006; Jain et al., 2006; El-Sayed et al.,2005). The diverse functional possibilities of GNPs allow a varietyof approaches for drug delivery system design. Hydrophobic drugscan be loaded onto GNPs through noncovalent interactions,requiring no structural modification to release drug. Likewise,covalent conjugation to the GNPs through cleavable linkages canbe used to deliver drugs to diseased cells, then the drug can bereleased by an external or internal stimuli (Nikunj et al., 2012).

Accordingly, a promise combination of gold and magneticnanoparticles in core–shell magnetic nanocarriers, magnetic goldnanoparticles (MGNPs), will provide a reasonable chemistrysurface for biological application (Verma et al., 2013), owing topresence of gold shell, in addition to active magnetic targeting ofiron oxide core. Interestingly, gold shell did not degrade themagnetic properties of iron oxide core (Elbialy et al., 2014; Kimet al., 2001b).

The anthracycline doxorubicin (DOX) is a highly efficient anti-neoplastic agent commonly used in the treatment of variouscancers including leukemia, ovarian cancer and especially latestage breast cancer (Chu and DeVita, 2007). The clinical use of DOXis 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 pumpon cell plasma membrane (Wong et al., 2006; Xiong et al., 2010).

The effectiveness of drug depends not only on the properties ofdrug itself, but also on the way of its delivery. Thus, the currentwork aims to enhance the therapeutic performance of DOX bysolving some of the problems that were offered by free DOX such asundesirable serious cardiac toxicity, short blood circulation time,low solubility in aqueous solution, lack of targeting, and widebiodistribution. As the loading of DOX on stabilized MGNPs mayprovide a better active tumor-targeting upon the application of anexternal magnetic field, thereby greatly improve the efficacy of thedrug and reduce its side effects.

In this study, DOX loaded magnetic gold nanoparticles (MGNPs-DOX) nanoconjugates were prepared and injected into bloodstream of tumor-bearing mice and were targeted using externalmagnetic field. Then, the therapeutic efficacy of the developedformulation was assessed. Furthermore, the DOX biodistributionfor different organs were measured and many biochemicalanalyses were also determined. Importantly, a strong magnet(1.14 T of surface strength of magnetic field) has been appliedexternally to ensure the deep penetration of the magnetic field linethroughout the body.

2. Material and methods

2.1. Materials

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 andthiolated polyethylene glycol(PEG-SH, MW5000) were purchasedfrom Sigma–Aldrich (St. Louis, MO, USA).

2.2. Synthesis of MNPs

MNPs were synthesized according to the previously de-scribed method (Elbialy et al., 2014). FeCl3�6H2O and FeCl2�4H2Owith a ratio of 1:0.62 g respectively, were dissolved in 40 mldeionized water then 5 ml of ammonia solution (28% w/v %)was added. Ten minutes later, 4.4 g of sodium citrate was addedand the reaction temperature was raised to 90 �C withcontinuous stirring for 30 min. After cooling, the precipitaterinsed with acetone two times to remove extra free citrate.During rinsing, the sample was separated from the supernatantusing a permanent magnet. Finally, the sample was dried invacuum pump without heating.

2.3. Preparation of MGNPs

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

2.4. Preparation of MGNPs-DOX

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

2.5. Nanoparticles characterization

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

192 N.S. Elbialy et al. / International Journal of Pharmaceutics 490 (2015) 190–199

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 sizerNICOMP TM 380 ZLS, USA) was used to measure the zeta-potentialfor the prepared MNPs, MGNPs and MGNPs-DOX nanoparticles.

2.6. Fourier transform infrared (FTIR) spectroscopy

The binding of PEG and DOX with MGNPs was studied by FTIRspectroscopy. The lyophilized samples of PEG, DOX, MGNPs andMGNPs-DOX were deposited in KBr disks and were recorded on aNICOLET 6700 FTIR Thermo scientific spectrometer, England. Thescanning was done in the range 400–4000 cm�1 at roomtemperature.

