A pharmaceutical study of doxorubicin-loaded PEGylated nanoparticles for.pdf

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ARTICLE IN PRESSG ModelIJP-11978; No. of Pages 10International Journal of Pharmaceutics xxx (2011) xxx xxx

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International Journal of Pharmaceutics

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A pharmaceutical study of doxorubicin-loaded PEmagne

J. Gautier ouzM. Souca Universit Fra Nanovb Institut Fdr

a r t i c l

Article history:Received 17 FeReceived in reAccepted 6 JunAvailable onlin

Keywords:Doxorubicin (DOX)Superparamagnetic iron oxidenanoparticles (SPION)Polyethylene glycolRelease kineticsIntracellular distribution

e canperped wer p

ent aeters

ical media and decreased elimination by the immune system. At physiological pH of 7.4, 60% of theloaded drug is gradually released from the DLPS in 2 h. The intracellular release and distribution ofDOX is followed by means of confocal spectral imaging (CSI) of the drug uorescence. The in vitrocytotoxicity of the DLPS on MCF-7 breast cancer cells is equivalent to that of a DOX solution. Thereversible association of DOX to the SPION surface and the role of polymer coating on the drug load-ing/release are discussed, both being critical for the design of novel stealth magnetic nanovectors forchemotherapy.

1. Introdu

Doxorubcycline famtreat a largand particuThe clinicalgerous beinet al., 2009)that is assotion on the Such a minforms commPtz et al., concentratiaccumulateenhanced p

Corresponcie, 31 avenuefax: +33 24736

E-mail add

0378-5173/$ doi:10.1016/j. this article in press as: Gautier, J., et al., A pharmaceutical study of doxorubicin-loaded PEGylated nanoparticles for magnetic drugInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

2011 Elsevier B.V. All rights reserved.

ction

icin (DOX) is an antineoplastic agent of the anthra-ily frequently used in association with other drugs toe number of cancers, like leukaemia, ovarian cancerslarly last stage breast cancers (Lankelma et al., 1999).

use of DOX is limited by its side effects, the most dan-g a cumulative dose-dependent cardiotoxicity (Leonard. To minimize the side effects, DOX can be vectorized,ciated to drug carriers that will favour its accumula-site of action and limit its dispersion in healthy tissues.imization has already been obtained with liposomalercialized for nearly ten years (Leonard et al., 2009;

2009). These forms increase the drug intratumouralon mainly due to their nanometric size: liposomes

in tumours by a passive mechanism known as theermeation and retention effect (EPR effect). Indeed,

ding author at: Laboratoire de Pharmacie Galnique, UFR de Pharma- Monge, 37200 Tours, France. Tel.: +33 247367201;7198.ress: [email protected] (E. Munnier).

the leaky fenestrations of neovasculature in tumours, combinedwith their inefcient lymphatic drainage, enable extravasa-tion and accumulation of such small objects (Veiseh et al.,2010).

In addition to this passive targeting, nanovectors of a new gen-eration are developed to permit an active targeting of tumours.Several concepts of active targeting are being investigated inthe literature, namely at a molecular scale, as the functionaliza-tion of vector surfaces with targeting ligands complementary tospecic or overexpressed receptors on cancer cells (Fan et al.,2010; Veiseh et al., 2010), or at a macroscopic scale, for exam-ple by means of an external magnetic eld (Douziech-Eyrolleset al., 2007; Lbbe et al., 1996a,b, 2001; Maeng et al., 2010; Yanget al., 2010). Magnetic drug targeting is thus based on the associ-ation of a drug and magnetic nanoparticles. These magnetic drugdelivery systems are essentially based on iron oxides (Aqil et al.,2008; Sabat et al., 2008; Wassel et al., 2007; Ying et al., 2011),known to be non-toxic (Weissleder et al., 1989; Ying and Hwang,2010).

To benet from the EPR effect, the size of the nal systems hasto be below the dimensions of vascular permeativity in tumours,i.e. 150 nm (Veiseh et al., 2010). The size of initial iron oxidenanoparticles is also determining for their magnetic properties:

see front matter 2011 Elsevier B.V. All rights reserved.ijpharm.2011.06.010tic drug targetinga,b, E. Munniera,b,, A. Paillarda,b, K. Herva,b, L. Da,b, P. Duboisa,b, I. Chourpaa,b

nc ois-Rabelais, EA 4244 Physico-Chimie des Matriaux et des Biomolcules, quipe atif de Recherche 135 Imagerie Fonctionnelle, Tours F-37000, France

e i n f o

bruary 2011vised form 25 May 2011e 2011e xxx

a b s t r a c t

One of the new strategies to improvlike the polyethylene glycol-coated sucalled PS). In this study, PS are loadDOXFe2+ complex reversible at low(DLPS, 3% w/w DOX/iron oxide) preszero at physiological pH, both param/ locate / i jpharm

Gylated nanoparticles for

iech-Eyrollesa,b,

ecteurs Magntiques pour la Chimiothrapie, Tours F-37200, France

cer chemotherapy is based on new drug delivery systems,aramagnetic iron oxide nanoparticles (PEG-SPION, thereafterith doxorubicin (DOX) anticancer drug, using a pre-formedH of tumour tissues and cancer cells. The DOX loaded PS

hydrodynamic size around 60 nm and a zeta potential near being favourable for increased colloidal stability in biolog-

Please cite xorutargeting.

ARTICLE IN PRESSG ModelIJP-11978; No. of Pages 102 J. Gautier et al. / International Journal of Pharmaceutics xxx (2011) xxx xxx

below 30 nm diameter, the particles are superparamagnetic, i.e.highly magnetisable in the presence of magnetic elds but voidof magnetic memory (remanence) (Mahmoudi et al., 2009). This isa critical requirement to avoid thrombotic risk related to magneticaggregationfor these puiron oxide niron oxide),20 nm (Roc

The surffor their bioand/or charadsorption recognitionreach their opsonisatiosion, the suhydrophilicFor exampldextran arelengthen thChess, 2003groups to co2010), in oing. Nevertand releaseThe activitythrough theloading on covalent bi(Mahmoudbe cleaved is hinderedentrapmeneffect (fast of stimuli (oped a novSPION (Musurface afteiron binds trole of an ithis modelcomplex diThis releaseenvironmenet al., 2011reversible lsystems weagainst MC2008).

