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Biomaterials 31 (2010) 5588e5597

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Biomaterials

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Superparamagnetic iron oxide e Loaded poly (lactic acid)–D-a-tocopherolpolyethylene glycol 1000 succinate copolymer nanoparticles as MRIcontrast agent

Chandrasekharan Prashant a, Maity Dipak b, Chang-Tong Yang c, Kai-Hsiang Chuang c, Ding Jun b,Si-Shen Feng a,d,e,*

aDepartment of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576bDepartment of Materials Science and Engineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574c Laboratory of Molecular Imaging, Singapore Bioimaging Consortium, Agency for Science, Technology and Research Singapore, 11 Biopolis Way,#02-02 Helios, Singapore 138667dDivision of Bioengineering, Faculty of Engineering, National University of Singapore, Block E3A #04-15, 7 Engineering Drive 1, Singapore 117574eNUS Nanoscience & Nanotechnology Initiative (NUSNNI), National University of Singapore, Blk E3-05-29, 2 Engineering Drive 3, Singapore 117581

a r t i c l e i n f o

Article history:Received 15 February 2010Accepted 26 March 2010Available online 29 April 2010

Keywords:Biodegradable polymersBiocompatibilityCellular and molecular imagingMicroencapsulationNanoprecipitationNanocomposite

* Corresponding author. Department of Chemical aFaculty of Engineering, National University of SingaSingapore 117576. Tel.: þ65 6516 3835; fax: þ65 677

E-mail address: chefss@nus.edu.sg (S.-S. Feng).

0142-9612/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.biomaterials.2010.03.070

a b s t r a c t

We developed a strategy to formulate supraparamagnetic iron oxides (SPIOs) in nanoparticles (NPs) ofbiodegradable copolymer made up of poly(lactic acid) (PLA) and D-a-tocopherol polyethylene glycol 1000succinate (TPGS) for medical imaging by magnetic resonance imaging (MRI) of high contrast and low sideeffects. The IOs-loaded PLAeTPGS NPs (IOs-PNPs) were prepared by the single emulsion method and thenanoprecipitation method. Effects of the process parameters such as the emulsifier concentration, IOsloading in the nanoparticles, and the solvent to non-solvent ratio on the IOs distribution within thepolymeric matrix were investigated and the formulation was then optimized. The transmission electronmicroscopy (TEM) showed direct visual evidence for the well dispersed distribution of the IOs within theNPs. We further investigated the biocompatibility and cellular uptake of the IOs-PNPs in vitrowith MCF-7breast cancer cells and NIH-3T3 mouse fibroblast in close comparison with the commercial IOs imagingagent Resovist�. MRI imaging was further carried out to investigate the biodistribution of the IOsformulated in the IOs-PNPs, especially in the liver to understand the liver clearance process, which wasalso made in close comparison with Resovist�. We found that the PLAeTPGS NPs formulation at theclinically approved dose of 0.8 mg Fe/kg could be cleared within 24 h in comparison with several weeksfor Resovist�. Xenograft tumor model MRI confirmed the advantages of the IOs-PNPs formulation versusResovist� through the enhanced permeation and retention (EPR) effect of the tumor vasculature.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Versatility in the use of supraparamagnetic iron oxide nano-crystals (SPIOs or IOs) has gained tremendous importance in thefield of biomedical application. In cancer research, for example, IOshave been used for molecular imaging, tumor imaging, cancerhyperthermia therapy, and other techniques [1e4]. IOs have beenextensively investigated as a contrast agent for MRI, which areeasily assimilated by the human body [5e7]. IOs are primarily used

nd Biomolecular Engineering,pore, 4 Engineering Drive 4,9 1936.

All rights reserved.

in MRI as liver signal erasers since they create a reduction in signalintensity in the reticuloendothelial tissues, in which they aregreatly concentrated and thus result in greater T2 shortening, toproduce more conspicuous area of liver not containing the reticu-loendothelial tissues. However, long term retention of IOs in liver isa concern for the side effects of IOs imaging agent [8]. Several otherside effects are also associated with administering of the IOs, whichmay create clinical complications [9].

Such problems could be addressed by two strategies. One is bycoating of the IOs core and another is by formulating of the IOs innanoparticles (NPs) of biodegradable polymers. Various coatingtechniques have been suggested to provide IOswith functionality ofdesired surface properties. For instance, thermally sensitive coat-ings provide the IOs with dual functionality for treatment of cancerby hyperthermia and through a release of the entrapped

C. Prashant et al. / Biomaterials 31 (2010) 5588e5597 5589

anti-cancer agent [10]. In another study, IOs with their surfacecoated with PEG and further linked to biotin or neutravidin alongwith a peptide enabled detection of specific cancer type thatproduces a proteolytic enzyme which enabled the peptide blockedbiotin and neutravidin to self assemble and thus influences the T2relaxivity in MRI. Such a coating strategy thus enables detection ofthe tumor as well as specification of the tumor type [11]. Moreover,coating IOs by hydrophilic or amphiphilic biodegradable polymerprovides them with other functionality such as biocompatibilityand long circulation in plasma.