2.7. Cell culture and tumor Inoculation.

As described previously (Elbialy et al., 2013), Ehrlich ascitescarcinoma 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 mlsaline. Female Balb/c mice of 22–25 g body weight and 6–8 weeksold (obtained from the animal house of NCI) were then injectedsubcutaneously in their right flanks where the tumors weredeveloped in a single and solid form. Tumor growth wasmonitored post-inoculation until the desired volume was about0.3–0.6 cm3.

Then, mice were divided into four groups (20 mice each): (cont-group) negative control injected with saline, (DOX-group) treatedwith free DOX (10 mg/kg), (MGNPs-DOX-group) treated withMGNPs-DOX (10 mg/kg free DOX equivalent) in the absence ofexternal magnetic field and (MGNPs-DOX-M-group) mice treatedwith MGNPs-DOX (10 mg/kg free DOX equivalent) followed byimmediate external application of neodymium–iron–boron mag-netic disc (1.14 T) at tumor site for 3 h (Table 1). Interestingly, theadministrated dose of the MGNPs was extremely safe for in vivoapplication (Li et al., 2011). All drugs were injected intravenouslyvia mice tail. All animal procedures and care were performed usingguidelines for the Care and Use of Laboratory Animals andapproved by the Animal Ethics Committee at Cairo University(National Research Council, 1996).

2.8. Intratumoral accumulation of MGNPs-DOX

Prior to in vivo application, both passive and magnetic targetingeffects were qualitatively examined using silver enhancementstaining experiment. One day post intravenous injection ofMGNPs-DOX, one mouse from each treated group MGNPs-DOX-group and MGNPs-DOX-M-group were sacrificed. Tumor tissueswere excised and fixed in 10% formalin for 24 h then they weresectioned, with thickness 5 mm, and stained with silver (accordingto the manufacturer’s instructions of Silver Enhancer Kit SE-100) tovisualize the accumulation of MGNPs in tumor tissues (Dickersonet al., 2008).

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

Group name Treatment regimen

Cont-group Saline

DOX-group Free DOX

MGNPs-DOX-group MGNPs-DOX

MGNPs-DOX-M-group MGNPs-DOX + external magnet (1.14 T) was applied on

2.9. Histopathological examination

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

2.10. Tumor size measurements

Due to the high growth rate in Ehrlich tumor model, change intumor volume (DV) was monitored over 18-day period for the fourtreated groups: cont-group, DOX-group, MGNPs-DOX-group andMGNPs-DOX-M-group. Ellipsoidal tumor volume (V) was assessedevery three days and calculated using the formula

V ¼ P6

� �ðd 2ÞðDÞ

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

2.11. Quantitative determination of doxorubicin amount in differentorgans

In vivo DOX biodistribution was assessed in different organs forthe three treated mice groups DOX-group, MGNPs-DOX-group, andMGNPs-DOX-M-group. Heart, liver, spleen, lung, kidney, brain andtumor were collected at 1, 3, and 24 h post single intravenousinjection of different formulations. Then, the collected tissues werewashed, 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 ofDOX was measured using spectrofluorometer (Shimadzu, RF-5301PC, Japan). The concentrations of DOX were calculated fromthe calibration curve at an excitation wave length of 480 nm andemission wave length of 585 nm. The final doxorubicin concen-trations were expressed as the microgram DOX per gram of tissue

2.12. Serum biochemical analysis

For mice groups (cont-group, DOX-group, MGNPs-DOX-group,and MGNPs-DOX-M-group), blood samples were collected 2 weekspost 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 andwere centrifuged for 10 min at 5000 rpm to separate serum(Lenaerts et al., 2005). Using a Konilab PRIME 30 fully automatedclinical chemistry analyzer, serum biochemical analysis was

Equivalent dose of DOX Equivalent dose of MGNPs

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Fig. 2. Zeta potential of the prepared MNPs, MGNPs, and MGNPs-DOX with averagezeta potential of about �45.1 �5 mV, �31.1 � 2.5 mV, and �25.9 � 2.4 mV respec-tively (n = 5). All zeta potential measurements were performed at 25 �C and at pH7.4.

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carried out. The levels of aspartate aminotransferase (AST) andalanine transaminase (ALT) were measured to evaluate liverfunctions. Serum LDH and CK-MB levels were used as markers forthe diagnosis of cardiac toxicity. Moreover, levels of urea, uric acidand creatinine were measured to assess the kidney functions. Allthe chemicals used for biochemical measurements were pur-chased from ELI Tech (Paris, France).