The aimdoxorubicinet al., 2008(Herv et athe loadingtity of doxomethod. Thcharacterizin vitro dohow PEGylexplored. Ccellular disnanovectorof the drucancer activectorizatio

2. Materials and methods

2.1. Nanoparticle preparation

Mateorubtical(DPBferril) we). Fecs ((APThlor

acidsed

acrovi

mmo Reu

PEGy PEGed p

c iros co

m. In wered re

hen sn wat wites w

Doxoylate

ex, asex w.5 M ted wugated in

se p PEG

anop

Morp moed u

oper([Fe]id, ths weas ba

hyd (Dy

alvernizeed

) opelydidistr this article in press as: Gautier, J., et al., A pharmaceutical study of doInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

of injectable delivery systems. The initial particles usedrposes are commonly called SPION (superparamagneticanoparticles) or USPIO (ultrasmall superparamagnetic

the latter term being reserved for particle sizes belowh et al., 2005).ace properties of the nanovectors are also essentialcompatibility. The presence of hydrophobic functionsges favours nanoparticle opsonisation, i.e. nonspecicof plasma proteins (opsonins), which accelerates their

and elimination by the immune system before theytarget tissue (Veiseh et al., 2010). In order to reduce then and to ensure a colloidal stability by sterical repul-rface of the magnetic systems must be coated with a

and neutral moiety, such as a biocompatible polymer.e, polymer molecules like polyethylene glycol (PEG) or

known to reduce the opsonisation phenomenon and toe duration of circulation of the nanovectors (Harris and). In addition, the coating can provide various chemicalnjugate drugs or targeting ligands (Wang and Thanou,

rder to combine macroscopic and microscopic target-heless, the coating may lead to modied drug loading

that must be taken into account (Zhu et al., 2010). of the drug loaded on nanovectors must be respected

formulation steps. The most common protocols of drugthe magnetic systems in the literature imply eithernding or entrapment of the drug into a polymer layeri et al., 2010). Covalent linkage can be too strong toby cellular enzymes, in particular if access to the drug

by the polymeric coating (Shkilnyy et al., 2010). Thet of the drug within a polymer often leads to a burstinitial release) and/or to a low release in the absenceDilnawaz et al., 2010; Yang et al., 2010). We devel-el method of reversible association of doxorubicin tonnier et al., 2008): DOX is adsorbed on the iron oxider being chelated with a Fe(II) ion (Fig. 1). The chelatedo OH groups on the SPION surface and thus plays thentermediary between the drug and nanoparticles. In, the drug release is pH-dependent as the DOXFe2+

ssociates in acidic conditions (Munnier et al., 2008). is expected to be tumour-specic, since the tumourt is known to be more acidic than blood (Greulich; Medeiros et al., 2011). In addition, this method ofoading protects the drug activity: the DOXFe2+SPIONre as active or even more so than a doxorubicin solutionF-7 human breast cancer cells in vitro (Munnier et al.,

of the present study is the loading of theiron (II) pre-formed complex (DOXFe2+, Munnier) on the PEGylated SPION developed by our groupl., 2008). We propose a complete study of our model:

process is optimized, in order to maximize the quan-rubicin bound, with a simple and easily transposablee optimized DOX-loaded PEGylated SPION are thened in terms of morphology, size and zeta potential. Thexorubicin release is investigated, in order to observeation modies the release. Biological aspects are alsoonfocal spectral imaging is used to follow the sub-tribution of doxorubicin after a treatment with thes and to determine the intracellular kinetics of actiong. Cytotoxicity assays permit to evaluate the anti-vity of the nanovectors and to notice the effect ofn.

2.1.1. Dox

maceusaline drous (titrisoFranceOrganisilane hydrocpionicpurchaSodiumwere prous a(Val deused.

2.1.2. The

describagnetiaqueoumediuSPIONacid anwere ttized icontacparticl

2.1.3. PEG

complcompltion (1incubacentrifmodi3.1.

Theloaded

2.2. N

2.2.1. The

examin(TEM),water TEM grsampletion w

Theby DLS2c (Min deioperform(4 mWThe poa size data.bicin-loaded PEGylated nanoparticles for magnetic drug

rialsicin hydrochloride was purchased from TEVA Phar-

s Ltd. (Puteaux, France). Dulbeccos phosphate bufferS), ferric nitrate nonahydrate (Fe(NO3)39H2O), anhy-c chloride (FeCl3) and iron standard solution 1 g/Lre purchased from Fisher Bioblock Scientic (Illkirch,

rrous chloride (FeCl2, 4H2O) was obtained from AcrosNoisy Le Grand, France). 3-AminopropyltrimethoxyES), N-(3-dimethylaminopropyl)-N-ethylcarbodiimideide (EDC) and methoxypoly(ethylene glycol) 5000 pro-

N-succinimidyl ester (activated PEG, aPEG) werefrom Sigma Aldrich (Saint-Quentin-Fallavier, France).etate and tris-(hydroxymethyl)-aminomethane (Tris)ded by Merck (Fontenay-sous-Bois, France), and fer-nium sulphate ((NH4)2Fe(SO4)26H2O) by Carlo Erbail, France). In all the experiments, deionized water was

lated SPIONylated ferrouids were prepared according to a methodreviously (Herv et al., 2008). Briey, superparam-

n oxide nanoparticles (SPION) were synthesized byprecipitation of ferric and ferrous chlorides in alkaline

order to stabilize the surface chemical composition, oxidized by ferric nitrate and nally peptized in nitric-suspended in a determined volume of water. SPIONilanized by a 12 h contact with APTES, washed and pep-ter at pH 3. Finally, SPION were PEGylated by a 24 hh aPEG, and puried by dialysis against water. Theseill be further mentioned as PS for PEGylated SPION.

rubicin (DOX) loading on PEGylated SPIONd SPION (PS) were loaded with DOX via a DOXFe2+

described elsewhere (Munnier et al., 2008). DOXFe2+

as pre-formed by contact between DOX and a Fe2+ solu-excess of Fe2+ over DOX) in Tris buffer pH 7.6. PS wereith DOXFe2+ complex in the dark, and harvested by

ion at 19,000 g for 1 h at 4 C. Several parameters were order to optimize drug loading, as described in Section

articles will be further mentioned as DLPS for DOX-ylated SPION.

article characterization

hology and sizerphology of nanoparticles and their diameters weresing a Philips CM20 electronic transmission microscopeating at 200 kV. The samples were diluted in deionized

103 g/L), then deposited on a carbon-coated coppere excess of solvent was removed with lter paper, andre left to air-dry before TEM viewing. The size estima-sed on 30 nanoparticles on 3 different images.rodynamic diameter of the particles was determinednamic Light Scattering) technique with an Autosizern Instruments, Orsay, France) after sample dilutiond water ([Fe] 2 103 g/L). Each measurement wasat 25 C, at least in triplicate, with a HeNe laserrating at 633 nm, with the scatter angle xed at 173.spersity index PDI is a measure of the broadness ofibution derived from the cumulative analysis of DLS


ARTICLE IN PRESSG ModelIJP-11978; No. of Pages 10J. Gautier et al. / International Journal of Pharmaceutics xxx (2011) xxx xxx 3

2.2.2. Zeta pThe zeta

Malvern Nasurement wlaser (4 mWof 17. Zetaing from 4.0Titrator, Ma0.1 M.

2.2.3. Iron dThe over

absorption moFisher Scwith HCl 6 Nsolution. Earesults permferrouid suof SPION (C

2.2.4. DrugDLPS we

sonic bath this article in press as: Gautier, J., et al., A pharmaceutical study of doxoruInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

Fig. 1. Schematic diagram of loading of pre-formed DOXFe2+ complex

otential measurements potential of the particles was determined using anoZ (Malvern Instruments, Orsay, France). Each mea-as performed at 25 C, at least in triplicate, with a HeNe) operating at 633 nm, with a light scattering angle

potential was determined as a function of pH (rang- to 10.0) by means of a MPT-2 Titrator (Multi Purposelvern Instruments, Malvern, UK) using HNO3 and KOH

eterminationall iron content in the ferrouids is measured by atomicspectrometry (iCE 3000 Series AA Spectrometer, Ther-ientic, France), after mineralization by a 12 h contact. A calibration curve was obtained with titrisol standardch determination was performed in triplicate. Theseit to estimate iron oxide concentration and mass inspensions, considering that iron represents 71.5% w/w

hourpa et al., 2005).

loading determinationre suspended for 1 h in acetate buffer pH 4 in an ultra-to permit the total release of DOX, as the DOXFe2+

complex diple was thconcentratispectrophomam, FrancEach determing is expriron oxide the non-PE2008).