Formulation of the IOs in nanoparticles (NPs) can be realized byusing various FDA-approved biodegradable polymers such as poly(lactic acid) (PLA) andpoly(lactic-co-glycolic acid) (PLGA) and variousnovel biodegradable copolymers such as poly(lactic acid)-poly(ethylene glycol) (PLEA) copolymer and poly(lactic acid)-D-a-tocopherol polyethylene glycol 1000 succinate (PLAeTPGS) copoly-mer [12,13,28]. Encapsulation by using preformed polymer can bedone by several methods such as the micro/nanoemulsion method[14,15], the nanoprecipitation method [16,17] and the micelleformation [18e20]. Emulsion polymerization makes use of mono-mers that polymerize over the encapsulant, which is stabilized byusing a surfactant [21e23], which is of great concern due to thepresence of certain chemical precursors that were not removed andcould have some cytotoxic effect [24]. The solvent displacementtechnique was used in the preparation of nanocapsules [24].The nanocapsules were formed when the non-solvent replaces thesolvent in the presence of a surfactant followed by deposition of thepreformedpolymerat the interface. Theencapsulantwas remained inan oily phase within the polymer layer. This process has beenextended to the formationof nanosphereswithout the surfactant [16]and ithasbeengaining increasing importanceover time forpreparingnanoparticles. Various parameter optimization of this method hasbeen done. For instance, it was suggested that the diffusion of thesolvent into the non-solvent results in the phase transformation ofthe polymer and thus in the formation of the nanoparticles [25]. Itwas also shownhowthenanoparticles formation couldbeaffected bychanging the concentrationof thepolymer in theorganicphaseor theconcentration of the surfactant in the aqueous phase, which mayinfluence the size of the formed nanoparticles [26]. Other research inthenanoprecipitationmethod included the choiceof the solvent [27].It was suggested that the solubility parameter of the solvent and thenon-solvent system strongly influence the particle formation, inwhich the particle size was usually used as the primary criteria foroptimization in the literature.

In the present research, we developed a strategy to formulateIOs in the NPs of PLAeTPGS copolymer, for enhanced MRI of higherimage contrast and less system side effects. The amphiphilic natureand enhanced biocompatibility of this copolymer make it prom-ising for nanoparticle delivery of diagnostic and therapeutic agents[28,29]. We have exploited the different treatment between drugand the IOs in the nanoparticle encapsulation process. A crystallinedrug after encapsulation could become in an amorphous state,resulting in approved solubility [30e32], whereas the IOs do notundergo a phase transition after encapsulation process. Ratherthese nanoparticles get entrapped within the polymeric matrix andretain their crystalline state. Thus diffusion of the solvent into thepolymeric matrix plays a key role during the preparation of thenanoparticle encapsulation.

Two commonly used methods for polymeric nanoparticlepreparation, namely the single emulsion method and the nano-precipitation method, are employed in this research for the nano-particle preparation in close comparison with optimization of thevarious synthesis parameters such as the emulsifier concentrationin the aqueous phase, solvent/non-solvent ratio, loading of IOs/drug and more. The effect of synthesis parameters on the size and

size distribution of the polymeric nanoparticles and the distribu-tion of IOs within the polymeric matrix were studied by laser lightscattering and transmission electron microscopy (TEM). The opti-mally synthesized nanoparticles were further investigated for invitro cytotoxicity and cellular uptake of the IOs-loaded nano-particles in close comparison with the commercial Resovist�. Dueto their desired particles size and their long half-life in the plasma,the IOs-loaded PLAeTPGS nanoparticles should have the propertyto accumulate in regions of leaky and high permeable vasculatureand lymphatic vessels such as those in tumor, which is also referredto as the enhanced permeability and retention (EPR). The nano-particle formulation of IOs can thus be exploited for passively tar-geted delivery of the imaging agent for clinical MRI with bettercontrast and less side effects [33].

2. Materials and methods

2.1. Materials

Lactide (3,6-dimethyl-1,4-dioxane-2,5-dione, C6H8O4) was purchased fromAldrich, which was recrystallized twice from ethyl acetate before use. Vitamin ETPGS (D-a-tocopherol polyethylene glycol 1000 succinate, C33O5H54(CH2CH2O)23)was from Eastman chemical company (USA), which was freeze dried for two daysbefore use. Stannous octoate (Sn(OOCC7H15)2) was purchased from Sigma and wasused as 1% distilled toluene solution. Millipore water was prepared by a Milli-Q PlusSystem (Millipore Corporation, Breford, USA). Oleylamine (OM, �70%), Oleic acid(OA, 90%), Iron(III) acetylacetonate (Fe(acac)3, 97%) and Nitric acid (�69%) werepurchased from SigmaeAldrich. All chemicals including absolute ethanol,dichloromethane (DCM), tetrahydrofuran (THF) and ethyl acetate were HPLC grade.They were used without further purification.

2.2. Synthesis of PLAeTPGS polymer

The PLAeTPGS copolymer of 90:10 PLA:TPGS w/w ratio was synthesized bya process discussed in our earlier publication [28]. In brief, weighted amounts oflactide, TPGS and 0.5 wt% stannous octoate (in distilled toluene) were added in anampoule. The ampoule was evacuated in liquid nitrogen for 45 min. After that theampoule was sealed by butane burner and reacted in silicone oil bath at 145 �C. After12 h, the reaction product was dissolved in DCM and then precipitated in excess coldmethanol to remove unreacted lactide monomers and TPGS. The final product wascollected by filtration and vacuum dried at 45 �C for two days. The product formedwere analyzed and confirmed by FTIR and 1H NMR at 500 Hz (Bruker AMX500). Theweight-average molecular weight and molecular weight distribution were deter-mined by gel permeation chromatography (GPC, Waters GPC analysis system withRI-G1362A refractive index detector).