3. Results and discussion

The physicochemical properties of nanomaterials are highlydependent on particle parameters such as size, shape and surfacecoating. Hence, we need to characterize the prepared nano-particles to confirm that they have the desired properties allowingthem to be used in drug delivery applications. In this study, wehave used various characterization techniques to study morphol-ogy, size, composition, and surface properties of the prepared NPs.

The morphologies and the size of the prepared MNPs andMGNPs were investigated by TEM (Fig. 1A,B). TEM micrographclearly shows that MNPs are almost spherical in shape with a sizeof about 10 nm (Fig. 1A). TEM image of MGNPs revealed that mostprepared nanoparticles were spherical shapes and they have lessaggregation with an average diameter about 22 nm (Fig. 1B).

In general, TEM images cannot show the core–shell structure ofMGNPs, since the electronic density of gold is much higher thanthat of iron oxide. For this reason, energy dispersive X-rayspectroscopy (EDX) is a complementary evidence needed tocharacterize the composite nanoparticles of this kind (Brownet al., 2000). Fig. 1C, shows the EDX spectrum of the preparedMGNPs. The spectrum validates that the prepared nanoparticlescontained the elements of Fe, Au, and O. The detected signals ofcopper and carbon arise from the TEM grid.

The magnitude of the zeta potential gives an indication of thepotential stability of the colloidal systems. The average zetapotential of the prepared MNPs and MGNPs was found to be�45.1 �5 mV and �31.1 � 2.5 mV respectively (Fig. 2). These highnegative values of zeta-potential confirmed the presence ofnegatively charged carboxylate groups on the surface of theprepared MGNPs due to the absorption of citrate onto theirsurfaces.

When MGNPs loaded with DOX the average zeta potential of theMGNPs-DOX was reduced to �25.9 � 2.4 (Fig. 2). It was reportedthat the majority of DOX molecules carry positive charges at pH 7.4(Anderson et al., 2002; Raghunand et al., 2003; Kataoka et al.,

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

2000). Consequently, the reduction in zeta potential valueindicated that a fraction of negative charges was neutralizeddue to the electrostatic interaction between (NH3+) of DOXmolecules and (COO�) groups of NPs surfaces (Kayal andRamanujan, 2010). Also it was found that coating the surfaces ofnanoparticles with PEG reduces the zeta-potential value (Englandet al., 2013).

Fourier transform infrared (FTIR) spectroscopy was used tomonitor different types of interactions. So, the binding of DOX andPEG with MGNPs surfaces was investigated by FTIR analysis. FTIRspectra of PEG, DOX, MGNPs and MGNPs-DOX were shown inFig. 3a–d respectively.

FTIR spectrum of PEG revealed various bands at, 2885 cm�1

(asymmetric stretching vibrations of the ��CH2), 1347 cm�1(C��Hbending of ��CH2 and ��CH3), 1108 cm�1 (C��O��C stretching) and600–900 cm�1 (N��H wagging) (Fig. 3a) (Manson et al., 2011).

Fig. 3b shows FTIR spectrum of pure DOX. The characteristicbands at 1620 cm�1, 1734 cm�1 and 2917 cm�1 were attributed toN��H bending, C¼O stretching vibration and C–H stretchingvibration respectively. The bands at 1000–1620 cm�1 are corre-sponding to the quinine and ketone groups of the DOX (Rana et al.,2007). Importantly, for pure DOX, the characteristic band at3430 cm�1 was due to N��H stretching vibrations for primaryamine structure.

For MGNPs FTIR spectrum, the IR band observed at 578 cm�1 inMGNPs spectrum can be attributed to the Fe��O stretchingvibrational mode of Fe3O4 (Zhou et al., 2012) (Fig. 3c). Also theobserved peaks at 3465 cm�1 (H��O stretching) and 1626 cm�1

(H��O��H bending) are due to adsorbed water molecules on theNPs surfaces (Kayal and Ramanujan, 2010).