2.2.5. In vitPEGylated S

Aliquotswere madethermostatcentrifugednanoparticlwas determdrug uoretrometer, ecurve.bicin-loaded PEGylated nanoparticles for magnetic drug

and of release of DOX from the DLPS.

ssociates at low pH (Munnier et al., 2008). The sam-en centrifuged at 19,000 g for 1 h at 15 C. DOXon was determined in the supernatant by UVvisibletometry (Anthlie Advanced Spectrophotometer, Seco-e), using its molar absorptivity determined at 500 nm.ination was performed at least in triplicate. Drug load-

essed as the ratio of DOX mass over the mass of thecore of the nanovectors to permit a comparison withGylated particles previously described (Munnier et al.,

ro release kinetics of drug from DOX-loadedPION (DLPS)

of DPLS suspensions, containing 104 g of iron, up to 2 mL with DPBS, continuously shaken anded at 37 C. At given time intervals, aliquots were

1 h at 19,000 g and 4 C in order to separate thees of the release medium. The drug concentrationined in the supernatant from the intensity of the

scence at 557 nm (Hitachi F-4500 uorescence spec-xcitation wavelength 500 nm), using a calibration

Please cite xorubicin-loaded PEGylated nanoparticles for magnetic drugtargeting.


2.3. Biological evaluation of DOX-loaded PEGylated SPION

For the biological evaluation, MCF-7 human breast carcinomacells (American Type Culture Collection, LGC Promochem, Mol-sheim, Fran(DMEM) supenicillin GCO2 atmospScientic (I

2.3.1. CytotFor cyto

24-well pla1 104 cellthe cells wing differenDOX solutio333 mg/L). (MTT) assayHanks Buffin 1 mL of was replacemazan crysat 540 nm uBioblock, Illwas determreduction insextuplicate

2.3.2. ConfoCover g

5 g/mL inplate. MCF-24 h. The mthe cultureThis incubaCO2. After tthe cover ging observawere carrieter (Horibaan automa(600 grooveorescence wuorescencthrough thea closed micat 37 C. Foated at halfthat providspectrum). were perfobution mapand shape (Munnier etted usingence spectrwere belowcompletelyorescence opower 5 Wtodegradati(tting scorsented as h

Table 1Physico-chemical properties of colloids PS and DPLS (n 3).

PEGylated SPION(PS)

DOX loadedPEGylated SPION

dynamspersiotenti

ctric pg (% w

ults

ptimi

inied pSecti

a siy in + grosee F

witrder

witovid

at than am008stabil overeme

PS aed as incdroglled cratio

DOXeousraturadinS rate of

are vious

(Munecautrast,PION

2008t (upreciayer. ex isitionected

adsorption on the iron oxide surface still takes place under-the PEG layer (Fig. 1). Therefore, the drug is loaded on thewo mechanisms: capture in the PEG layer (Dilnawaz et al.,Lbbe et al., 1996a; Yallapu et al., 2010) and iron-mediatedtion on the SPION surface (Munnier et al., 2008).dulating DOXFe2+ complex/PS iron ratio has an inuence onX loading (Fig. 3A). Indeed, the loading is 1% for a DOXFe2+ex/PS iron ratio of 0.05, but increases to 3% for a ratio of 0.1. this article in press as: Gautier, J., et al., A pharmaceutical study of doInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

ce) were grown in Dulbeccos Modied Eagle Mediumpplemented with 5% foetal bovine serum and 100 UI/mL

and 100 g/mL streptomycin at 37 C in a humidied 5%here. All reagents were purchased from Fisher Bioblockllkirch, France).

oxicity evaluation of DOX-loaded PEGylated SPIONtoxicity assays, cells were seeded for 48 h in standardtes (Cellstar, Greiner Bio-One, Courtaboeuf, France) ats per well. Then the culture medium was discarded andere treated for 96 h with 500 L of medium contain-t doxorubicin concentrations (0.00130 M), either asns or as DLPS suspensions (iron content from 1 g/L toCell viability was determined using a tetrazolium dye

(Mosmann, 1983). The cells were rinsed thrice withered Salt Solution (HBSS) pH 7.4 and incubated for 4 hmedium containing 0.5 g/L of MTT. Then the mediumd by 500 L of dimethylsulfoxide to dissolve the for-tals formed by viable cells. Absorbance was measuredsing a multiwell plate reader (ELX800, BioTek, Fisherkirch, France). The 50% inhibitory concentration (IC50)ined as the drug concentration that resulted in a 50%

cell viability. All the experiments were performed in.

cal spectral imaginglasses of 1.4 cm2 were coated with poly-d-lysine at

water for 1 h then placed in the wells of a 24-well7 cells were plated (2 104 cells/well) and cultured foredium was then replaced by a suspension of DLPS in

medium at the concentration of 1 M of doxorubicin.tion was made for 5, 30, 60, 120, 180 min at 37 C/5%reatment, MCF-7 cells were washed twice in HBSS andlasses were then mounted for confocal spectral imag-tion on live cells at 37 C. Fluorescence measurementsd out using an XploraINV confocal microspectrome-

Jobin Yvon, Villeneuve dAscq, France) equipped withted XYZ scanning stage, a low dispersion gratings/mm) and an air-cooled CCD detector. The DOX u-as excited using a 532 nm line of an Ar+ laser. The

e spectra were excited and collected in confocal mode, 60 LWD objective. Live treated cells were placed inroscopy chamber (Harvard Instruments) thermostated

r each cell analysis, an optical section (xy plane) situ--thickness of the cell was scanned with a step of 0.7 med maps containing typically 900 spectra (0.05 s perBoth acquisition and treatment of multispectral mapsrmed with LabSpec software. Subcellular drug distri-s were established via analysis of both the intensityof DOX uorescence spectra, as described previouslyt al., 2011). Briey, each experimental spectrum was

the least-squares method to a sum of the three refer-a of doxorubicin (see the Section 3). The tting errors

5% (typically 24%). The cellular autouorescence was neglected, because of the absence of any signicant u-f the untreated cells under the conditions used (laser

on the sample, 0.05 s per spectrum). No sample pho-on was observed. The average quantitative informationes) was extracted from each spectral map and repre-istograms.

