Nbc

MMa

b

c

d

a

ARRAA

KDPNMST

1

ibmptetootp

(

0h

International Journal of Pharmaceutics 448 (2013) 221– 230

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

jo ur nal homep a ge: www.elsev ier .com/ locate / i jpharm

ovel core–shell magnetic nanoparticles for Taxol encapsulation iniodegradable and biocompatible block copolymers: Preparation,haracterization and release properties

aria Filippousia,∗, Sofia A. Papadimitrioub, Dimitrios N. Bikiarisb,∗∗, Eleni Pavlidouc,avroeidis Angelakerisc, Dimitris Zamboulisd, He Tiana, Gustaaf Van Tendelooa

EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, BelgiumLaboratory of Polymer Chemistry and Technology, Department of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, GreeceSolid State Physics Section, Physics Department, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, GreeceLaboratory of General and Inorganic Chemical Technology, Department of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece

r t i c l e i n f o

rticle history:eceived 31 January 2013eceived in revised form 12 March 2013ccepted 13 March 2013vailable online xxx

eywords:rug deliveryolyesteranocarriers

a b s t r a c t

Theranostic polymeric nanocarriers loaded with anticancer drug Taxol and superparamagnetic iron oxidenanocrystals have been developed for possible magnetic resonance imaging (MRI) use and cancer therapy.Multifunctional nanocarriers with a core–shell structure have been prepared by coating superparamag-netic Fe3O4 nanoparticles with block copolymer of poly(ethylene glycol)-b-poly(propylene succinate)with variable molecular weights of the hydrophobic block poly(prolylene succinate). The multifunctionalpolymer nano-vehicles were prepared using a nanoprecipitation method. Scanning transmission electronmicroscopy revealed the encapsulation of magnetic nanoparticles inside the polymeric matrix. Energydispersive X-ray spectroscopy and electron energy loss spectroscopy mapping allowed us to determinethe presence of the different material ingredients in a quantitative way. The diameter of the nanoparti-

agnetic nanoparticlesEMEM

cles is below 250 nm yielding satisfactory encapsulation efficiency. The nanoparticles exhibit a biphasicdrug release pattern in vitro over 15 days depending on the molecular weight of the hydrophobic partof the polymer matrix. These new systems where anti-cancer therapeutics like Taxol and iron oxidenanoparticles (IOs) are co-encapsulated into new facile polymeric nanoparticles, could be addressed aspotential multifunctional vehicles for simultaneous drug delivery and targeting imaging as well as real

peuti

time monitoring of thera

. Introduction

Nanotechnology is at the leading edge of the rapidly develop-ng new therapeutic and diagnostic schemes in diverse areas ofiomedicine. Different materials from natural to synthetic poly-ers as well as inorganic materials with variable structural and

hysical properties are being used as building blocks of bioma-erials. Recently, a new term ‘theranostics’ is used in order toncompass two distinct definitions, which is the combination ofherapeutic and diagnostic agents on a single platform. The devel-pment of theranostic nanoparticles is emerging as a new form

f “smart” nano-materials that may simultaneously monitor andreat diseases (Janib et al., 2010; Shubayev et al., 2009) offering theotential to personalize and advance medicine. A possible approach

∗ Corresponding author. Tel.: +32 3 265 35 29; fax: +32 3 265 33 18.∗∗ Corresponding author. Tel.: +30 2310 997812; fax: +30 2310 997667.

E-mail addresses: Maria.filippousi@ua.ac.be (M. Filippousi), dbic@chem.auth.grD.N. Bikiaris).

378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2013.03.025

c effects.© 2013 Elsevier B.V. All rights reserved.

of this concept is the use of different polymers as vehicles for theencapsulation of magnetic nanoparticles. The polymeric nanopar-ticles (NPs) system can be designed and functionalized in differentways to incorporate a wide variety of chemotherapeutics as well asmagnetic nanoparticles (MNPs) for the monitoring and eventuallydelivery of therapeutic agents to the tumor cells, which makes thecancer theranostics possible (Ling et al., 2011).

Among them MNPs, with the appropriate surface chemistry,are major classes of nanoscale inorganic materials with the poten-tial to revolutionize current clinical diagnostic and therapeutictechniques (Kwon et al., 2011; McCarthy and Weissleder, 2008;Rösler et al., 2001; Sun et al., 2008, Veiseh et al., 2010). MNPs areemployed as magnetic carriers allowing them to be manipulated,tracked, imaged and remotely heated. Due to these unique physi-cal properties and ability to function at the cellular and molecularlevel of biological interactions, MNPs are actively investigated as

the next generation of magnetic resonance imaging (MRI) contrastagents and as carriers for targeted drug delivery (Corot et al., 2006;Cherukuria et al., 2010). Since the particles must be effectively con-trolled by the applied magnetic field, their magnetic properties,

2 nal of

dnose

aopawtnM2AmctZme

vpoptmli(capTwah

tmsedS(fotP

Pttai(

2

2

(p

22 M. Filippousi et al. / International Jour

ispersion and degree of agglomeration are important. Iron oxideanoparticles meet these requirements. The advantage they havever other magnetic nanoparticles is that they present low toxicity,ince Fe exists in the human body; e.g. in porphyrin and a lack of Feven provokes anemia (Calmona et al., 2012; Mornet et al., 2004).

A wide variety of different biodegradable polymers alreadypproved by FDA can be used for the incorporation of ironxide nanoparticles (IOs). Polymers such as poly(caprolactone),oly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA)s well as various novel biodegradable copolymers decoratedith poly(ethylene glycol) (PEG) in order to overcome and avoid

he reticuloendothelial system creating long-circulating “stealth”ano-carriers (Couvreur and Vauthier, 2006; Kataoka et al., 2001;a et al., 2008; Romberg et al., 2008; Stolnik et al., 1995; Torchilin,

002; Van Butsele et al., 2007) are used as IOs nano-carriers.lso such types of polymers may be functionalized with certainolecules like folic acid, in order to target the nano-carrier to spe-

ific cancer cells, or Vitamin E TPGs as a new strategy to overcomehe multi-drug resistance phenotype (Dong and Mumper, 2010;hang et al., 2012; Wang et al., 2011), can and have been used asain host materials for IOs nanoparticles (Guo et al., 2011; Prashant

t al., 2010; Tan et al., 2011).Compared to other aliphatic polyesters that were used pre-

iously, poly(propylene succinate) (PPSu) is a relatively newolyester with higher biodegradation rate due to its lower degreef crystallinity. Moreover, it is a very soft material with a meltingoint close to body temperature (Tm = 44 ◦C) and a glass transi-ion temperature of −36 ◦C, properties that favors the use of these

aterials as potential multi-functional nano-carriers for encapsu-ation of iron nanoparticles and anticancer drug and applicationn MRI as well as deliver of therapeutic agent. A previous studyVassiliou et al., 2010) reported a new type of block copolymersonsisting namely of A–B–A triblock copolymers, obtained through

relatively simple one-pot reaction, where A is the hydrophilicolymer part (PEG) and B represents a hydrophobic part (PPSu).hese copolymers form self-assembled core–shell nanoparticlesith hydrophobic PPSu and hydrophilic PEG forming the core

nd the shell respectively; they are capable of encapsulating bothydrophilic and hydrophobic model drugs.