In case of MGNPs-DOX complex, the amine peak of pure DOX at3430 cm�1 was broadened and shifted to 3440 cm�1, indicating theformation of electrostatic interaction between protonated aminegroups of the doxorubicin molecule with the surface of MGNPs(Fig. 3d) (Mirza and Shamshad, 2011). Also surface modification ofMGNPs with PEG and DOX resulted in the appearance of newlycharacteristic peaks at (621 cm�1, 1070 cm�1, 1270 cm�1, and1400 cm�1). According to FTIR analysis, it could be suggestedthat: (1)PEG and DOX molecules were successfully attached to NPssurfaces. (2) ��NH2 group of DOX is the active site for theattachment 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. 3. FTIR spectra of (a) polyethylene glycol (PEG), (b) DOX, (c) MGNPs and (d) MGNPs-DOX.

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) inpresence of external magnetic field (active targeting). The nanoparticles appear as dark small dots or aggregates (�300).

194 N.S. Elbialy et al. / International Journal of Pharmaceutics 490 (2015) 190–199

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Fig. 5. The average changes in Ehrlich tumor volume as a function of time for thetreated groups (DOX-group, MGNPs-DOX-group, and MGNPs-DOX-M-group) as thecontrol group (n = 7).

N.S. Elbialy et al. / International Journal of Pharmaceutics 490 (2015) 190–199 195

silver enhancement staining. The mechanism of nanoparticlesvisualization was taken place when the silver stain enlarges theaccumulated nanoparticles by precipitation of metallic silver ongold surface. This silver coating increases photoemission and givesa high contrast signal visible under a light microscope (Birrel et al.,1986), so the NPs seen as dark small dots or aggregates (Elbialyet al., 2013). Histological examination of tumor tissue, using silverenhancement staining, clearly showed a higher accumulation ofmagnetically targeted MGNPs-DOX (Fig. 4B) over that of passivelytargeted MGNPs-DOX (Fig. 4A). These passively targeted NPs totumor were owing to the enhanced permeability and retention

Fig. 6. Section of Ehrlich tumor tissues, stained with H&E for (A) cont-group, (B) DOX-g

(EPR) effect of tumor vasculature regardless of the absence orpresence of external magnet.

The antitumor activity of the developed nanocarrier (MGNPs-DOX) was assessed by following up the average change in tumorvolume (DV) over 18 days for the four experimental animal groups(cont-group, DOX-group, MGNPs-DOX-group, and MGNPs-DOX-M-group).

Fig. 5, revealed a pronounced inhibition in tumor growth forMGNPs-DOX-M-group compared with MGNPs-DOX-group andCont-group. This marked decrease in Ehrlich tumor volume, forMGNPs-DOX-M-group, was attributed to the implementation ofMTD protocol that achieved maximum attraction of MGNPs-DOXinto tumor site. Consequently, the therapeutic index of the drugwas improved by increasing DOX concentration in the targetedregion (tumor) (Bajaj and Yeo, 2010; Lencioni, 2010). The obvioustumor regression in MGNPs-DOX-group, compared with that ofCont-group and DOX-group, corroborated that the MGNPs-DOXcould be passively targeted to tumor by EPR effect leading to therelease of DOX at tumor site. Hence, MGNPs-DOX-group showed adelay in the tumor growth rate compared with the cont-group. Oneway analysis of variance (ANOVA) was used to test the effect oftime on the tumor growth. Additionally, the least significantdifference was performed to compeer between different treatmentregimens and controls.

Histopathological examination was performed for the treatedand control experimental groups in order to assess the degree ofcell necrosis (Fig. 6A–D). Examination of the entire tumor sectionsfor the various groups (Cont-group, DOX-group, MGNPs-DOX-group, and MGNPs-DOX-M-group) revealed marked differences inthe cellular features with varying degrees in tumor cell necrosis.Tumor section of cont-group showed a neoplastic feature withnormal necrosis percentage of focal and diffuse necrosis (Fig. 6A).

roup, (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-DOX-group, and MGNPs-DOX-M-group. (A) one hour post drugs injection, (B) Threehours post drugs injection and (C) 24 h post drugs injection. Data representmean � standard deviation (n = 4).