HydroPolydiZeta p

IsoeleLoadin

3. Res

3.1. O

Thedescrib2008; sentedstabilitFeOH2value (coating

In otreatedtant prgroupscreate et al., 2loidal neutrameasu

Thepreparbicin ithe hycontromolar

Thean aqutempeDOX loDOX/Pthe timresults

PreSPIONDOX, bIn conrated Set al., nicanan appPEG lacomplno addbe expchelateneath PS by t2010; adsorp

Mothe DOcompl(DLPS)

ic diameter (nm) 68.0 (2.4) 62.3 (2.5)ty index (PDI) 0.174 (0.018) 0.140 (0.030)al (mV), pH 411 16 (7.2) to 17

(4.6)21 (6.3) to 21(3.4)

oint (IEP) pH 7.65 (0.14) pH 7.28 (0.10)/w DOX/iron oxides) 3.07% (0.04)

and discussion

zation of DOX loading to PS

tial SPION were prepared according to a methodreviously to obtain a cationic aqueous sol (Herv et al.,on 2.1). SPION obtained in this manner typically pre-ze of 8 2 nm in TEM (data not shown). The colloidalacidic medium is provided by electrostatic repulsion ofups on the surface of SPION and is dependent on pHig. 2). However, for neutral particles around pH 7.4, ah PEG can provide stability due to steric hindrance.

to bind the PEG chains to SPION, the nanoparticles wereh 3-aminopropyltrimethoxysilane (APTES). This reac-es (i) methoxy silane groups that react with the hydroxyle surface of SPION and (ii) amino groups necessary toide bond with the activated ester group of aPEG (Herv

). These PEGylated SPION (PS) present an excellent col-lity due to steric repulsion, their surface being nearlyr a wide range of pH as demonstrated by zeta potentialnts (Fig. 2 and Table 1).re loaded with a pre-formed DOXFe2+ complex (Fig. 1),s described elsewhere (Munnier et al., 2008): doxoru-ubated with Fe2+ ions to form a chelate by replacingen of the C11 phenolic group of DOX under strictlyoncentration and pH conditions (pH 7.6, 1.5 iron/DOX).Fe2+ complex solution is then incubated with PS in

buffer pH 7.6 at room temperature, since increasinge up to 50 C during incubation shows no incidence ong (data not shown). In contrast, loading depends on theio (w/w ratios of DOX/PS iron varied from 0.05 to 1) and

incubation (30 min to 24 h). The most representativepresented in Fig. 3.ly described assays on non-PEGylated, citrate stabilizednier et al., 2008) showed no signicant loading of free

se of a lack of afnity of the drug to the surface of SPION. using DOXFe2+ chelates, the drug loading on the cit-

attained 14.6 0.5% DOX/iron oxide w/w (Munnier). In the present study, free DOX loading on PS is sig-

to 1.82 0.19% DOX/iron oxide w/w). It indicates thatble amount of drug is diffused and captured in theUnder similar conditions, the loading with DOXFe2+

higher: up to 3% DOX/iron oxide w/w (Fig. 3). Sinceal interaction between DOXFe2+ chelate and PEG can, this increase in DOX loading should indicate that the



Fig. 2. Zeta po ed SPhydrodynamic

On the contameliorate 27%, 7% anratios of 0.1

The decrSPION (14%hydroxyl grand/or silansurface of SSPION surfa

In order meric layer,shown) to 2stabilized fodrug loadin

ociat

Fig. 3. DOX loDOX/iron ratiotential versus pH for a batch (average of ve determinations) of SPION and PEGylat size distribution of DLPS (n = 3).

rary, increasing the ratio to 0.5 and 1 does not seem to to diss this article in press as: Gautier, J., et al., A pharmaceutical study of doxoruInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

the loading. The loading efciency exhibits saturation:d 3% of incubated drug was loaded respectively with, 0.5 and 1 DOX/PS iron (w/w).eased DOX loading in DLPS (3%) compared to citrated, Munnier et al., 2008) is expected, since a part of theoups on the surface of SPION are occupied by silaneses-PEG and the steric hindrance of PEG chains on thePION limits the access of the DOXFe2+ complex to thece.to study the diffusion time necessary to cross the poly-

we increased the incubation time from 15 min (data not4 h. The drug loading increased between 15 and 30 min,r 2 h and decreased for 24 h (Fig. 3B). This decrease ing may be due to a tendency of the complex in solution

librium betRazzano et permit the Dthat longer of saturatiobelow correi.e. 30 min iature. This for future th

3.2. Physico

Transmithe morpho

0

0.5

1

1.5

2

2.5

3

0.20 0. 4 0.6 0. 8 1incubated DOX/iron w/w ratio (%)

A B

Load

edD

OX

(%, w

/w d

rug/

iron)

0

20

40

60

80

100

0

Load

ed D

OX

(%of

max

imum

load

ing)

ading results under different conditions: (A) inuence of incubated DOX/iron ratio for 30 0.5.ION before (PS) and after DOX loading (DLPS). Insert: TEM image and

e or to precipitate over time, thus changing the equi-bicin-loaded PEGylated nanoparticles for magnetic drug

ween free and PS-bound complex (Fiallo et al., 1999;al., 1990). The results suggest that 30 min are enough toOXFe2+ complex to migrate through the PEG layer and

contact is useless to enhance loading, probably becausen of the PEG layers with the drug. The results describedspond to DLPS prepared under the optimal conditions,

ncubation with DOX/PS iron ratio of 0.1 at room temper-method is sufciently simple to be easily transposableerapeutic applications.

-chemical characterization of DLPS

ssion electron microscopy (TEM) permits to observelogy of the iron oxide nucleus and estimate its real

84 12 16 20 24incubation time(h)

min of incubation (n = 3); (B) inuence of incubation time (n = 3) for

Please cite xorubicin-loaded PEGylated nanoparticles for magnetic drugtargeting.


diameter. The DLPS appear to have a uniform roughly sphericalshape (see insert in Fig. 2), with an average size of 10 nm 2 nm.These data are similar to those for SPION and PS obtained in ourlaboratory (Herv et al., 2008; Munnier et al., 2008; Shkilnyy et al.,2010), and by coprecip2011; YallaPEGylated pthe drug cahave not suof drying duchemical coof PEG and uorescenc

On the coate the hydaccount theDLPS are for various The DLS meimages: theinsert in FigThis result suspension

The DLSin functionto zero overm prior onature of thDPLS suspeparticularlyhydrodynamin zeta poteof zeta potelayer, as the(see Fig. 2).in particulaposition C1ertheless, taits major pcharge cousupercial that the decpotential arin the prese

Finally, their small physiologictration in vet al., 2010)

3.3. Kinetic

The in vdonor/accethe suspenstemperaturpH to evaluacid/sodium

At pH 7.burst effectreaches a plThe resultsdrug to be pH.

0

20

40

60

80

100

120

0

ded

vitroium a

H 4,leasduramentas th008)

to ston th

kine a clin120

case dru

targanis

free raturtic nnawas loa

targeYan eut also of the entrapped DOX, which will then carry a positive

on the sugar moiety (see Fig. 1). Naturally, we are consciouse observed kinetics are only in vitro models and cannot bey extrapolated to intracellular or in vivo kinetics. Under themental conditions, the appearance of a plateau indicates thatf the loaded DOX is only slowly released from the DLPS. This

results is described in the literature for coated SPION. At pH% of adsorbed DOX are released within 24 h from PEO-coated