Transmission electron microscopy (TEM) is an essential tool forhe characterization of soft materials encapsulated with heavy ele-

ent materials at the nanoscale (Loos et al., 2009). Due to theirensitivity under the electron beam and the low contrast theyxhibit in traditional bright-field TEM imaging, high angle annularark field scanning transmission electron microscopy (HAADF-TEM) is an ideal technique to obtain nanoscale morphological dataPennycook and Jesson, 1991). This method is especially well suitedor the characterization of heavy elements on supports consistingf light atoms, such as polymers because the signal is proportionalo Zn (1.6 < n < 2) (Hawkes, 1992; Midgley and Weyland, 2003;ennycook, 1992b).

In the present study three different copolymers of the mPEG-PSu-mPEG structure consisted from different molecular weight ofhe hydrophobic part (PPSu) are used as polymeric carriers in ordero create nanoparticles of dual functionality capable of being useds theranostic systems where IOs and anticancer drug Taxol arencorporated. These copolymers, which were studied previouslyVassiliou et al., 2010) are used for first time in such application.

. Materials and methods

.1. Materials

Succinic acid (Su) (purum 99+%), 1,3-propanediol (PD)purity > 99.7%), titanium (IV) butoxide (≥97%) (TBT), triphenylhosphate (≥99%) (TPP) and poly(ethylene glycol) monomethyl

Pharmaceutics 448 (2013) 221– 230

ether (mPEG) with molecular weight 2000 g/mol were obtainedfrom Sigma–Aldrich and used as received. Paclitaxel (Taxol),a white, odourless, crystalline powder with melting point of210–220 ◦C and 853.92 g/mol molecular weight, was purchasedfrom Indena SPA, Italy. FeCl3·6H2O, FeSO4·7H2O (analytical grade)used without further purification. Doubly distilled water wasused for all solutions. During precipitation for the synthesis ofmagnetite nanoparticles solutions were stirred for about 20 minin an open beaker with a magnetic stirrer. All other materials andsolvents used in the analytical methods were of analytical grade.

2.2. Synthesis of PPSu and mPEG–PPSu block copolymers

Poly(propylene succinate) with different molecular weight wereprepared by a two-stage melt polycondensation method (ester-ification and polycondensation) in a glass batch reactor. Theprocedure has been presented in a previous publication (Vassiliouet al., 2010) (Papadimitriou and Bikiaris, 2009). In brief, the properamount of succinic acid and propanediol in an acid/diol molar ratio1/1.3 and the catalyst TBT (1 × 10−3 mol/mol Su) were charged intothe reaction tube of the polyesterification apparatus. The reactionmixture was heated at 190 ◦C under an argon atmosphere andconstant speed stirring (350 rpm). This first step (esterification)is considered to be completed after the collection of theoreticalamount of H2O (about 3 h), which was removed from the reac-tion mixture by distillation and collected in a graduated cylinder.In the second step of polycondensation, 0.3 wt% of TPP, was addedas heat stabilizer and a vacuum (5.0 Pa) was applied slowly over aperiod of 15 min. The temperature was slowly increased to 220 ◦Cwhile stirring speed was increased to 720 rpm. The polyconden-sation continued for about 60 min at 220 ◦C and after that time thetemperature was increased to 240 ◦C for all prepared polyesters.At 0, 30 and 60 min of vacuum application, samples were drawnthe reaction vessel in order to obtain PPSu of different molecularweights: PPSu1, PPSu2 and PPSu3, respectively.

For the preparation of mPEG–PPSu block copolymers a thirdstep was employed, during which each one of the samples col-lected previously was reacted at 240 ◦C for 30 min under vacuum(5.0 Pa) with 5 equiv. of mPEG of molecular weight 2000 g/mol.The polyesters were removed and purified (to remove unreactedmPEG) by repeated dissolution in chloroform and precipitation incold methanol (3 times).

2.3. Synthesis of IOs

In most applications reported in the literature, iron oxides, suchas magnetite and maghemite, were the magnetic material of choice.Among the most common synthesis route to produce magnetite(Fe3O4) is the coprecipitation of divalent and trivalent iron fromthe respective hydrated salts in an alkaline medium (Kang et al.,1996; Lu et al., 2007; Wan et al., 2007; Qu et al., 1999). Mag-netic iron oxide nanoparticles were synthesized by this method:2.0 mmol FeCl3·6H2O were dissolved in 30 mL doubly distilledwater in a 50 mL conical flask at room temperature. Then 1.0 mmolFeSO4·7H2O was added into the solution. 5 mL of a 5 N NaOHsolution was added under stirring. A black precipitate appearedimmediately.

Fe2+ + 2Fe3+ + 8OH− → Fe3O4 ↓ + 4H2O

During precipitation for the synthesis of magnetite nanoparti-

cles solutions were stirred for about 20 min in an open beaker witha magnetic stirrer. The resulting black precipitates were collectedby centrifugation, washed several times with double distilled waterand finally dried in a freeze-dryer.

nal of

2n

ctnstgsssatma

2

ipeHo4s

swaood(go

opadpwa2wd

2n

ndFmtcdGTwed

M. Filippousi et al. / International Jour

.4. Preparation of IOs-Taxol encapsulated in the polymericanoparticles

The mPEG–PPSu copolymer nanoparticles were prepared by aoprecipitation method. Copolymer (100 mg) was dissolved in 2 mLetrahydrofurane (THF) and 2 mg superparamagnetic iron oxideanoparticles were transferred to the organic phase and probeonicated until homogeneity was achieved; Taxol (10 mg) was alsoransferred to the organic phase. The resulting solution was pouredradually into 30 mL of aqueous phase to aid diffusion of the organicolvent and precipitation of the nanosized particles. The resultantolution was stirred continuously overnight to allow the organicolvent (THF) to vaporize. The particle suspension was centrifugedt 9500 rpm for 20 min to obtain the NPs. The NPs were washedhrice with deionized water and the suspension was filtered by a

icrofilter with a pore size of 1.2 �m in order to remove polymerggregates. The samples were freeze-dried (Tan et al., 2011).

.5. Polymer characterization

The polymers prepared were fully characterized in terms ofntrinsic viscosity and molecular weight determination using gelermeation chromatography as previously reported (Vassiliout al., 2010). Moreover the chemical structure was determined.ydrogen-1 NMR (1H NMR) spectra of the prepared materials werebtained using a Bruker spectrometer operating at a frequency of00 MHz for protons. Deuterated chloroform (CDCl3) was used asolvent in order to prepare solutions of 5% w/v.

The thermal measurements were carried out using a differentialcanning calorimeter (Setaram DSC141). Samples of about 10 mgere heated from −80 ◦C up to 100 ◦C at a heating rate of 10 ◦C/min

nd the melting temperatures (Tm) were recorded as the maximumf the peak. The samples remained at this temperature for 5 min inrder to erase any thermal history and afterwards they were cooledown to −80 ◦C at a cooling rate of −150 ◦C/min and scanned againsecond heating) up to 100 ◦C at a heating rate of 10 ◦C/min. Thelass transition temperatures (Tg) were recorded as the midpointsf the parallel shift lines.