196 N.S. Elbialy et al. / International Journal of Pharmaceutics 490 (2015) 190–199

Tumor sections of the DOX-group and MGNPs-DOX-group oftreated mice revealed mild and moderate cell coagulative necroticregions respectively (Fig. 6B,C). The mild tumor cell necrosisobserved in DOX-group is due to the normal distribution of DOX byblood circulation. While, the moderate tumor cell necrosis ofMGNPs-DOX-group was attributed to the passively targetedMGNPs-DOX at tumor site induced by EPR effect. Ehrlich tumorsection of MGNPs-DOX-M-group, treated with MGNPs-DOX inpresence of external magnetic field, showed an extensive necrosis,complete loss of cellular details “ghosts”, with scattered residual ofviable tumor cells (Fig. 6D).

Biodistribution studies will be at the core of any safetyevaluation of products containing nanomaterials. Biodistributionis an imperative study to know where in the body the nano-therapeutics are distributed, how long they remain at differentorgans, and how it is cleared from these organs. This informationwould allow researchers to more accurately interpret anytoxicological finding that might be observed in the preclinicalstudies.

Studies have proven that the most important factors formagnetic drug targeting are magnetic flux density and magneticfield exposure time (Alexiou et al., 2002). It was found thatApplication of 0.5 T permanent magnet on liver cause anenrichment of drugs in the targeted region of liver. Thus theconcentration of drug was reduced in other organs (Chao et al.,2011). Also previous results shown that the drug concentrations intumors of mice in presence of 0.5 T permanent magnet were higherthan those from the passively targeted group (Chao et al., 2012). Inthis study stronger magnet (1.14 T) was used to enhance thetargeting of drugs and reducing their side effects. Hence, in vivostudies were carried out to investigate the time dependentmagnetic targeting of DOX for three experimental treated groupsof mice DOX-group, MGNPs-DOX-group, and MGNPs-DOX-M-group. DOX concentrations in different organs were measured atdifferent time intervals; 1 h, 3 h, and 24 h post intravenousinjection of drug (Fig. 7A–C).

For DOX-group, 1 h post drug administration, the DOX had awide distribution in tissues, and was mainly concentrated in theliver (10. 56 mg DOX/g liver), which is the major site for themetabolism 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 acomparatively low accumulation in the tumor tissues (1.2 mg DOX/g tumor) (Fig. 7A). No significant difference was observed betweenDOX levels, in almost all organs, at 1 h and 3 h post free DOXadministration (Fig 7A,B). Twenty four hours post free DOXadministration, the concentrations of DOX were (8.025 mg DOX/gliver), (6.6 mg DOX/g kidney), (5.2 mg DOX/g heart), and (2.0 mgDOX/g tumor) for liver, kidney, heart, and tumor respectively(Fig. 7C). This indicated that the concentration of DOX decreased inalmost all organs which attributed to the clearance of drug fromthe body (mainly by liver).

In case of MGNPs-DOX-group, 1 h post drug administration, theDOX concentrations in liver, spleen, lung, and heart were (9.1 mgDOX/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 suggestedthat the conventional i.v. administration of MGNPs-DOX dramati-cally captures the nanocarriers in liver, heart, lung, and spleen andsome of the nanocarriers could be passively accumulated in thetumor (Tietze et al., 2013).

Interestingly, for MGNPs-DOX-group, the DOX concentrationsin tumor tissues were (5.0 mg DOX/g tumor) and (5.7 mg DOX/gtumor), 3 h and 24 h post drug administration respectively. Thisrelatively higher accumulation, compared with DOX-group,assured the passive accumulation of NPs throughout tumor tissueswhich induced by the leaky nature of tumor vasculature.

Obviously, administration of MGNPs-DOX in presence of anexternal magnetic field (active targeting) dramatically changed theDOX concentrations in the different organs (MGNPs-DOX-M-group) (Fig 7A–C). In case of MGNPs-DOX-M-group, DOX concen-trations in tumor tissues were (5.2 mg DOX/g tumor) and (12.2 mgDOX/g tumor) at 1 h and 3 h post drug administration respectively.These results indicated that the application of magnetic field ontumor region leads to a selective biodistribution of DOX at tumorsite, while minimizing its concentration at other healthy tissues.For MGNPs-DOX-M-group, 24 h post drug administration, themeasured DOX concentration in tumor was (10.3 mg DOX/g tumor).This marked retention of DOX in tumor region, even though themagnetic field was removed, is due to the lake of lymphaticdrainage in tumor and the ability of tumor tissue to retain theaccumulated MGNPs-DOX according to their size.