(Maeng et al., 2010) and 31% in 48 h from composite poly-ated SPION (Rastogi et al., 2011). Our hypothesis is that the

of DOX is controlled by the drug diffusion through the poly- this case, the donor/acceptor volume ratio is decisive in thef the plateau, and we can imagine that it would be displacedcceptor volume were larger. This is not the only explanation,he ionic strength of the release medium may play a role ine of ion exchange at the surface of the SPION, as described inrature (Li et al., 2008). Further studies could shed a light one DOX release from DLPS is modied in vivo. We will presentesults of the intracellular release kinetics of the drug in thection. this article in press as: Gautier, J., et al., A pharmaceutical study of doInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

similar to those for other iron nanoparticles obtaineditation in the literature (Aqil et al., 2008; Rastogi et al.,pu et al., 2010). The TEM size of non-PEGylated andarticles is very similar because the polymer layer andnnot be visualised on TEM images: these compoundsfcient electron density and their layers fold becausering sample preparation (Mahmoudi et al., 2010). Themposition of the organic layers, namely the presenceDOX, was conrmed respectively by means of FTIR ande spectroscopy (data not shown).ntrary, dynamic light scattering (DLS) permits to evalu-

rodynamic diameter of nanoparticles, which takes into drug/polymer layer. As shown in Table 1, values for62 nm, which is close to those previously publishedPEGylated SPION (Herv et al., 2008; Xie et al., 2007).asurements conrm the impression given by the TEM

distribution of the nanoparticles is monomodal (see. 2) with a satisfactory PDI smaller than 0.2 (see Table 1).shows that no large aggregates are present in the DLPS.

results are completed by zeta potential measurements of the pH. For PS and DLPS, the zeta potential is closer the pH range from 4 to 10 (Fig. 2). These results con-bservations (Herv et al., 2008) and support the stericale colloidal stability of the suspensions. This implies thatnsions are physically stable in suspension at any pH, and

at physiological pH. We observe a slight decrease inic diameter (62 versus 68 nm, Table 1) and an increase

ntial for DLPS compared to PS. This slight modicationntial cannot be due to a signicant damage of the PEG

obtained prole is very far from the one of native SPION One hypothesis could be that DOX ionizable functions,r the amino group (pKa 8.2) and the phenolic group at1 (pKa 9.5) inuence the surface charge of DLPS. Nev-king into account the relatively low drug loading, and

resence at the surface of SPION, the increased surfaceld not be directly induced by the presence of DOX inPEG layers. The most plausible hypothesis seems to bereased hydrodynamic diameter and the increased zetae due to a change in spatial conformation of PEG chainsnce of DOX.the physical and chemical characteristics of DLPS likesize, surface neutrality and good colloidal stability atal pH make them compatible with a systemic adminis-ivo (Harris and Chess, 2003; Duan et al., 2008; Veiseh.

s of DOX release from DLPS

itro release of DOX from DLPS was studied in a 24-foldptor volume ratio to mimic the signicant dilution ofion in the organism under physiological conditions ofe and pH (37 1 C, DPBS buffer pH 7.4), as well as acidicate the pH dependence of the system (37 1 C acetic

acetate buffer pH 4).4, the kinetics observed with DLPS are progressive: no

appears, DOX is released continuously during 2 h thenateau equivalent to 70% of the loaded drug (see Fig. 4).

demonstrate that the PEG layer does not prevent thereleased but delays this phenomenon at physiological

% D

OX

rele

ased

/DO

X lo

a

Fig. 4. Inacid/sod

At pdrug reimum experifaster, et al., 2essarydiffusi

Theing. Infor 60In ourloadedpeuticthe orgby theto litemagnefor Dilvesicleon the2011; DOX, bchargethat thdirectlexperia part okind of7.4, 33SPIONmer coreleasemer. Invalue oif the asince tthe ratthe litehow thsome rnext se642 8

Time (h)

Acetate buffer pH 4

DPBS pH 7.4

release of doxorubicin from DLPS (DPBS pH 7.4, 37 C, n = 3 and aceticcetate buffer pH 4, 37 C, n = 3).

the release is considerably accelerated with 85% of theed in 1 h and practically total recovery within 2 h. A min-tion of 1 h is imposed by the centrifugation step of the, but our hypothesis is that the phenomenon is evene DOXFe2+ complex is not stable at pH 4 (Munnier. The time of release is then dependent on the time nec-abilization of the acidic pH near the SPION surface afterrough the PEG layer.tics at pH 7.4 seem adapted to magnetic drug target-ical study, Lbbe et al. (2001) applied a magnetic eld

min in order to accumulate their vectors in the tumour., the moderate release during the rst hour (40% ofg) should permit the nanoparticles to reach the thera-et without disseminating a high percentage of drug inm. This is important to limit the side effects generateddrug. In any case, this time seems reasonable comparede (2 weeks kinetics with glycerol monooleate coatedanoparticles loaded with paclitaxel and/or rapamycinz et al., 2010; 200 h kinetics with wormlike polymerded with SPION and DOX for Yang et al., 2010). Oncet, the more acidic pH of tumour tissue (Medeiros et al.,t al., 2007) should stimulate the release of the chelated



3.4. In vitro biological evaluation

3.4.1. Intracellular distribution and interaction of DOX deliveredwith DLPS

Intracellredistributiimaging (Cmicroscopyuorescencsion spectrto distinguuorescenc

Three sigDOX were dence spectrtwo more ointracellulareference sspecic maa video imcients can(Fig. 5C).

As descrtrum (hereanuclear DNcell membrnanoparticltra, denotedhigher polaspectra areet al. (2010)DOX was cowithin the Pinvolved wcles were scoated withbound throthe presentis not uoremolecules eand their lar environmPEG enviro(Shkilnyy etern of bothcreates a mparticles areven more are incubatsuch a blueincrease in the local entric constanin the litera(1990) PEG branes by dfrom them. the PEG-relPEG layers itral similaripreviously (Shkilnyy e

From thand of the fraction (spstill entrap

cytoplasmic fraction (spectrum cyt2) might be assigned to the drugmolecules leaving the PEG layers and thus exposed to an increaseof the polarity. As to a possible DOX fraction released from the par-ticles outside the cells, under the conditions used, it is negligible in

f thes, wellula

nce oly rer dru011)

the as delivercencclearafterintrauce rom

by i locahe inulatianopracelhese ced cenccenc

(2.2%se dae iny ca

of d.

Cytot aim

on desactioing t6 h inin thn of Mmparoluti. Weier ed fo

xicityboth

are c0.6 aresul

96 hmechNA

l min DOXt vell is presefree echa this article in press as: Gautier, J., et al., A pharmaceutical study of doInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

ular doxorubicin release and subcellular doxorubicinon are followed in living cells by confocal spectralSI). In opposition to classical confocal uorescence, the CSI collected not only the intensity of doxorubicine at a given wavelength, but rather the total emis-um from each point scanned. This approach permitsish very subtle modications of doxorubicin intrinsice (Fig. 5).nicantly different uorescence spectra of intracellularetected in different cellular compartments (see refer-

a in Fig. 5A): a red-shifted spectrum in the nucleus andr less blue-shifted ones in cytoplasmic regions. Eachr spectrum of DOX was tted as a weighted sum of thesepectra. The tting coefcients were used to generateps (Fig. 5B) that can be merged and superimposed onage of the cell (Fig. 5D). In addition, the tting coef-

be statistically treated and presented in histograms

ibed before (Munnier et al., 2011), the nuclear spec-fter denoted nuc) is assigned to DOX intercalated intoA. This corresponds only to DOX diffused through theane or intracellularly released from DLPS, since thees cannot enter the nucleus. The two cytoplasmic spec-

cyt1 and cyt2, correspond respectively to a lower andrity of the molecular environment. These cytoplasmic

very similar to those observed previously by Shkilnyy for the cytoplasmic location of SPIONDOXPEG wherevalently bound to the SPION surface, i.e. deeply buriedEG layer. In the work reported by Shkilnyy et al., cells

ere MCF-7 cells as in our experiment, and the parti-imilar, with a 73 nm hydrodynamic size and were