Enzymatic hydrolysis of the prepared samples was performedn films being 5.0 cm × 5.0 cm in size and ∼2 mm thickness, pre-ared in a hydraulic press, which were placed in petries containing

phosphate buffer solution (pH 7.2) with 0.09 mg/mL Rhizopuselemar lipase and 0.01 mg/mL of Pseudomonas Cepacia lipase. Theetries were then incubated at 37 ± 1 ◦C in an oven for several dayshile the media was replaced at predetermine time intervals. After

specific period of incubation (4 h for the first 24 h and then every4 h) the films were removed from the petri, washed with distilledater and weighted until constant weight. The degree of biodegra-ation was estimated from the mass loss.

.6. Characterization of magnetic nanoparticles and magneticanoparticles encapsulated in the polymeric matrix

The surface morphology of the polymer nanoparticles and mag-etic nanoparticles encapsulated in the polymer nano-matrix wasetermined by scanning electron microscopy SEM/EDS using theEI Helios NanoLab 650 operated at 5 kV. Samples suitable for trans-ission electron microscopy (TEM) were prepared by drop casting

he aqueous solution of the assemblies on holey, carbon-coatedopper grids. Bright field (BF) TEM images, selected area electroniffraction (SAED) and STEM images were acquired using a Tecnai2 electron microscope operated at 200 kV. The energy filtered

EM mapping and the electron energy loss spectroscopy (EELS)ere conducted using an UltraTwin CM30 Philips FEG instrument

quipped with a post column GIF200 detector. Crystallographicata on the iron oxide nanoparticles were obtained by X-ray

Pharmaceutics 448 (2013) 221– 230 223

powder diffraction analysis using a Huber G670 Guinier Camera (CuKa1 radiation transmission geometry, Ge (1 1 1) monochromator ona primary beam, Ge external standard) in the 2�-range 10◦–100◦.

Magnetic information of the iron oxide nanoparticles wasobtained by recording hysteresis loops with an Oxford 1.2 H/CF/HTVibrating sample magnetometer (VSM), at room temperature andwith applied fields as high as 1 T.

2.7. Characterization of drug-loaded nanoparticles

The prepared nanoparticles were freeze dried to obtain the finalproduct. Drug loading content was determined with high perfor-mance liquid chromatography (HPLC) analysis using a ShimadzuHPLC (model LC-20AD). 3 mg of nanoparticles were added in 50 mlof ACN/H2O 50/50 v/v and stirred with a magnetic stirrer till com-plete dissolution. A clear solution was obtained which was filteredthrough 45 �m ready for HPLC analysis. The column used wasa Eclipse XDB-C18, 5 �m, 250 mm × 4.6 mm. The flow rate was1 mL/min and the column temperature was 25 ◦C. A diode arraydetector was used at 227 nm, and quantification of the API wasbased on a calibration curve created by diluting with mobile phasea stock solution of 20 �g/mL paclitaxel in water/ACN 50/50 v/v toconcentrations 20, 10, 5, 2.5, 1 and 0.5 �g/mL. The nanoparticleyield, drug loading and drug entrapment efficiency were calculatedusing the following Eqs. (1)–(3), respectively:

Nanoparticles yield (%) = weight of nanoparticlesweight of polymer and drug fed initially

× 100 (1)

Drug loading content (%) = weight of drug in nanoparticlesweight of nanoparticles

× 100 (2)

Entrapment efficiency (%) = weight of drug in nanoparticlesweight of drug fed initially

× 100 (3)

Fourier transform infrared spectroscopy (FTIR) spectra of thefreeze dried nanoparticles were obtained using a Perkin-Elmer FTIRspectrometer, model spectrum 1000. In order to collect the spectraa small amount of each material was used (1 wt%) and compressedin KBr tablets. The IR spectra, in absorbance mode, were obtained inthe spectral region of 450–4500 cm−1 using a resolution of 4 cm−1

and 64 co-added scans.Particle size distribution of the prepared nanoparticles was

determined by dynamic light scattering (DLS) using a ZetasizerNano instrument (Malvern Instruments, Nano ZS, ZEN3600, UK)operating with a 532 nm laser. A suitable amount of nanoparticleswas dispersed in distilled water creating a total concentration 1‰and was kept at 37 ◦C under agitation at 100 rpm.

Wide angle X-Ray diffractrometry (WAXD) was used for theidentification of the crystal (structure and changes) of the iron oxidenanoparticles and also drug and polymer in the case of polymeric-MNPs samples. WAXD study was performed over the range 2� from5 to 60◦, using an automated powder diffractometer Rigaku MiniFlex II, from 5 to 60o,diffractometer with Bragg–Brentano geometry(�, 2�) and Ni-filtered CuKa radiation (� = 0.15406).

In vitro drug release studies were performed using the dis-

solution apparatus I basket method. Drug-loaded nanoparticlesuspensions corresponding to 2 mg of drug were placed in a dialysiscellulose membrane bag (Sigma), having a molecular weight cut-off12.400, tied and placed into the baskets. The dissolution medium

224 M. Filippousi et al. / International Journal of Pharmaceutics 448 (2013) 221– 230

EG–P

fstsfie

3

3

p2pvtwaam

fiwsot

Scheme 1. Synthesis route to mP

or Taxol consisted of 500 mL water (pH = 7.4, Tween 80 0.1%). Thetirring rate was kept constant at 100 rpm, as was the tempera-ure at 43 ◦C. At predetermined time intervals 2 mL of the aqueousolution was withdrawn from the release media. The samples wereltered and assayed for drug by HPLC as mentioned above. In eachxperiment the samples were analyzed in triplicate.

. Results and discussion

.1. Synthesis and characterization of polymers

The synthesis procedure followed in order to create the blockolymers as previously described with details (Vassiliou et al.,010) is shown in Scheme 1. In the first two steps the aliphaticolyester PPSu with different molecular weights was preparedia esterification and melt polycondensation procedure and in thehird step after the addition of mPEG, the final block copolymersere prepared. According to this procedure mPEG–PPSu diblock

nd mPEG-PPSu-mPEG triblock copolymers can be formed. To avoidny confusion in the text and for briefness all the prepared copoly-ers will be characterized as mPEG–PPSu.The chemical structure of the prepared co-polymers was identi-

ed with 1H NMR spectroscopy (Fig. 1). The peak at about 3.65 ppmas assigned to the methylene protons of the mPEG block (b). The

hift of the protons corresponding to the middle methylene groupf the monomer PD appeared at 1.95 ppm (d), while the protons ofhe other two methylene groups of PD appeared as a characteristic

Fig. 1. 1H NMR spectrum of mPEG–PPSu–mPEG.

PSu–mPEG triblock copolymers.

peak at 4.1–4.2 ppm (a). Finally, the protons of the two methy-lene groups corresponding to the two methylene groups of themonomer succinic acid gave a characteristic peak at ∼2.6 ppm (c).