2.3. Synthesis of IOs

Iron oxide nanoparticles (IOs) were prepared by thermal decomposition methodof Fe(acac)3 as done earlier [34]. In brief, 2 mmol of Fe(acac)3 was dissolved ina 20 ml surfactant mixture of oleic acid (OA) and oleylamine (OM) and magneticallystirred under a flowof argon. The solutionwas dehydrated at 120 �C for 1 h, and thenquickly heated to 300 �C temperature and kept at this temperature for 1 h. The blacksolution was cooled to room temperature by removing the heat source. A total of20ml of ethanol was added into the solution and the precipitated IOs were collectedby centrifugation at 10,000 rpm, followed by washing with ethanol three times. Theas-prepared IOs were dispersed in THF. The iron content and coating was confirmedby using inductively coupled plasma-mass spectrometry (ICP-MS).

2.4. Preparation of IOs-encapsulated polymeric nanoparticles

IOs-loaded PLAeTPGS nanoparticles (IOs-PNPs) were prepared by twomethods,namely the single emulsion solvent extraction/evaporation method and the nano-precipitation method. In the single emulsion method, 100 mg of the polymer alongwith known amount of IOs and 8 ml of DCM were added and the solution wasvortexed till the polymer is fully dissolved in the organic solvent and a clear solutionis obtained. The organic phase was then added into 120 ml of aqueous phase con-taining the emulsifier at a given concentration (TPGS of 15, 5 and 1% w/v) anda probe sonicator was used to disperse the emulsion for 90 s. The solution wasallowed to stir overnight to remove the solvent.

In the nanoprecipitation method, 100 mg of the polymer along with knownamount of IOs and 8 ml of THF were added and vortexed till the polymer was fullydissolved in the organic solvent and a clear solution is obtained. 30 ml of aqueousphase containing a known concentration of the emulsifier (TPGS of 15, 5 and 1%w/v)was well dispersed using a probe sonicator for 30 s. The organic phase was thenadded into the aqueous phase. The solutionwas vigorously stirred till uniformity and

C. Prashant et al. / Biomaterials 31 (2010) 5588e55975590

diluted with large amount of water. The solution was then allowed to be stirredovernight to evaporate the organic solvent. The nanoparticles were obtained aftercentrifugation at 10,000 rpm for 15 min. The duration of centrifugation wasincreased for samples with higher concentration of the emulsifier. Several washeswere done to remove the emulsifier. The nanoparticles were then characterized fordistribution of IOs within the polymer matrix by using TEM (JEM 2010F, JEOL, Japan)and for surface morphology by using FESEM (JEOL, JSM-6700F, Japan) as well as thesize and size distribution by using Zetasizer (Nano ZS, Malvern Instruments Ltd, UK).

2.5. X-ray photoelectron spectroscopy (XPS)

XPS was carried out for qualitative determination of the iron content and thecarbon contenton the surface of the IOs-PNPs. A small amount of thedry nanoparticlepowder was analyzed using XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu,Japan). Data were analyzed with the software provided by the manufacturer.

2.6. ICP-MS analysis

A known amount of IOs-PNPs were put in a glass test tube. 2 ml of concentratednitric acidwas added. The tubewas then heated to 110 �C for 45min and the sampleswere analyzed using ICP-MS (Agilent ICP-MS 7500 Series) after sufficient dilutionwith milli-Q water. The analysis of sample was done in comparisonwith the ICP-MSstandard (Sigma).

2.7. Magnetic property of IOs-PNPs

The superparamagnetic properties of the nanoparticle formulation of IOs wereinvestigated by the vibrating sample magnetometer (VSM, Lakeshore 7300 Series,USA).

2.8. In vitro cytotoxicity

MCF-7 breast cancer cells and NIH-3T3 mice fibroblast cells were seeded in 96-well plates (Costar, IL, USA) at the density of 3 � 105 viable cells/well using Dul-becco’s modified eagle medium (DMEM), containing 5% fetal bovine serum and 1%antibiotics, and incubated for 24 h with MCF-7 or 48 h with NIH-3T3 cells to allowcell attachment. Themediawas removed and the replacedwith freshmedia. To eachwell, 10 ml of the IOs-PNPs suspension or Resovist� at concentrations ranging from2.5 to 10 mmol [Fe]/L was added and incubated for 24 h. Untreated wells were usedas control. Five hours prior the time point, 10 ml of 5 mg/ml MTT (3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) in PBS was addedinto the 96-well plates. The cells were incubated till the designated time point. Afterincubation, 100 ml of a stop mix solution composing of 20% sodium dodecyl sulfate(SDS) in 50% dimethylformamide (DMF) was added into each well. The plate wasincubated for an hour to dissolve the formazan crystals that are formed and theabsorbance in each well was measured by microplate reader (GENios, Tecan,Switzerland) at 550 nm and 630 nm as reference wavelength.

2.9. In vitro cellular uptake of NPs

2.9.1. Quantitative evaluationMCF-7 breast cancer cells and NIH-3T3 mouse fibroblast cells were seeded in 24

well plates (Costar, IL, USA) at a density 1 � 105 viable cells/ml/well. The cells wereallowed to grow for 24 h. After 24 h, the medium in the wells were replaced withfresh medium and 100 ml of the IOs-PNPs suspension or the Resovist� at the variousdesignated [Fe] concentration were added to the wells. Wells with neither thenanoparticles suspension nor the Resovist� added were used as control. Afterincubation for 24 h with MCF-7 or 48 h with NIH-3T3 cells, the culture mediumwasremoved. The plates were washed 4 times with PBS, each time with fresh exchangeof PBS. The cells were then trypsinated and then centrifuged at 2000 rpm for 5 min.To the cell pellet, 2 ml of concentrated nitric acid was added and was heated to120 �C. This dissolved and converted the IOs-PNPs internalized by the cells into itscorresponding ions. The solution was diluted using Milli-Q water, and the sampleswere analyzed by inductively coupled plasma-mass spectrometer (ICP-MS, AgilentICP-MS 7500).