PEG-5000. DOX remained uorescent since it wasugh the amine group, outside of the uorophore. In

study, DOX bound to SPION by intermediate of Fe2+

scent (Munnier et al., 2008). In contrast, iron-free DOXntrapped/diffused inside the PEG layer are uorescentuorescence spectra are characteristic of their molecu-ent within the PEG. As we determined previously, the

nment changes once the nanoparticles are inside cellst al., 2010). According to the blue-shifted spectral pat-

cytoplasmic spectra, the PEG of intracellular particlesuch more apolar environment for DOX, than when thee in aqueous buffer. Furthermore, this environment isapolar (more hydrophobic) than in the case when cellsed with DOX solution (Munnier et al., 2011). In general,

shift of the emission spectrum is accompanied by anuorescent emission intensity. This kind of altering ofvironment around the uorophore to a lower dielec-t medium upon PEGmembrane interaction is knownture (Arnold et al., 1985). According to Ohki and Arnoldincreases the hydrophobicity of contacting lipidic mem-estabilizing their surface and by withdrawing free waterOn the other hand, the decrease in dielectric constant ofated environment can be related to condensation of thenside intracellular vesicles. This argument and the spec-ty are both in agreement with internalization observedfor DOX covalently bound to SPION covered with PEGt al., 2010).e logical consideration of the apolar environmentrelease kinetics, the major cytoplasmic uorescenceectrum cyt1) should correspond to DOX moleculesped in PEG (most apolar environment). The minor

view oThu

extraceorescetypicalcellulaet al., 2tion ofAs it walso deuoresthe nu22.8% of the we dedcome fcarried

Thewith taccummer nthe intfrom tis reduuoresuoresbation

Therm thdeliverreleasesection

3.4.2. The

loadedresultssite of Accordover 9duced divisioWe cobicin s(Fig. 6)(Munnadjustecytotostudy, valuesto be These withinmajor in the DSeverature asit is nothe wecould of the eral mbicin-loaded PEGylated nanoparticles for magnetic drug

recorded spectra (data not shown). are not able to exclude a small contribution of DOX ofr origin notably for nuclear uorescence. Indeed, u-f the doxorubicin administered as aqueous solutionsults in a strong nuclear staining (>90% of total intra-g) and a minor distribution in the cytoplasm (Munnier, which matches its main mechanism of action, inhibi-nuclear enzyme DNA topoisomerase II (Hande, 2006).termined before (Munnier et al., 2011) citrated SPION

most of doxorubicin to the nucleus (more that 90% ofe after 1 h incubation). However, when we used DLPS,

DOX uorescence represents only 2.7% after 5 min and 3 h of incubation. In the latter case, the main fractioncellular DOX is that of the spectrum cyt1 (Fig. 5). Thus,that the main cellular doxorubicin uorescence did notdoxorubicin released outside the cells, but from the drugnternalized nanoparticles.lisation of cyt1 uorescence (Fig. 5) is concomitantternalization of PEGylated SPION by endocytose andon inside intracellular vesicles like lysosomes as poly-articles (Harush-Frenkel et al., 2008). The kinetics oflular drug distribution indicate slow migration of DOXcytoplasmic vesicles to the nucleus (cyt1 uorescencefrom 97.5% after 5 min to 77.1% after 3 h, whereas nuce increases from 2.7% to 22.8%). Interestingly, the cyt2e increased temporarily during the rst 30 min of incu-) and then became negligible.ta on subcellular DOX interaction and distribution con-teresting potential of PEGylated SPION as controlledrriers for doxorubicin. The effect of this progressiveoxorubicin on cellular viability is described in the next

oxicity of this biological evaluation was to determine if DOXDLPS still presents an antineoplastic activity. The CSIcribed above showed that the entrance of DOX at itsn, the nucleus, is delayed when it is vectorized by DLPS.o these results, we chose to perform a cytotoxicity assay

order to study the activity of the whole drug intro-e medium. This duration corresponds to two cycles ofCF-7 cells, which guaranties the validity of the results.

ed the cytotoxicity on MCF-7 cancer cells of doxoru-on with DLPS with the same amount of loaded drug

demonstrated previously that neither drug-free SPIONt al., 2008) nor PEGylated SPION (Shkilnyy et al., 2010)r the same iron concentration produce any signicant

on MCF-7 cells. For all concentrations tested in this doxorubicin and DPLS have similar activity. The IC50omparable for the two treatments and were determinednd 0.9 M with doxorubicin and DLPS, respectively.ts show that our original loading process is reversible

and does not damage the DOX pharmacophore. Theanism of action of free doxorubicin is an intercalation

and an inhibition of the topoisomerase II (Hande, 2006).or mechanisms of action are described in the litera--induced production of ROS (Minotti et al., 2004). As

ry probable that the whole quantity of DLPS put intointernalized by the cell even in 96 h, the nanovectorsnt a higher anticancer activity than the same amountdrug. We utter the hypothesis that in this case, sev-nisms of action are involved, as DOX has a different



Fig. 5. Confocuorescence: spectra. (C) Staintensity scaleof the article.)

intracellulalated SPIONis a joint aclong incubabrane, and o this article in press as: Gautier, J., et al., A pharmaceutical study of doxoruInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

al spectral uorescence imaging results on the subcellular DOX distribution in live MCF-nuclear form (Nuc, red line) and two cytoplasmic forms (Cyt 1, blue line and Cyt 2, greentistically validated contribution to the cellular uorescence of the three forms of uoresc

for colocalisation and encoded with pseudo colors. (For interpretation of the references t

r distribution from free DOX and DOX-loaded non PEGy- (Munnier et al., 2011). The more plausible hypothesistion of DOX released in the culture medium within thistion time, which diffuses through the plasmic mem-f DOX released intracellularly. This hypothesis does not

exclude theFurther stuof these naantineoplassolution.bicin-loaded PEGylated nanoparticles for magnetic drug

7 cancer cells. (A) Reference spectra used to t the intracellular DOX line). (B) Typical subcellular distribution maps of the three referenceence of DOX (n 6). (D) Superposition of the maps using an extendedo color in this gure legend, the reader is referred to the web version

participation of supplementary mechanisms of action.dies can be useful to explore the mechanisms of actionnovectors, and to determine if they present a highertic activity than the drug administered to cells as a



0

20

40

60

80

100

120DLPS suspension

DOX solution

Cel

l via

bilit

y (%

)

Fig. 6. CytoxicMCF-7 cancer

4. Conclus

The origDOX to theis adapted ttion of DOXDOX/iron oseveral respple and rapand seems over, drug csecondary mlight on howpose of stearelease andwill explore

Acknowled

This stuNationale cGrand-Ouesthe Region

References

Aqil, A., VasseuJrme, C.,Eur. Polym

Arnold, K., Heof aqueousstructure.