The molecular weight of the prepared copolymers was mea-sured by gel permeation chromatography analysis. During the firststage of esterification (Scheme 1) oligomers having an averagemolecular weight between 1500 and 6000 g/mol are formed whilewater is removed as by-product. In the second step of polyconden-sation the molecular weight can be further increased due to thevacuum application. According to these procedures PPSu with dif-ferent molecular weights were prepared like 5800 (PPSu1), 13600(PPSu2) and 18900 g/mol (PPSu3). These polyesters were used forthe preparation of block copolymers with mPEG and the molecu-lar weight of block copolymers are presented in Table 1. As can beseen the addition of mPEG increases slightly the molecular weightof block copolymers, compared with neat PPSu.

Differential scanning calorimetry (DSC) was used to identify themain characteristic peaks of Tm and Tg values of the synthesizedpolymers (Fig. 2). The melting point (Tm) of the triblock copolymerswere recorded and are similar to that of pure PPSu. Neat PPSu has amelting point at 44 ◦C while mPEG melts at 58.7 ◦C. All copolymershave melting points close to 44–45 ◦C indicating that PEG can co-crystallize with PPSu. Similar are also the Tg temperatures, whichas can be seen are close to the Tg of neat PPSu. This is very importantsince as was found from a previous study both the Tm and Tg valuescould affect the drug release behavior (Karavelidis et al., 2010).

Enzymatic degradation of the copolymers was carried out at37 ◦C in phosphate buffer solution (pH 7.2) containing 0.09 mg/mLR. delemar lipase and 0.01 mg/mL of P. Cepacia lipase. In Fig. 3 theweight loss of pure PPSu and mPEG–PPSu copolymers of different

composition during enzymatic hydrolysis for several days are pre-sented. As can be seen PPSu has a characteristic high degradationrate and after 120 h it is completely degraded. The hydrolysis of the

Table 1Thermal properties of the prepared materials.

Sample Mn (g/mol)a Tg (◦C)b Tm (◦C)

mPEG–PPSu1 8700 −36.6 44.7mPEG–PPSu2 15 400 −37.7 44.5mPEG–PPSu3 20 200 −37.8 44.3PPSu 5600 −35.0 44.0mPEG 2000 ∼−60.0 58.7

a Measured by GPC.b Measured by DSC.

M. Filippousi et al. / International Journal of

-60 -30 0 30 60

0.0

0.6

1.2

1.8

2.4H

eat F

low

(W/g

) End

o U

p

Temperature (oC)

PPSu mPEG mPEG-PPSu2

58.7oC

44oC

44.5oC

Fig. 2. DSC Thermograms of PPSu, mPEG and its mPEG–PPSu2 copolymer.

0 20 40 60 80 100 1200

20

40

60

80

100

Wei

ght l

oss

(%)

PPSu mPEG-PPSu1 mPEG-PPSu2 mPEG-PPSu3

F

mbttfedswPd

FX

Enzymatic Hydrolysis Time (h)

ig. 3. Weight loss (%) of the prepared copolymers during enzymatic hydrolysis.

PEG–PPSu copolymers occurred at its PPSu block, due to esterond breakage by the enzymes. The different PPSu composition ofhe copolymers had only little effect on the enzymatic degrada-ion in our experiments, similarly to previously reported resultsor PCL–PEG copolymers (He et al., 2003; Zhao et al., 2006). Nev-rtheless as the molecular weight of the PPSu segment increased,egradation rates decreased slightly (Fig. 3). The hydrophilic mPEG

egments facilitate the penetration of the water through them andater comes easier in contact with the hydrophobic segments of

PSu promoting copolymer degradation. Thus, the decrease of theegradation rate as the molecular weight of PPSu block increased

ig. 4. (a) Low-resolution TEM image of an aggregation of nanoparticles (b) the corresp-ray powder diffraction pattern of MNPs.

Pharmaceutics 448 (2013) 221– 230 225

is probably associated with the decreased proportion of PEG in thecopolymers with increasing the molecular weight of PPSu block.However, all copolymers can be hydrolysed during time. Further-more, as was found from our previous study these copolyestersexhibited low toxicity against HUVEC cells, with appreciable cyto-toxicity (higher than 20% reduction of cell viability) being observedonly after exposing the cells at high nanoparticles concentrations,i.e. from 800 �g/ml and higher (Vassiliou et al., 2010). In termsof polymer toxicity on HUVEC cells, the mPEG–PPSu copolymerswere comparable to PLA, a polymer of high biocompatibility that iswidely used in drug nanoencapsulation.

3.2. Characterization of IOs nanoparticles

Bright field TEM (BF-TEM) (Fig. 4a) reveals the tendency of theindividual particles to aggregate (Marques et al., 2008; Wan et al.,2007). The corresponding electron diffraction (ED) pattern from theregion of Fig. 4a,b and the derived interplanar distances are in verygood agreement with the XRPD results, confirming the presence ofmagnetite (Fe3O4) or maghemite (�-Fe2O3) nanoparticles. An X-raydiffraction pattern of the as synthesized iron oxide nanoparticles isshown in Fig. 4c. X-ray diffraction pattern were characterized bythe typical reflections (1 1 1), (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1),(5 3 3), (7 1 1) and (7 3 1). Based on the inverse spinel structure ofthe iron oxide nanoparticles, those reflections are in accordancewith the data reported in the literature (Lu et al., 2007; Liu et al.,2003). Phase identification was done by comparing the experimen-tal XRPD patterns with PDF database (PDF#39-1346, 85-1436).

Fig. 5(a) shows a high-resolution TEM image of a single Fe3O4nanoparticle together with the corresponding Fourier transform.Both demonstrate a perfect crystalline structure, free of defects.Particle size distributions were obtained by using the BF-TEMimages, presented in Figs. 4a and 5a. The MNPs nanoparticles canbe observed as small dark spots. The average diameter of the IOsnanoparticles is 9 nm and is presented in the corresponding his-togram in Fig. 5c.

The results of the TEM study combined with the elementalmap for the iron and the oxygen are presented in Fig. 6a–d. Togain complementary information about the oxidation state of iron,EELS was used for compositional quantification (Fig. 6e). The Fe/Oratio, which was derived from EELS quantification is 0.76 ± 0.02(Dumitrache et al., 2004; Golla-Schindler et al., 2006) and corre-sponds to the magnetite (Fe3O4) form of iron oxide.

The magnetic properties of the MNPs were measured at roomtemperature using a vibrating sample magnetometer in a magnetic

field applied up to 1 T. The hysteresis loop was made to determinethe saturation magnetization (Ms) and coercivity (Hc) (Fig. 7). TheMs (47 emu/g) is lower than the value (90 emu/g) of bulk mag-netite (Simeonidis et al., 2008) probably due to the small size of

onding electron diffraction pattern confirming the presence of IO crystals and (c)

226 M. Filippousi et al. / International Journal of Pharmaceutics 448 (2013) 221– 230

Fig. 5. (a) High-resolution TEM image along a {1 1 0} zone axis, (b) the corresponding Fourier transform and (c) size distribution of the IOs.