2.9.2. Qualitative analysisTo assess the cellular uptake of the IOs-PNPs qualitatively, theMCF-7 cancer cells

were grown in 175 cm2 culture flask at a cell density of 1 �106 cells/ml. After 24 h,the medium was replaced by fresh medium containing the IOs-PNPs at 2 mmol of[Fe]/L equivalent concentration. After 4 h of incubation, the medium was removed;the cells were washed 4 times with PBS, and fixed with 2.5% glutaraldehyde at 37 �C.After 2 h, the glutaraldehyde solution was removed and the cells were washed withPBS. The cells were then scrapped and pelleted at 3500 rpm for 10 min. The pelletwas post-fixed using 1% Osmium tetroxide at PH 7.4 for 1 h, followed by washingwith PBS two times. The cell pellet was then fixed in 6% gelatine, followed by a seriesof dehydration process using ethanol and acetone, and finally embedded in aralditeresin. The sections were stained using lead citrate prior to viewing. The sampleswere viewed using Transmission Electron Microscope (TEM, (JEOL, JEM-1220)).

2.10. MRI

Transverse relaxivity measurement and in vivoMRI were conducted on a Varian9.4T MRI system (Palo Alto, CA, USA).

2.10.1. Calculation of relaxivityThe transverse relaxation time T2 of the IOs-PNPs were measured in 1% Agarose

phantoms with concentration from 0.4 to 0.0066 mM [Fe], using multiple spin echosequence at TR¼ 10,000ms and TE¼ 5.69 ms. Transverse relaxivity (r2 in mM

�1 s�1)were obtained from linear least-squares determination of the slopes of 1/T2 versusthe concentration of [Fe] plots. The r2* measured by multiple gradient echosequence with same concentrations with those measured by T2. The longitudinalrelaxation time (T1) of the IOs-PNPs were measured by using inversion recoveryspin echo sequence.

2.10.2. In vivo MRI studyIn vivo study was conducted on Wistar rats (male, weight 320e340 g) under 2%

isoflurane anesthesia using 72 mm volume coil. The animal protocol was approvedby the Institutional Animal Care and Use Committee (IACUC), National University ofSingapore (#802/05(A10)09). The polymeric contrast agents were injected ata dosage of 0.8 mg [Fe]/kg body weight through tail vein. T2-weighted images wereacquired every 48 s for 32 min with T2-weighted fast spin echo sequence (TR/TE ¼ 1000/28.24 ms, flip angle ¼ 90� , field of view ¼ 8 cm, thickness ¼ 2 mm,intersection gap ¼ 0.3 mm, matrix ¼ 256 � 192, under fat saturation and externaltrigger. Respiration rate¼ 60/min). The coronal and axial sections of the whole bodyof the rat were imaged at different time periods. The SNR (signal to noise ratio) fromthe liver were measured and plotted against time.

SNR ¼ ROI mean signalBackground standard deviation

% Contrast change ¼ SNR at any time tSNR at time zero before contrast

� 100

Xenograft model was developed using severe combined immune deficiency(SCID)mice (female, 20 g). A 5�106 number ofMCF-7 cancer cells were injected intothe subcutaneous part of themicenear the rightflank. The tumorwas thendevelopedand allowed to grow until a volume of 150e200 mm3. The SCID mice were imagedusing a Bruker 7T Clinscan MRI system. The protocol was approved by the A*STARInstitutional Animal Care andUse Committee (IACUC) (#060188). Contrast agentwasinjected (dosage: 5mgof [Fe]/kg bodyweight) through tail veins of themice under 1%isofluraneanesthesia. T2-weighted imageswere acquiredat various timepoints usingT2-weighted turbo spin echo sequence (TR/TE ¼ 1500/36 ms, resolution ¼ 100 mm,thickness ¼ 1 mm). Signals from region of interest (ROI) of the tumor was taken andnormalized with the signal of the phantom filled with saline.

3. Results and discussion

3.1. Synthesis of PLAeTPGS copolymer

PLAeTPGS (with a ratio 90:10 of PLA:TPGS) was successfullysynthesized by ring opening polymerization. The polymer wascharacterized using FTIR and 1H NMR as shown in Fig. 1. Thecarbonyl band shift from 1730 cm�1 for TPGS to 1755 cm�1 forPLAeTPGS polymer and also the CH stretching at 2945 cm�1 for PLAto at 2880 cm�1 for that for TPGS can be observed from the FTIRspectra (Fig. 1A). The successful synthesis of PLAeTPGS copolymerwas further verified by 1H NMR for the presence of CH proton peakat 5.2 ppm for PLA-TPGS compared with 5.07 ppm for PLA. Thesignals at 5.2 and 1.69 ppm were assigned to the PLA segment andthat at 3.65 ppm was assigned to the TPGS segment (Fig. 1B). Thenumber-averaged molecular weight of the PLAeTPGS copolymerwas determined to be Mn ¼ 17,027 by NMR and Mn ¼ 9856 by GPCrespectively. The weight-averaged molecular weight was found byGPC to be Mw ¼ 13559 with polydispersity of 1.36. Our results arequite close to those given in our earlier work in the literature forPLAeTPGS of 88:12 PLA:TPGS component ratio [28].