Chourpa, I., DJonathan, of iron oxiterized by

Dilnawaz, F., Smagnetic i3694370

Douziech-EyroDubois, P.,paramagn

Duan, H.W., Kuthe affectsiron oxideJ. Phys. Ch

Fan, C., Gao, Wity of stealPharm., do

Fiallo, M.M.L., Ganthracyclsphinx. J. I

Greulich, C., Diendorf, J., Simon, T., Eggeler, G., Epple, M., Kller, M., 2011. Uptake andintracellular distribution of silver nanoparticles in human mesenchymal stemcells. Acta Biomater. 7, 347354.

Hande, K.R., 2006. Topoisomerase II inhibitors. Update Cancer Ther. 1, 315.Harris, J.M., Chess, R.B., 2003. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug

ov. 2, FrenkparticK cell., Douimelelopmicatioa, J., Dt, P.J.,. Cance, R.C.Fhe thrubic

Wonginteraloped0..S., Aleting. .S., Bea. Pre. Canc.S., B

thias, , R., Sical exoxoru469

J.H., Le, W., Ktionalothe506di, Me nans in chdi, Mafeli,

of iro3, 232s, S.F.,icles f

G., Meculariotoxin, T.,icatio3., E., Crpa, magntoxici, E., Soucrubicm. 36

Arnobrane

Schmduled30 10 1 0,1 0,01DOX concentration (M)

ity of DLPS versus DOX at equal drug concentration as measured oncells (96 h, MTT assay).

ion

inal approach of binding and pH-sensitive release of SPION surface using a pre-formed DOXFe2+ complexo PEGylated SPION. This approach allows the prepara--loaded SPION carrying modest amounts of drug (3%xide, w/w). Nevertheless, the model is interesting inects. The drug is loaded on the particles following a sim-id protocol. The drug release kinetics is pH-dependent,to be favourable for magnetic drug targeting. More-ytotoxicity is preserved, and we suppose that severalechanisms of action are involved. This study sheds a

PEGylation of the particles, often realized for the pur-lth, has an important inuence on drug loading, drug

nanoparticle distribution into the cell. Further studies the cytotoxic potential of these DLPS in vivo.

gments

dy was supported in part by grants from the Ligueontre le Cancer (Conseil Scientique Inter-Rgionalt (CSIRGO), Dlgation Indre-et-Loire, France) and fromCentre, France (NANOMAG Project).

r, S., Duguet, E., Passirani, C., Benot, J.P., Roch, A., Mller, R., Jrme, R., 2008. PEO coated magnetic nanoparticles for biomedical application.. J. 44, 31913199.rrmann, A., Pratsch, L., Gawrisch, K., 1985. The dielectric properties

solutions of poly(ethylene glycol) and their inuence on membraneBBA Biomembranes 815, 515518.

DiscHarush-

nanoMDC

Herv, KH., Ldeveappl

LankelmDiesClin

Leonarding tdoxo

Li, Y., lar deve607

Lbbe, Atarg

Lbbe, A1996cacy

Lbbe, AMatSohrClinepid4686

Maeng, Kimfuncchem4995

Mahmouoxidtion

MahmouH., HicityC 11

Medeiropart

Minotti,molcard

Mosmanappl556

MunnierChouparacyto

MunnierH., doxoPhar

Ohki, S.,mem

Ptz, G.,sche this article in press as: Gautier, J., et al., A pharmaceutical study of doxoruInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010

ouziech-Eyrolles, L., Ngaboni-Okassa, L., Fouquenet, J.-F., Cohen-S., Souc, M., Marchais, H., Dubois, P., 2005. Molecular compositionde nanoparticles, precursors for magnetic drug targeting, as charac-

confocal Raman microspectroscopy. Analyst 130, 13951403.ingh, A., Mohanty, C., Sahoo, S.K., 2010. Dual drug loaded superpara-ron oxide nanoparticles for targeted cancer therapy. Biomaterials 31,6.lles, L., Marchais, H., Herv, K., Munnier, E., Souc, M., Linassier, C.,

Chourpa, I., 2007. Nanovectors for anticancer agents based on super-etic iron oxide nanoparticles. Int. J. Nanomed. 2, 541550.ang, M., Wang, X.X., Wang, Y.A., Mao, H., Nie, S.M., 2008. Reexamining

of particle size and surface chemistry on the magnetic properties of nanocrystals: new insights into spin disorder and proton relaxivity.em. C 112, 81278131.., Chen, Z., Fan, H., Li, M., Deng, F., Chen, Z., 2010. Tumor selectiv-

th multi-functionalized superparamagnetic iron nanoparticles. Int. J.i:10.1016/j.ijpharm.2010.10.038.arnier-Suillerot, A., Matzanke, B., Kozlowski, H., 1999. How Fe3+ bindsine antitumour compounds. The myth and the reality of a chemicalnorg. Biochem. 75, 105115.

dose limit393397.

Rastogi, R., GEvaluationposites for167.

Razzano, G., Rstants of achromoph

Roch, A., Gosspensions: 532539.

Sabat, R., Barrich, J., 200Int. J. Phar

Shkilnyy, A., Limelette,uation of nmagnetic 114, 5850bicin-loaded PEGylated nanoparticles for magnetic drug

214221.el, O., Rozentur, E., Benita, S., Altschuler, Y., 2008. Surface charge ofles determines their endocytic and transcytotic pathway in polarizeds. Biomacromolecules 9, 435443.ziech-Eyrolles, L., Munnier, E., Cohen-Jonathan, S., Souc, M., Marchais,tte, P., Warmont, F., Saboungi, M.L., Dubois, P., Chourpa, I., 2008. Theent of stable aqueous suspensions of PEGylated SPION for biomedicalns. Nanotechnology 19, 465608, 7 pp.ekker, H., Luque, R.F., Luykx, S., Hoekman, K., van der Valk, P., van

Pinedo, H.M., 1999. Doxorubicin gradients in human breast cancer.r Res. 5, 17031707.., Williams, S., Tulpule, A., Levine, A.M., Oliveros, S., 2009. Improv-erapeutic index of anthracycline chemotherapy: focus on liposomalin (MyocetTM). J. Breast 18, 218224., H.L., Shuhendler, A.J., Rauth, A.M., Wu, X.Y., 2008. Molecu-ctions, internal structure and drug release kinetics of rationally

polymerlipid hybrid nanoparticles. J. Control. Release 128,

exiou, C., Bergemann, C., 2001. Clinical applications of magnetic drugJ. Surg. Res. 95, 200206.rgemann, C., Huhnt, W., Fricke, T., Riess, H., Brock, J.W., Huhn, D.,clinical experiences with magnetic drug targeting: tolerance and ef-er Res. 56, 46944701.ergemann, C., Riess, H., Schriever, F., Reichardt, P., Possinger, K.,M., Drken, B., Herrmann, F., Grtler, R., Hohenberger, P., Haas, N.,ander, B., Lemke, A.J., Ohlendorf, D., Huhnt, W., Huhn, D., 1996b.periences with magnetic drug targeting: a phase i study with 4-bicin in 14 patients with advanced solid tumors. Cancer Res. 56,3.e, D.H., Jung, K.H., Bae, Y.H., Park, I.S., Jeong, S., Jeon, Y.S., Shim, C.K.,im, J., Lee, J., Lee, Y.M., Kim, J.H., Kim, W.H., Hong, S.S., 2010. Multi-

doxorubicin loaded superparamagnetic iron oxide nanoparticles forrapy and magnetic resonance imaging in liver cancer. Biomaterials 31,6.., Sant, S., Wang, B., Laurent, S., Sen, T., 2010. Superparamagnetic ironoparticles (SPION): development, surface modication and applica-emotherapy. Adv. Drug Deliv. Rev., 006, doi:10.1016/j.addr.2010.05.., Shokrgozar, M.A., Simchi, A., Imani, M., Milani, A.S., Stroeve, P., Vali,U.O., Bonakdar, S., 2009. Multiphysics ow modelling and in vitro tox-n oxide nanoparticles coated with poly(vinyl alcohol). J. Phys. Chem.22331.