F cles. Inc ectrut

tv(c(

ig. 6. (a) Bright field image and EFTEM image of oxygen (b) and iron (c) of the partiorresponds to iron oxide nanoparticles. (e) The O–K edge and Fe–L2,3 edge EELS sphe reader is referred to the web version of this article.)

he IO particles. But according to the literature, even small Ms

alues (7–22 emu/g) are reported as adequate for bioapplicationsArsalani et al., 2010). For biomedical use, magnetic nanoparti-les are preferred to be superparamagnetic at room temperatureAkbarzadeh et al., 2012). This result indicates that the magnetic

-1.0 -0.5 0.0 0.5 1.0

-40

-20

0

20

40

Field ( T)

Mag

netiz

atio

n (e

mu/

g)

Fig. 7. Room temperature hysteresis loop of the magnetic nanoparticles.

the combined color map (d) of the O (red) and Fe (green) elements, the yellow aream of magnetite. (For interpretation of the references to color in this figure legend,

materials can be aligned under an external magnetic field but theywill not retain any residual magnetism when the external field isremoved.

3.3. Characterization of MNPs encapsulated in the polymericmatrix

SEM micrographs from the polymer nanoparticles and the MNPsencapsulated in the polymer nanoparticles are shown in Fig. 8.These images are also representative for the rest of the samples.SEM images reveal that the size of the nanoparticles does notexceed 250 nm, which is in agreement with our previous study(Vassiliou et al., 2010). Furthermore, in all cases the nanoparticlesexhibit a narrow and unimodal size distribution. Concerning theeffect of PPSu molecular weight in the particle size it was foundthat as the size of the hydrophobic PPSu segment increases so doesthe overall particle size. This result is in agreement with our previ-ous study (Vassiliou et al., 2010), as well as other studies usingblock copolymers, where the longer the hydrophobic block, thebigger the micelles (Chen et al., 2006a,b; Hu et al., 2007; Gaumetet al., 2008). However, the ability of SEM to study in detail the

nanoparticle structure is limited and for this reason HAADF-STEMmeasurements were also performed.

In the HAADF-STEM technique, the signal is proportional tothe atomic number of the elements Zn (1.6 < n < 2) and therefore

M. Filippousi et al. / International Journal of Pharmaceutics 448 (2013) 221– 230 227

mPEG

tePnpttpctcmc

FSic

Fig. 8. SEM micrographs (a) of mPEG–PPSu–mPEG 3 and (b) of mPEG–PPSu–

he heavier element regions are imaged brighter than the lighterlement regions (Hawkes, 1992; Midgley and Weyland, 2003;ennycook, 1992b). Fig. 9(a) and (c) shows iron oxide Fe3O4anoparticles (bright region with high Z) encapsulated inside aolymeric matrix (darker region with lower Z surrounding the par-icles) from different regions. It should be noticed that there is aendency of the iron oxide nanoparticles to aggregate even in theolymer matrix. STEM-EDX mapping was performed in order toonfirm the presence of the different materials in the polymer par-

icle system. Elemental maps of C, O and Fe are shown in Fig. 9b. It islear that the Fe3O4 nanoparticles are dispersed in the mPEG–PPSuatrix, indicating that magnetic nanoparticles are homogeneously

overed by the polymer.

ig. 9. (a) HAADF-STEM image showing the iron oxides nanoparticles embedded in a polyTEM EDX mapping of mPEG–PPSu–mPEG–MNPs. The blue region corresponds to carbon,n the polymer (less bright thin layer surrounding the bright particles). A color map is preolor in this figure legend, the reader is referred to the web version of this article.)

3–MNPs embedded in the polymer prepared on a carbon coated TEM grid.

In order to have more information about the encapsulation andconfirm that the particles are surrounded by polymer, tilting wasperformed in high angles (Fig. 10). From these images it is clearthat the encapsulation of iron oxide nanoparticles into mPEG–PPSucopolymers can form core–shell nanoparticles. It is well knownthat in aqueous media, the AB or ABA type copolymers, where Arepresents the hydrophilic segment and B the hydrophobic core,may form simple micelles with the hydrophilic part to be the outerpart and the hydrophobic the inner (He et al., 2007). In our block

copolymers PPSu units will form the core and the hydrophilic PEGtails projecting out into the water forming the shell of nanopar-ticles. Furthermore, it is clear that MNPs are mainly located intocore part of nanoparticles leading to the preparation of dual

mer matrix (less bright region around the magnetic nanoparticles) and (b) HAADF- the green to iron and the red to oxygen. (c) HAADF STEM image of IOs encapsulatedsented in (d) to highlight the encapsulation. (For interpretation of the references to

228 M. Filippousi et al. / International Journal of Pharmaceutics 448 (2013) 221– 230

Fig. 10. HAADF STEM images of MNPs encapsulated in the polyme

Table 2Nanoparticle yield, drug loading content and entrapment efficiency, of mPEG–PPSunanoparticles loaded with Taxol.

Np’s mPEG-PPSu-mPEG

Polyester 1 2 3Yield (%) 73.58 ± 1.11 78.21 ± 2.87 70.57 ± 8.41

fb

3p

bndnacc

iiFoHism

F

Drug loading (%) 7.2 ± 0.6 8.3 ± 0.2 8.5 ± 0.3Entrapment efficiency (%) 53 ± 3.67 65 ± 1.33 60 ± 2.43

unctional nano-carriers. This is because iron oxides are hydropho-ic materials.

.4. Characterization of Taxol-MNPs encapsulated in theolymeric matrix

The prepared block copolymers are consisted from a hydropho-ic part (PPSu) and a hydrophilic (PEG) and were used for Taxolanoencapsulation. Table 2 summarizes the nanoparticle yield,rug loading content and the entrapment efficiency of mPEG–PPSuanoparticles with the Taxol. It must be noted that the yield as wells the encapsulation efficiency of anticancer drug Taxol may beharacterized as satisfactory. Drug loading is also similar or higherompared with similar formulations (Niu et al., 2013).

The prepared nanoparticles loaded with Taxol were character-zed by XRPD, in order to identify the physical state of the drugncorporated in the polymeric nanoparticles. As can be seen fromig. 11 the WAXD pattern of pure taxol shows a large numberf sharp diffraction peaks with the most characteristic at 12.68◦.owever, this peak was not recorded in the prepared nanoparticles

ndicating that the drug was encapsulated in the amorphous state,ince in all nanoparticles the characteristic peaks of the polymeratrix were recorded.

10 20 30 40 50 600

1000

2000

3000

4000

5000

6000

7000

nanoparticles mPEG-PPSu-mPEG 2 MNPs taxol

Inte

nsity

(cou

nts)

2 theta (degree)

nanoparticles mPEG-PPSu-mPEG 1 MNPs taxolTaxol

ig. 11. XRPD patterns of Taxol-MNPs encapsulated in the polymeric matrix.

ric matrix at different tilt angles: (a) 0◦ , (b) 30◦ and (c) 60◦ .