3.2. Synthesis of hydrophobic IOs

We have successfully synthesized hydrophobic IOs whichcoated with oleic acid (OA) and oleylamine (OM). Fig. 2A showsa TEM image of the hydrophobic IOs, fromwhich it can be seen that

Fig. 1. (A) Fourier Transformation Infrared Spectroscopy (FTIR) of amphiphilic D-a-tocopherol polyethylene glycol 1000 succinate (TPGS) and PLAeTPGS copolymer, (B) 1H NMRspectrum of PLAeTPGS copolymer.

C. Prashant et al. / Biomaterials 31 (2010) 5588e5597 5591

the IOs are highly monodispersed and non-agglomerated. Theparticle size is found to be 10 nmwhich is in the superparamagneticsize range. The OA and OM coating was confirmed by FTIR as shownin Fig. 2B. The peaks at 2926 and 2854 cm�1 are associated with theCeH stretching vibration, which arises from the surface adsorbedOA and OM coatings respectively. The peaks at 1562 and 1440 cm�1

are due to the COO� stretching vibration and the peak at 1411 cm�1

(which is merged with the 1440 cm�1 peak) is due to the CeNbending vibration, which arises from the surface adsorbed OA andOM coatings, respectively. The broad band between 3600 and3000 cm�1 centered at about 3400 cm�1 is due to the OeHstretching vibration arising from surface adsorbed water. In addi-tion, the strong absorption band at 584 cm�1 is due to the FeeOstretching vibration of Fe3O4 IOs.

3.3. Synthesis of IOs-encapsulated polymeric nanoparticles(IOs-PNPs)

The IOs-PNPs were synthesized in this research by the singleemulsion method and the nanoprecipitation method, respectively.Table 1 summarizes the synthesis parameters and the resultsobtained from the IOs-PNPs prepared by the single emulsionmethod. Fig. 3AeC show the TEM image of the IOs-PNPs prepared

Fig. 2. (A) TEM image of the as-synthesized hydrophobic IOs (scale ¼ 10 nm), (B) FTIR confir

by the emulsion method at emulsifier concentration of 1%, 5% and15% respectively. It can be seen that for the IOs-PNPs prepared bythe single emulsion method, the distribution of the IOs within thepolymer matrix is not significantly affected by the increase inthe emulsifier concentration. Moreover, there seems a trend thatthe IOs are distributed more towards the periphery. This kind of IOsdistribution in the polymeric matrix was seen previously in otherswork as well [35,36]. However the size of the IOs-PNPs was foundreduced with increase of the surfactant concentration at the sameIOs loading. This is understandable sincemore surfactantmoleculescould cover larger surfaces of the nanoparticles of smaller size asdescribed in the literature [27]. There was also considerablereduction in the IOs encapsulation efficiency (EE) of the IOs-PNPswith increase in the emulsifier concentration. This can be due to thestabilization of the hydrophobic IOs by the micelles of the surfac-tant which might be formed and then washed away in the prepa-ration process. It should be noticed that the surfactantconcentration used in the IOs-PNPs preparation process fell in themicelle forming region of TPGS which is between the criticalmicelle concentration (CMC) 0.03e20%.

The characters of the IOs-PNPs prepared by the nanoprecipitationmethod are summarized inTable 2. The TEM images of such IOs-PNPsprepared with the emulsifier concentration of 0%, 1%, 5% and 15% are

mation of the oleic acid and oleylamine coating on the as-synthesized hydrophobic IOs.

Table 1Characters of the IOs-loaded PLAeTPGS nanoparticIes (IOs-PNPs) prepared by the single emulsion method.

Polymer % Emulsifier (w/v) in aqueous phase IO Loading EE%a % Fe per IOs-PNPsb Size PDI

PLAeTPGS 1 2% 36.67 � 3.51 0.815 � 0.078 352.03 � 18.28 0.379 � 0.058PLAeTPGS 5 2% 24.02 � 2.33 0.600 � 0.0.58 307.44 � 16.46 0.236 � 0.044PLAeTPGS 15 2% 16.03 � 3.31 0.464 � 0.096 282.22 � 14.70 0.153 � 0.043

a EE% ¼ Amount of iron oxide present in the IOsePNP obtainedAmount of iron oxide used in the preparation of IOsePNP� 100.

b % Iron content ¼ Weight of iron in X grams of IOsePNP ðin gramsÞX ðin gramsÞ � 100.

Fig. 3. TEM images of IOs-loaded PLAeTPGS nanoparticles (IOs-PNPs) prepared by the single emulsion method at the emulsifier concentration of (A) 1%, (B) 5% and (C) 15% and bythe nanoprecipitation method at the emulsifier concentration of (D) 0%, (E) 1%, (F) 5% and (G) 15% respectively (scale bar ¼ 100 nm).

C. Prashant et al. / Biomaterials 31 (2010) 5588e55975592

Table 2Characters of the IOs-loaded PLAeTPGS nanoparticIes (IOs-PNPs) prepared by the nanoprecipitation method.