Santos, A.M., Fessi, H., Elaissari, A., 2011. Stimuli-responsive magneticor biomedical applications. Int. J. Pharm. 403, 139161.enna, P., Salvatorelli, E., Cairo, G., Gianni, L., Anthracyclines:, 2004.

advances and pharmacologic developments in antitumor activity andcity. Pharmacol. Rev. 56, 185229.

1983. Rapid colorimetric assay for cellular growth and survival:n to proliferation and cytotoxicity assays. J. Immunol. Methods 65,

ohen-Jonathan, S., Herv, K., Linassier, C., Souc, M., Dubois, P.,I., 2011. Doxorubicin delivered to MCF-7 cancer cells by super-etic iron oxide nanoparticles: effects on subcellular distribution andty. J. Nanopart. Res. 13, 959971.Cohen-Jonathan, S., Linassier, C., Douziech-Eyrolles, L., Marchais,, M., Herv, K., Dubois, P., Chourpa, I., 2008. Novel method ofinSPION reversible association for magnetic drug targeting. Int. J.3, 170176.ld, K., 1990. Surface dielectric constant, surface hydrophobicity and

fusion. Membr Biol. 114, 195203.ah, O., Eckes, J., Hug, M.J., Winkler, K., 2009. Controlled application and

removal of nanoparticle based chemotherapeutics (CARL) will reduceing adverse events in anticancer chemotherapy. Med. Hypotheses 72,

ulati, N., Kotnala, R.V., Sharma, U., Jayasundar, R., Koul, V., 2011. of folate conjugated PEGylated thermosensitive magnetic nanocom-

tumor imaging and therapy. Colloids Surf. B: Biointerfaces 82, 160

izzo, V., Vigevani, A., 1990. Determination of phenolic ionization con-nthracyclines with modied substitution pattern of anthraquinoneore. Farmaco 45, 215222.uin, Y., Muller, R.N., Gillis, P., 2005. Superparamagnetic colloid sus-water magnetic relaxation and clustering. J. Magn. Magn. Mater. 293,

nadas-Rodrguez, R., Callejas-Fernndez, J., Hidalgo-lvarez, R., Estel-8. Preparation and characterization of extruded magnetoliposomes.

m. 347, 156162.Munnier, E., Herv, K., Souc, M., Benoit, R., Cohen-Jonathan, S.,

P., Saboungi, M.L., Dubois, P., Chourpa, I., 2010. Synthesis and eval-ovel biocompatible super-paramagnetic iron oxide nanoparticles as

anticancer drug carrier and uorescence active label. J. Phys. Chem. C5858.



Veiseh, O., Gunn, J.W., Zhang, M., 2010. Design and fabrication of magnetic nanopar-ticles for targeted drug delivery and imaging. Adv. Drug Deliv. Rev. 62, 284304.

Wang, M., Thanou, M., 2010. Targeting nanoparticles to cancer. Pharmacol. Res. 62,9099.

Wassel, R.A., Grady, B., Kopke, R.D., Dormer, K.J., 2007. Dispersion of super paramag-netic iron oxide nanoparticles in poly(d,l-lactide-co-glycolide) microparticles.J. Colloids Surf. 292, 125130.

Weissleder, R., Stark, D.D., Engelstad, B.L., Bacon, B.R., Compton, C.C., White, D.L.,Jacobs, P., Lewis, J., 1989. Superparamagnetic iron oxide: pharmacokinetics andtoxicity. Am. J. Roentgenol. 152, 167173.

Xie, J., Xu, C., Kohler, N., Hou, Y., Sun, S., 2007. Controlled PEGylation of monodisperseFe3O4 nanoparticles for reduced non-specic uptake by macrophage cells. Adv.Mater. 19, 31633166.

Yallapu, M.M., Othman, S.F., Curtis, E.T., Gupta, B.K., Jaggi, M., Chauhan, S.C., 2010.Multifunctional magnetic nanoparticles for magnetic resonance imaging andcancer therapy. Biomaterials, doi:10.1016/j.biomaterials.2010.11.028.

Yan, G.P., Robinson, L., Hogg, P., 2007. Magnetic resonance imaging contrast agents:overview and perspectives. J. Radiol. 13, e5e19.

Yang, X., Grailer, J.J., Rowland, I.J., Javadi, A., Hurley, S.A., Steeber, D.A.,Gong, S., 2010. Multifunctional SPIO/DOX-loaded wormlike polymervesicles for cancer therapy and MR imaging. Biomaterials 31, 90659073.

Ying, E., Hwang, H.M., 2010. In vitro evaluation of the cytotoxicity of iron oxidenanoparticles with different coatings and different sizes in A3 human T lym-phocytes. J. Sci. Total Env. 408, 44754481.

Ying, X.Y., Du, Y.Z., Hong, L.H., Yuan, H., Hu, F.Q., 2011. Magnetic lipid nanoparticlesloading doxorubicin for intracellular delivery: preparation and characteristics.J. Magn. Magn. Mater. 323, 10881093.

Zhu, S., Hong, M., Tang, G., Qian, L., Lin, J., Jiang, Y., Pei, Y., 2010. Partly PEGy-lated polyamidoamine dendrimer for tumor-selective targeting of doxorubicin:the effects of PEGylation degree and drug conjugation style. Biomaterials 31,13601371. this article in press as: Gautier, J., et al., A pharmaceutical study of doInt J Pharmaceut (2011), doi:10.1016/j.ijpharm.2011.06.010bicin-loaded PEGylated nanoparticles for magnetic drug

A pharmaceutical study of doxorubicin-loaded PEGylated nanoparticles for magnetic drug targeting1 Introduction2 Materials and methods2.1 Nanoparticle preparation2.1.1 Materials2.1.2 PEGylated SPION2.1.3 Doxorubicin (DOX) loading on PEGylated SPION

2.2 Nanoparticle characterization2.2.1 Morphology and size2.2.2 Zeta potential measurements2.2.3 Iron determination2.2.4 Drug loading determination2.2.5 In vitro release kinetics of drug from DOX-loaded PEGylated SPION (DLPS)

2.3 Biological evaluation of DOX-loaded PEGylated SPION2.3.1 Cytotoxicity evaluation of DOX-loaded PEGylated SPION2.3.2 Confocal spectral imaging

3 Results and discussion3.1 Optimization of DOX loading to PS3.2 Physico-chemical characterization of DLPS3.3 Kinetics of DOX release from DLPS3.4 In vitro biological evaluation3.4.1 Intracellular distribution and interaction of DOX delivered with DLPS3.4.2 Cytotoxicity

4 ConclusionAcknowledgmentsReferences

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