This amorphisation can be attributed to the evolved interactionsbetween polymer and drug. For this reason all nanoparticles werestudied by FTIR spectroscopy in order to reveal if there are anyinteractions between drug and polymer matrix or drug and MNPs.However, as can be seen from Fig. 12, in the recorded spectra thecharacteristic peaks of Taxol were not identified separate from thepolyesters groups. These experimental results respond to the factthat the amount of drug incorporated into the polyester nanopar-ticles is relatively low in comparison with the amount of polyesterused in order to create the nanoparticles, which eventually leadsto the domination of the main polyester absorbance groups overthe characteristic groups of Taxol. This is more characteristic in1650–1750 cm−1 where the carbonyl peaks of Taxol and polyesterare recorded and in the drug loaded nanoparticles it is not possibleto recognize this peak. For this reason it is not possible to identifyany interaction between drug and carrier.

The mean particle size of the polymeric nanoparticles, as wellas their distribution, was measured by dynamic light scattering(Fig. 13). It is very important to know the particle size of theprepared nanoparticles since it is one of the most important param-eters that can affect the drug release, the physical stability and thecellular uptake (Liggins et al., 1997). Especially for the purposeof this study it was desired to develop nanoparticulate systemswith a mean particle size above 100 nm, in order to take advan-tage of the physical changes which occur in the tumor tissue whenthey are topically heated. As can be seen from Fig. 13 the preparednanoparticles show a unimodal size distribution for all polyesters.The mean nanoparticle diameter varied from 160 to 250 nm, whichis desired for the application in targeted delivery of taxol. In the

present study it was found that increasing the molecular weight ofthe copolymers led to an increase of the mean particle size, which isin accordance with a study previously reported using PLGA (Mittal

4000 3500 3000 2500 2000 1500 1000 5000.0

0.2

0.4

0.6

0.8

1.0mPEG-PPSu-Fe3O4nanoparticles

Abs

orba

nce

wavenmber (cm-1)

mPEG-PPSu-Fe3O4-Taxol nanoparticles

Taxol

Fig. 12. FTIR spectra of Taxol and mPEG–PPSu nanoparticles loaded with Taxol.

M. Filippousi et al. / International Journal of

10 10 0 100 0 1000 0-202468

10121416182022

Inte

nsity

(%)

Particle Size Di stribu tion (nm)

mPE G-PPS u1 mPE G-PPS u2 mPE G-PPS u3

Fd

ettwi

3

nnpAtdaaaoet(waft

F

ig. 13. Particle size distribution of prepared nanoparticles using copolymers withifferent molecular weight.

t al., 2007). Comparing the measured particle sizes from DLS withhe micrographs recorded from SEM and TEM it can be concludedhat the addition of taxol drug has no effect on the particle size. Thisas expected since only a small amount of the drug is encapsulated

nside the nanoparticles.

.5. Drug release

Fig. 14 shows the release profile of Taxol from the mPEG–PPSuanoparticles. As previously reported, the release of a drug fromanoparticles of biodegradable polymers is a rather complicatedrocess, where different parameters can affect the release profile.s a result, many different factors may play a contradictory role in

he drug release (Vassiliou et al., 2010). Such factors are polymeregradation rate, molecular weight, crystallinity, glass transitionnd melting temperature, binding affinity between the polymernd the drug, the capability of the polymer to incorporate a highmount of the drug, the size of the nanoparticles, the hydrophilicityf the drug and so on (Gref et al., 1994; Hu et al., 2003; Karavelidist al., 2011; Zhang et al., 2004). In the case of block copolymershe ratios between the different block plays also an important roleNanaki et al., 2011). In the studied nanoparticles a burst releaseas observed at early times, which was followed by a phase of rel-

tively slow drug release. The burst effect is probably due to theraction of drug located on (or close to) the surface of the nanopar-icles (Ge et al., 2002).

0 50 100 150 200 250 300 350 4000

20

40

60

80

100

120

mPEG-PPSu-mPEG 3

mPEG-PPSu-mPEG 2

time (hrs)

% D

rug

Rel

ease

mPEG-PPSu-mPEG 1

ig. 14. Drug release profile pattern of Taxol from the mPEG–PPSu nanoparticles.

Pharmaceutics 448 (2013) 221– 230 229

Taxol release from the nanoparticles may be characterized assustained over many hours and the rate of release (percent releaseat specific times) drops with an increase in the molecular weight(length) of the PPSu block in the copolymer, especially in case ofmPEG–PPSu–mPEG3. This may partly be attributed to the increasedhydrophobic interactions between the nanoparticle core and thedrug and partly to the increased nanoparticle size with increas-ing PPSu length as previously mentioned (Vassiliou et al., 2010).Gref et al. (1994) reported that crystallization of the hydropho-bic drug occurs inside the nanoparticles at relatively high drugloadings, which explained the lower release rates observed withthe nanoparticles having a relatively high drug content. However,in our case, since there was no evidence of drug crystalliza-tion, the differences in the release profiles between the differentmPEG–PPSu compositions should be mainly attributed to the dif-ferent PPSu block length. Furthermore, the initial (burst) release ofTaxol appears to decrease with an increase of the molecular weight(length) of PPSu block in the copolymer. When the PPSu segmentbecomes longer, a higher fraction of Taxol is entrapped inside thehydrophobic core of the nanoparticles and, as a result, less drugcan be released in the initial stage (He et al., 2007; Hu et al., 2007).Based on the release data obtained, it can be concluded that the dis-tribution of the drug content into mPEG–PPSu–mPEG nanoparticlesis influenced by the hydrophobicity of the drug and this distribu-tion affects in turn the release behavior. One part of Taxol wasentrapped into the hydrophobic core (PPSu block) of the nanopar-ticles and only a certain amount was located at (or close to) thehydrophilic shell (mPEG block). This amount, as the release profileclearly demonstrates, is depending on the length of the hydropho-bic macromolecular part of the polyester (PPSu). For this reason therelease rate at the early stages of the release process is much higherfor Taxol when a polymer with higher mass ratio of mPEG is used.

4. Conclusions

In this work, nanoparticles with an Fe3O4 core and amphiphilictriblock copolymers of poly(propylene succinate) (PPSu) andpoly(ethylene glycol) (PEG) shell with different hydropho-bic/hydrophilic ratios have been successfully synthesized andfully characterized. Electron microscopy observations confirm theencapsulation of the nanosystem of MNPs inside the polymericmatrix. The nanoparticle yield drug loading and entrapment effi-ciency are considered to be satisfactory, while the in vitro releaseprofile of the drug confirmed that it was mainly affected by thedifferences of the hydrophobic part of the copolymers. Therefore,our nanosystem, in which magnetic nanoparticles and anticancerdrug Taxol are incorporated in the polymeric matrix can be poten-tially developed to create dual functional nanoparticles capable tobe used as theranostic systems.

Acknowledgement

GVT and MF acknowledge funding from the European ResearchCouncil under the 7th Framework Program (FP7), ERC grant No.246791 – COUNTATOMS.

References

Akbarzadeh, A., Samiei, M., Davaran, S., 2012. Magnetic nanoparticles: preparation,physical properties, and applications in biomedicine. Nanoscale Res. Lett. 7, 144.

Arsalani, N., Fattahi, H., Nazarpoor, M., 2010. Synthesis and characterization of PVP-functionalized superparamagnetic Fe3O4 nanoparticles as an MRI contrast agent.

eXPRESS Polym. Lett. 4, 329–338.