Polymer % Emulsifier IO loading EE%a % Iron contentc Sizeb PDI

PLAeTPGS 0 2% 16.58 � 1.90 0.368 � 0.042 356.77 � 07.34 0.321 � 0.063PLAeTPGS 1 2% 54.33 � 3.33 0.987 � 0.061 302.53 � 15.83 0.283 � 0.022PLAeTPGS 5 2% 58.17 � 3.60 1.292 � 0.080 272.80 � 02.53 0.128 � 0.016PLAeTPGS 15 2% 64.17 � 2.32 1.283 � 0.046 249.43 � 19.89 0.093 � 0.008

a EE% ¼ Amount of iron oxide present in the IOsePNP obtainedAmount of iron oxide used in the preparation of IOsePNP� 100.

b Hydrodynamic diameter measurement, n ¼ 3.c % Iron content ¼ Weight of iron in X grams of IOsePNP ðin gramsÞ

X ðin gramsÞ � 100.

C. Prashant et al. / Biomaterials 31 (2010) 5588e5597 5593

shown in Fig. 3DeG, respectively. From these figures, the effect ofemulsifier concentration on the IOs distributionwithin the polymericmatrix can be observed, which shows that with increase of thesurfactant concentration in the aqueous phase, the IOs were distrib-utedmore andmore to the interior of the polymer matrix, leading toalmost homogenous distribution at high emulsifier concentration.Moreover, both of the size of the IOs-PNPs and the polydispersityindex (PDI) were greatly decreased with increase of the emulsifierconcentration at the same IOs loading. The EE increased as well.Fig. 4AeC show the FESEM images of the IOs-PNPs synthesized by thenanoprecipitation method at emulsifier concentration of 1%, 5% and15%, fromwhich the size of the IOs-PNPs was reconfirmed.

The ratio of the solvent to non-solvent and IOs loading levelcould be important factors that may affect the size of the IOs-PNPsand the IOs distribution within the polymeric NPs. In one set of theexperiment, IOs-PNPs were prepared with different loading level of1%, 2% and 3% IOs (w/w based on polymer) were prepared by thenanoprecipitation method at 15% emulsifier concentration. It wasobserved that the distribution of IOs in the polymeric matrix wasnot affected (TEM not shown). However, the size did increasewith the IOs loading in the order of 225.60 � 15.037 nm (PDI:0.073 � 0.055) for 1% IOs loading, 249.43 � 19.89 nm(PDI: 0.093� 0.008) for 2% IOs loading, and 448.6� 20.03 nm (PDI:0.497 � 0.044) for 3% IOs loading respectively. This finding agreeswith that observed from the nanoparticle formulation of hydro-phobic drugs, in which the nanoparticle size was also found toincrease with the drug loading [17].

The ratio of the solvent to non-solvent and IOs loading levelcould jointly affect the size of the IOs-PNPs and the IOs distributionwithin the polymeric NPs. It seems that increase in the formerparameter and/or decease in the later parameter would lead tosmaller particle size. We found that the solvent/non-solvent ratio of1:15, 4:15 and 3:5 yielded nanoparticles of iron content of 1.1%, 1.2%and 0.8% and size of 283 � 5.80, 249 � 19.9 and 203 � 8.93 nmrespectively, and the IOs-PNPs prepared at the 1:15 solvent/non-

Fig. 4. Field emission scanning electron microscopy (FESEM) images of IOs-loaded PLAeTPGconcentration of (left) 1%, (middle) 5% and (right) 15%, respectively.

solvent ratio were polydispersed while IOs distribution in the NPsprepared at the 4:15 solvent/non-solvent ratio looked moreuniform and had a higher iron content, thus was consideredoptimal (the TEM images not shown).

We can thus conclude that the nanoprecipitation preparationmethod seems preferable to prepare the IOs-PNPs of more desiredparticle size and IOs distribution, for which the surfactant concen-tration, the solvent to non-solvent ratio and the IOs loading level arethree important parameters that may determine the particle sizeand size distribution as well as the IOs distribution within thepolymeric NPs. Taking into consideration the size of the IOs-PNPsand the iron content, the IOs-PNPsprepared at the4:15 solvent/non-solvent ratio and 2% IOs loading level was found to be optimal.

3.4. XPS analysis

XPS can qualitatively and quantitatively determine the chemicalcomposition up to a depth of 5e10 nm. In the XPS spectrum, a peakcharacterizes a corresponding chemical bond and the area-under-the curve at that peak gives information about the relative % of thatchemical bond. 5e10 nm is the depth to which the X-ray canpenetrate under the surface and thus the XPS peak obtained is forthe chemical structure of the surface up to a 5e10 nm depth. TheXPS spectra of the IOs-PNPs prepared by the nanoprecipitationmethod at the various emulsifier concentrations are shown inFig. 5. It can be found from this figure that with increase in theemulsifier concentration in the NPs preparation process, the ironcontent in the IOs-PNPs, which is denoted by the peaks at bindingenergy 708 eV (2p3/2) and 722 eV (2p1/2), diminished. This meansthat all loaded IOs have been located within the polymeric matrix.The iron content on the surface becomes almost nil for the IOs-PNPssynthesized at 15% emulsifier concentration. The IOs-PNPsprepared with no emulsifier showed the highest content of iron onthe surface. On the other hand, the area-under-the curve at theCeOeC peak 286.1 eV, which denotes the PEG element of the TPGS

S nanoparticles (IOs-PNPs) prepared by the nanoprecipitation method at the emulsifier

A B

C

290292 288 286 284 282Binding Energy (eV)

290292 288 286 284 282Binding Energy (eV)