Intriguingly, 24 h post injection, our results demonstrated thatthe measured DOX concentrations in tumor of MGNPs-DOX-M-group showed 2-folds and 5-folds increase over that of MGNPs-DOX-group and DOX-group respectively. This indicated the

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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 oflactate 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) Thelevel of enzymes (U/L). Data represent mean � standard deviation (n = 4).

N.S. Elbialy et al. / International Journal of Pharmaceutics 490 (2015) 190–199 197

effectiveness of the active targeting using an external magneticfield.

In summary we can conclude that,

� DOX-group showed the highest DOX accumulation in heart andliver suggesting its toxicity to these organs are more thanMGNPs-DOX-group and MGNPs-DOX-M-group.

� Utilizing MGNPs as DOX carriers that interact with an externalmagnetic field (MTD) offers the chance to selective targeting ofthe chemotherapeutic agent in tumor tissues in comparison toother modalities of treatment.

In vivo toxicity of the prepared formulations was assessed bymeasuring various biochemical parameters for heart, liver andkidney. Serum LDH and CK-MB levels were extensively used inclinical practice as markers for the diagnosis of cardiac necrosisand toxicity (Andreadou et al., 2007). Likewise, the level of ALT andAST (liver enzymes) is an indicator for the proper performance ofliver. Any damage in liver tissue may lead to a disturbance in thesecreted amount of these enzymes in blood stream (Shrestha et al.,2007). Furthermore, the levels of urea, creatinine and uric acid inblood 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 doseadministration of free DOX compared with their levels in thecontrol 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 tothe hepatic and cardiac toxicity of free DOX (Injac and Strukelj,2008; Iqbal et al., 2008). Such DOX toxicity is generally mediatedthrough the generation of free radicals (Bulucu et al., 2009). Inaddition to such oxidative damage, it was found that DOX toxicityhas 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-DOX-group showed a significant decrease in LDH and CK-MB levels ascompared to that administrated with free DOX (p < 0.01andp < 0.05 respectively). Hence, the loading of DOX on magneticgold nanocarriers resulted in lower cardiac toxicity indicated thereduction of the accumulated MGNPs-DOX in heart. Importantly,for MGNPs-DOX-M-group, no significant changes were observed inLDH, CK-MB, AST and ALT serum levels compared to their normallevels in control mice group. This indicated that the activetargeting of drug nanocarriers increases the selective accumula-tion and maintenance of the chemotherapeutic agent into thetarget region. Providing vital organs (heart and liver) protectionfrom excessive dose of drug. The levels of urea, creatinine and uricacid values were changed in a non significant manner for groupsDOX-group, MGNPs-DOX-group and MGNPs-DOX-M-group com-pared with the cont-group (Fig. 8C–E). However, these observedchanges in the kidney of urea, creatinine and uric acid might be dueto the highest clearance of different drug formulations by thekidney. Interestingly, previous studies reported that renal destruc-tion and apoptosis were observed in animals administrated withDOX 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 differentorgans after repeated administration did not produce anymortality or any indication of toxicity as assessed by animalbehavior, tissue morphology, serum biochemistry, hematologicalanalysis, and histopathological examination (Lasagna-Reeves et al.,2010).

These results were similar to tumor treatment with liposomalDOX where drug toxicity to heart, liver and kidney is minimized

198 N.S. Elbialy et al. / International Journal of Pharmaceutics 490 (2015) 190–199

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

4. Conclusions

This study reports a simple method for the preparation andcharacterization of doxorubicin-loaded paramagnetic gold coatediron oxide nanoparticles. Loading of DOX was confirmed by FTIRmeasurements. Biodistribution studies revealed that the MGNPs-DOX could be successfully retained throughout tumor in thepresence of suitable external magnetic field. Administration ofMGNPs-DOX, in presence of external magnetic field, showed thebest therapeutic anticancer activity and lowest systemic toxicitycompared to that of free DOX.

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