Calmona, M.F., Souzab, A.T., Candido, N.M., Bartolomeu Raposo, M.I., Taboga, S.,Rahal, P., Nery, J.G., 2012. A systematic study of transfection efficiency and cyto-toxicity in HeLa cells using iron oxide nanoparticles prepared with organic andinorganic bases. Colloid Surf. B: Biointerf. 100, 177–184.

2 nal of

C

C

C

C

C

D

D

G

G

G

G

G

H

H

H

H

H

J

K

K

K

K

K

L

L

L

L

L

30 M. Filippousi et al. / International Jour

hen, C., Yu, C.H., Cheng, Y.C., Yu, P.H.F., Cheung, M.K., 2006a. Biodegrad-able nanoparticles of amphiphilic triblock copolymers based on poly(3-hydroxybutyrate) and poly(ethylene glycol) as drug carriers. Biomaterials 27,4804–4814.

hen, C., Yu, C.H., Cheng, Y.C., Yu, P.H.F., Cheung, M.K., 2006b. Preparationand characterization of biodegradable nanoparticles based on amphiphilicpoly(3 hydroxybutyrate)-polyethylene glycol)-poly(3 hydroxybutyrate) tri-block copolymer. Eur. Polym. J. 42, 2211–2220.

herukuria, P., Glazera, E.S., Curleya, S.A., 2010. Targeted hyperthermia using metalnanoparticles. Adv. Drug Deliv. Rev. 62, 339–345.

orot, C., Robert, P., Idee, J.M., Port, M., 2006. Recent advances in iron oxide nanocrys-tal technology for medical imaging. Adv. Drug Deliv. Rev. 58, 1471–1504.

ouvreur, P., Vauthier, C., 2006. Magnetic nanoparticles: prospects in cancer imag-ing and therapy. Pharm. Res. 23, 1417–1450.

ong, X., Mumper, R.J., 2010. Nanomedicinal strategies to treat multidrug-resistanttumors: current progress. Nanomedicine 5, 597–615.

umitrache, F., Morjan, I., Alexandrescu, R., Morjan, R.E., Voicu, I., Sandu, I., Soare,I., Ploscaru, M., Fleaca, C., Ciupina, V., Prodan, G., Rand, B., Brydson, R., Wood-word, A., 2004. Nearly monodispersed carbon coated iron nanoparticles for thecatalytic growth of nanotubesynanofibres. Diamond Rel. Mater. 13, 362–370.

aumet, M., Vargas, A., Gurny, R., Delie, F., 2008. Nanoparticles for drug delivery: theneed for precision in reporting particle size parameters. Eur. J. Pharm. Biopharm.69, 1–9.

e, H., Hu, Y., Jiang, X., Cheng, D., Yuan, Y., Bi, H., Yang, C., 2002. Preparation,characterization and drug release behaviors of drug nimodipine-loaded poly(�-caprolactone)–poly(ethylene oxide)–poly(�-caprolactone) amphiphilic triblockcopolymer micelles. J. Pharm. Sci. 91, 1463–1473.

olla-Schindler, U., Hinrichs, R., Bomati-Miguel, O., Putnis, A., 2006. Determinationof the oxidation state for iron oxide minerals by energing–filtering TEM. Micron37, 473–477.

ref, R., Minamitake, Y., Peracchia, M.T., Trebetskoy, V.S., Torchilin, V.P., Langer,R., 1994. Biodegradable long-circulating polymeric nanospheres. Science 263,1600–1603.

uo, M., Que, C., Wang, C., Liu, X., Yan, H., Liu, K., 2011. Multifunctional super-paramagnetic nanocarriers with folate-mediated and pH-responsive targetingproperties for anticancer drug delivery. Biomaterials 32, 185–194.

awkes, P.W., 1992. In: Frank, J. (Ed.), In the Electron Microscope as a StructureProjector. Plenum Press, New York, USA (Chapter 2).

e, F., Li, S., Vert, M., Zhuo, R., 2003. Enzyme catalyzed polymerization and degra-dation of copolymers prepared from �-caprolactone and poly(ethylene glycol).Polymer 44, 5145–5151.

e, G., Ma, L.L., Pan, J., Venkatraman, S., 2007. ABA and BAB type triblock copolymersof PEG and PLA: a comparative study of drug release properties and “stealth”particle characteristics. Int. J. Pharm. 334, 48–55.

u, Y., Jiang, X., Ding, Y., Zhang, L., Yang, C., Zhang, J., Chen, J., Yang,Y., 2003. Preparation and drug release behaviors of nimodipine-loadedpoly(caprolactone)–poly(ethylene oxide)–polylactide amphiphilic copolymernanoparticles. Biomaterials 24, 2395–2404.

u, Y., Xie, J., Tong, Y.W., Wang, C.H., 2007. Effect of PEG conformation and particlesize on the cellular uptake efficiency of nanoparticles with the HepG2 cells. J.Control. Release 118, 7–17.

anib, S.M., Moses, A.S., MacKay, J.A., 2010. Imaging and drug delivery using thera-nostic nanoparticles. Adv. Drug Deliv. Rev. 62, 1052–1063.

ang, Y.S., Risbud, S., Rabolt, J., Stroeve, P., 1996. Synthesis and characterization ofnanometer-size Fe3O4 and gamma-Fe2O3 particles. Chem. Mater. 8, 2209–2211.

aravelidis, V., Giliopoulos, D., Karavas, E., Bikiaris, D., 2010. Nanoencapsulation ofa water soluble drug in biocompatible polyesters. Effect of polyesters meltingpoint and glass transition temperature on drug release behavior. Eur. J. Pharm.Sci. 41, 636–643.

aravelidis, V., Karavas, E., Giliopoulos, D., Papadimitriou, S., Bikiaris, D., 2011. Eval-uating the effects of crystallinity of new biocompatible polyester nanocarrierson drug release behavior. Int. J. Nanomed. 6, 3021–3032.

ataoka, K., Harada, A., Nagasaki, Y., 2001. Block copolymer micelles for drug deliv-ery: design, characterization and biological significance. Adv. Drug Deliv. Rev.47, 113–131.

won, Oh, J., Myung, Park, J., 2011. Iron oxide-based superparamagnetic polymericnanomaterials: design, preparation, and biomedical application. Prog. Polym.Sci. 36, 168–189.

iggins, R.T., Hunter, W.L., Burt, H.M., 1997. Solid-state characterization of paclitaxel.J. Pharm. Sci. 86, 1458–1463.

ing, Y., Wei, K., Luo, Y., Gao, X., Zhong, S., 2011. Dual docetaxel/superparamagneticiron oxide loaded nanoparticles for both targeting magnetic resonance imagingand cancer therapy. Biomaterials 32, 7139–7150.

iu, K., Zhao, L., Klavins, P., Osterloh, F.E., Hiramatsu, H., 2003. Extrinsic magneto-resistance in magnetite nanoparticles. J. Appl. Phys. 93, 7951–7953.

oos, J., Sourty, E., Lu, K., de With, G., Bavel, S., 2009. Imaging polymer systems

with high-angle annular dark field scanning transmission electron microscopy(HAADF-STEM). Macromolecules 42, 2581–2586.

u, A.H., Salabas, E.L., Schuth, F., 2007. Magnetic nanoparticles: synthesis,protection, functionalization, and application. Angew Chem. Int. Ed. 46,1222–1244.