O-C=O

O-C=O

C-O-C=O

C-O-C=O

C-C/C-H

C-C/C-H

C-O-C

C-O-C

2P1/22P3/2

No Emulsifier

1% TPGS

5% TPGS

15% TPGS

740 730 720 710 700Binding Energy (eV)

Fig. 5. (A) X-ray photoelectron spectroscopy (XPS) spectrum of iron bond on the surface of the IOs-loaded PLAeTPGS nanoparticles prepared by the nanoprecipitation method atthe concentration of emulsifier of 0% (i.e. no emulsifier), 1%, 5% and 15%, respectively, (B) XPS spectrum of carbon bond on the surface of the IOs-loaded PLAeTPGS nanoparticlesprepared by the nanoprecipitation method without emulsifier, (C) XPS spectrum of carbon bond on the surface of the IOs-loaded PLAeTPGS nanoparticles prepared by thenanoprecipitation method at 15% emulsifier concentration.

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macromolecule, increases from 22.56% for the IOs-PNPs synthe-sized without emulsifier (Fig. 5B) to 33.73% for those prepared with15% TPGS emulsifier concentration (Fig. 5C). This shows that theemulsifier macromolecules do remain on the surface of the poly-meric NPs to stabilize the NPs formulation. Such observation wasnot as significant for the IOs-PNPs prepared with emulsifierconcentration lower than 15%. This can be understandable since thecarbon peak nowcould be contributedmore from the oleic acid andoleylamine coating of the IOs near the surface than from theemulsifier macromolecules.

Fig. 6. Hysteresis curve of IOs and IOs-loaded PLAeTPGS nanoparticles (IOs-PNPs)prepared by the nanoprecipitation method at the emulsifier concentration of 0%, 1%, 5%and 15%, respectively.

3.5. Hysteresis

Fig. 6 shows the hysteretic MeH curves of the IOs-PNPsprepared by the nanoprecipitation method at the various emulsi-fier concentrations. The normalized saturation magnetization (ss)of the hydrophobic IO crystals is 84.5 emu/g while that of the IOs-PNPs were 10.26, 44.46, 70.11, and 79.23 emu/g when prepared atthe 0%, 1%, 5% and 15% emulsifier concentration, respectively. Thess values of the IOs-PNPs are found to be always lower than that ofhydrophobic IO crystals due to the encapsulation within the non-magnetic polymer matrix [37]. Moreover, the ss values of the IOs-PNPs are found to increase with increasing the emulsifierconcentration in the NPs preparation process. This could be due tothe increment of the iron content within the IOs-PNPs withincreasing the emulsifier concentration. Conversely, the ss valuesare found to decrease with the increase of the non-magneticpolymer content over the IOs in the NPs (i.e. decrease in IOsencapsulation in the IOs-PNPs) with decreasing the emulsifierconcentration. The zero coercivity and remenance of the MeHcurves indicate that all of the encapsulated IOs are super-paramagnetic in nature [37].

3.6. Cytotoxicity

Cytotoxicity of the IOs-PNPs was measured by the MTT assay24 h after incubation with MCF-7 breast cancer cells and NIH-3T3cells in close comparisonwith the clinical IOs agent Resovist� at theequivalent 2.5, 5, 10 mmol [Fe]/L, respectively. The results areshown in Fig. 7, fromwhich it can be seen that both formulation ofsupraparamagnetic IOs, the IOs-PNPs and Resovist�, have littletoxicity on MCF-7 cancer cells at least at the designated equivalentIO concentration ranged (Fig. 7A). However, Resovist� has much

Fig. 7. Cytotoxicity of the IOs-PNPs was measured by the MTT assay 24 h after incubation with (A) MCF-7 breast cancer cells and (B) NIH-3T3 cells in close comparison with theclinical IOs agent Resovist� at the equivalent 2.5, 5, 10 mmol [Fe]/L, respectively (n ¼ 8).

Fig. 8. Shows the TEM image of MCF-7 cancer cell line incubated with IOs-PNPs; A.image shows that the IOs-PNPs are confined to a vesicle in the cytoplasm of the cell(Scale ¼ 200 nm); B. image shows a magnified view of the vesicle marked with themiddle arrow in image A. The vesicle carries the IOs-PNPs into the cell. The walls of theparticle seem to be disturbed, indicating the process of metabolism by the cell(scale ¼ 100 nm).

C. Prashant et al. / Biomaterials 31 (2010) 5588e5597 5595

higher cytotoxicity for the NIH-3T3 mouse fibroblast cells than theIOs-PNPs, which demonstrates the advantages of the latter over theformer (Fig. 7B). It was found that the IOs-PNPswere approximately15, 17 and 10 fold less toxic than Resovist� at the same ironconcentration of 10, 5 and 2.5 mmol Fe/L, respectively.

3.7. Cellular uptake of IOs-PNPs

3.7.1. Quantitative investigationCellular uptake of the IOs formulated in the PLAeTPGS NPs (the

IOs-PNPs) was measured by ICP-MS, which was conducted in closecomparison with the IOs formulated in the Resovist�. For MCF-7breast cancer cells, the cellular uptake of IOs formulated in the IOs-PNPs were found 1.2, 16.4, 20.4 fold higher than that formulated inResovist� at the equivalent iron concentrations of 2.5, 1.25 and0.75 mmol Fe/L, respectively. For the NIH-3T3 mouse fibroblastcells, the cellular uptake of IOs formulated in the PLAeTPGS NPswas found 16.23 and 6.5 fold higher than the Resovist� formulationat the equivalent iron concentrations of 2.5 and 0.75 mmol Fe/L,respectively although Resovists� showed higher cellular uptakethan the IOs-PNPs formulation at the equivalent iron concentrationof 1.25 mmol Fe/L due to the irregular cellular behaviour.