Pharmaceutics 448 (2013) 221– 230

Ma, L.L., Jie, P., Venkatraman, S.S., 2008. Block copolymer ‘Stealth’ nanoparticlesfor chemotherapy: interactions with blood cells in vitro. Adv. Funct. Mater. 18,716–725.

Marques, R.F.C., Garcia, C., Lecante, P., Ribeiro, S.L.J., Noe, L., Silva, N.J.O., Amaral,V.S., Millan, A., Verelst, M., 2008. Electro-precipitation of Fe3O4 nanoparticlesin ethanol. J. Magn. Magn. Mater. 320, 2311–2315.

McCarthy, J.R., Weissleder, R., 2008. Multifunctional magnetic nanoparticles fortargeted imaging and therapy. Adv. Drug Deliv. Rev. 60, 1241–1251.

Midgley, P.A., Weyland, M., 2003. 3D electron microscopy in the physical sciences:the development of Z-contrast and EFTEM tomography. Ultramicroscopy 96,413–431.

Mittal, G., Sahana, D.K., Bhardwaj, V., Ravi Kumar, M.N.V., 2007. Estradiol loadedPLGA nanoparticles for oral administration: effect of polymer molecular weightand copolymer composition on release behavior in vitro and in vivo. J. Control.Release 119, 77–85.

Mornet, S., Vasseur, S., Grasset, F., Duguet, E., 2004. Magnetic nanoparticle designfor medical diagnosis and therapy. J. Mater. Chem. 14, 2161–2175.

Nanaki, S.G., Pantopoulos, K., Bikiaris, D.N., 2011. Synthesis of biocompatible poly(�-caprolactone)-block-poly(propylene adipate) copolymers appropriate for drugnanoencapsulation in the form of core–shell nanoparticles. Int. J. Nanomed. 6,2981–2995.

Niu, C., Wang, Z., Lu, G., Krupka, T.M., Sun, Y., You, Y., Song, W., Ran, H., Li, P., Zheng, Y.,2013. Doxorubicin loaded superparamagnetic PLGA-iron oxide multifunctionalmicrobubbles for dual-mode US/MR imaging and therapy of metastasis in lymphnodes. Biomaterials 34, 2307–2317.

Papadimitriou, S., Bikiaris, D., 2009. Novel self-assembled core–shell nanoparticlesbased on crystalline amorphous moieties of aliphatic copolyesters for efficientcontrolled drug release. J. Control. Release 138, 177–184.

Pennycook, S.J., Jesson, D.E., 1991. High-resolution Z-contrast imaging of crystals.Ultramicroscopy 37, 14–38.

Pennycook, S.J., 1992b. Z-contrast transmission electron microscopy: direct atomicimaging of materials. Annu. Rev. Mater. Sci. 22, 171–195.

Prashant, C., Dipak, M., Yang, C.T., Chuang, K.H., Jun, D.S., Feng, S.S., 2010.Super-paramagnetic iron oxide-loaded poly(lactic acid)-d-alpha-tocopherolpolyethylene glycol 1000 succinate copolymer nanoparticles as MRI contrastagent. Biomaterials 31, 5588–5597.

Qu, S., Yang, H., Ren, D., Kan, S., Zou, G., Li, D., Li, M., 1999. Magnetite nanoparti-cles prepared by precipitation from partially reduced ferric chloride aqueoussolutions. J. Colloid Interface Sci. 215, 190–192.

Romberg, B., Hennink, W.E., Storm, G., 2008. Sheddable coatings for long-circulatingnanoparticles. Pharm. Res. 25, 55–71.

Rösler, A., Vandermeulen, G.W.M., Klok, H., 2001. Advanced drug delivery devicesvia self-assembly of amphiphilic block copolymers. Adv. Drug Deliv. Rev. 53,95–108.

Shubayev, V.I., Pisanic, I.I., Jin, T.R.S., 2009. Magnetic nanoparticles for theragnostics.Adv. Drug Deliv. Rev. 61, 467–477.

Simeonidis, K., Mourdikoudis, S., Tsiaoussis, I., Angelakeris, M., Dendrinou-Samara,C., Kalogirou, O., 2008. Structural and magnetic features of heterogeneouslynucleated Fe-oxide nanoparticles. J. Magn. Magn. Mater. 320, 1631–1638.

Stolnik, S., Illum, L., Davis, S.S., 1995. Long circulating microparticulate drug carriers.Adv. Drug Deliv. Rev. 16, 195–214.

Sun, C., Lee, J.S.H., Zhang, M., 2008. Magnetic nanoparticles in MR imaging and drugdelivery. Adv. Drug Deliv. Rev. 60, 1252–1265.

Tan, Y.F., Chandrasekharan, P., Maity, D., Yong, C.X., Chuang, K., Zhao, Y., Wang, S.,Ding, J., Feng, S., 2011. Multimodal tumor imaging by iron oxides and quantumdots formulated in poly(lactic acid)-d-alpha-tocopheryl polyethylene glycol1000 succinate nanoparticles. Biomaterials 32, 2969–2978.

Torchilin, V.P., 2002. PEG-based micelles as carriers of contrast agents for differentimaging modalities. Adv. Drug Deliv. Rev. 54, 235–252.

Van Butsele, K., Jerome, R., Jerome, C., 2007. Functional amphiphilic and biodegrad-able copolymers for intravenous vectorisation. Polymer 48, 7431–7443.

Vassiliou, A.A., Papadimitriou, S.A., Bikiaris, D.N., Mattheolabakis, G., Avgoustakis,K., 2010. Facile synthesis of polyester-PEG triblock copolymers and preparationof amiphilic nanoparticles as drug carriers. J. Control. Release 148, 388–395.

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, 284–304.

Wan, J., Tang, G., Qian, Y., 2007. Room temperature synthesis of single-crystal Fe3O4

nanoparticles with superparamagnetic property. Appl. Phys. A 86, 261–264.Wang, J., Liu, W., Tu, Q., Wang, J., Song, N., Zhang, Y., Nie, N., Wang, J., 2011.

Folate-decorated hybrid polymeric nanoparticles for chemically and physi-cally combined paclitaxel loading and targeted delivery. Biomacromolecules 12,228–234.

Zhang, L., Hu, Y., Jiang, X., Yang, C., Lu, W., Yang, Y.H., 2004. Camptothecinderivative-loaded poly(caprolactone-co-lactide)-b-PEG-b-poly(caprolactone-co-lactide) nanoparticles and their biodistribution in mice. J. Control. Release96, 135–148.

Zhang, Z., Tan, S., Feng, S., 2012. Vitamin E TPGS as a molecular biomaterial for drugdelivery. Biomaterials 33, 4889–4906.

Zhao, Q., Cheng, G., Song, C., Zeng, Z., Tao, J., Zhang, L., 2006. Crystallizationbehaviour and biodegradation of poly(3-hydroxybutyrate) and poly(ethyleneglycol) multiblock copolymers. Polym. Degrad. Deliv. 91, 1240–1246.