3.7.2. Qualitative investigationQualitatively investigation of cellular uptake of the IOs-PNPs in

MCF-7 breast cancer cells was visualized by TEM. Fig. 8 shows TEMimages of the MCF-7 cells 4 h after incubation with the IOs-PNPs atequivalent 2 mmol Fe/L NP concentration. It can be seen fromFig. 8A that the IOs-PNPs are confined within a meso vesicle in thecytoplasm of the cell (scale bar ¼ 200 nm). Fig. 8B shows the mesovesicle in Fig. 8A at higher magnification (scale ¼ 100 nm). In Fig. 8A and B, the arrow marks the meso vesicle. It seems that the IOs-PNPs were internalized by a mechanism called endocytosis(penocytosis), in which the NP sacrifice its interfacial energy tobend a small piece of the lipid bilayer membrane to form a smallmeso vesicle in a scale of 200e300 nm that will bring the IOs-PNPsinto the cells. Moreover, the surface of the nanoparticle seem tohave been disturbed, indicating the process of metabolism by thecell.

3.8. In vivo MRI

3.8.1. Relaxivity studiesThe IOs-PNPs had a relaxivity of r2 ¼ 164.09, r2* ¼ 280.73 and

r1 ¼ 0.3372 (L mmol Fe�1 s�1). This indicated that the IOs-PNPs canbe used for T2-weightedMRI imaging. All in vivo experiments werecarried out under T2-weighted sequence.

3.8.2. In vivo liver clearanceAsmentioned before, themostly used formulation of IOs used in

current MRI is Resovist�, which has raised concerns for its lowimagining effect and toxicity due to their accumulation in thevisceral organs. As a preliminary study, we investigated the

Fig. 9. The signal trend in the liver obtained after i.v. injection of the IOs-PNPssuspension at an equivalent iron dose of 0.8 mg Fe/kg of body weight of Wistar rats,which was imaged by a 9.4 T MRI Varian scanner. The insert shows the MRI images (theaxial section) of the liver taken at various designated time. The arrow indicates theliver of the rat.

C. Prashant et al. / Biomaterials 31 (2010) 5588e55975596

biodistribution of the IOs-PNPs in the various organs 1 h afterintravenous (i.v.) injection of the IOs-PNPs suspension at theequivalent iron dose of 0.8 mg Fe/kg through the tail of rats. Sucha dose was determined from a report in the literature, in which theaverage concentration of IOs used on human MRI was around0.54 mg Fe/Kg [38]. Fig. 9 shows the signal trend in the liverobtained before 0.6, 24, 48, and 120 h after i.v. injection of the IOs-PNPs suspension at an equivalent iron dose of 0.8 mg Fe/kg of bodyweight of Wistar rats, which was imaged by a 9.4 T MRI Varianscanner. The insert shows the MRI images (the axial section) of theliver taken at various designated time. The arrow indicates the liverof the rat. It can be seen from the Fig. 9 inset that the IOs-PNPs areaccumulated to large extent in the liver. Kidney and other organshad a reduced uptake (images not shown). However, the signal

Fig. 10. Xenograft tumor model of the signal trend in the liver obtained after i.v.injection of the IOs-PNPs suspension at an equivalent iron dose of 5 mg Fe/kg of bodyweight of SCIF mice in which a tumor has been induced, which was imaged by a 7 TMRI Bruker scanner. The insets show the MRI images (the axial section) of the tumorswhich were taken off from the animal before and 5 h after the injection of the contrast,respectively. The arrow indicates the tumor.

intensity of the liver is lost within 24 h as shown by the % contrastchange curve versus time in Fig. 9. This indicates the advantages ofthe IOs-PNPs formulation over Resovist� for earlier clearance of theIOs from the animal that can thus greatly reduce the IOs toxicity tothe organs.

3.8.3. In vivo tumor MRIWe further investigated the potential of the IOs-PNPs for MRI

imaging on a xenograft model, which is shown in Fig. 10. The signaltrend shown in this figure was from the tumor obtained before and0.33, 2, 5 and 12 h after i.v. injection of the IOs-PNPs suspension atan equivalent iron dose of 5 mg Fe/kg of body weight of SCID mice,inwhich a tumor has been induced. TheMRI was conducted by a 7 TMRI Bruker scanner. The insets show the MRI images (the axialsection) of the tumors which were taken off from the animal beforeand 5 h after the injection of the contrast, respectively. The arrowindicates the tumor. It can be seen from the inserts that the IOs-PNPs have been internalized in the tumor developed in SCID miceas shown by the heterogeneity developed in the tumor after 5 hcompared to the tumor scanned before administration of the IOs-PNPs at time zero. This indicates that the IOs-PNPs can be used topassively target tumor by EPR.

4. Conclusion

The IOs-loaded PLAeTPGS NPs (IOs-PNPs) prepared by thesingle emulsion method and the nanoprecipitation method atthe various process parameters, especially the IOs loading level inthe NPs and the solvent/non-solvent ratio used in the process, werecharacterized for their physicochemical and supraparamagneticproperties for an optional formulation using nanoprecipitationmethod. We found that the IOs-PNPs prepared by the nano-precipitation method at the 4:15 solvent/non-solvent ratio and 2%IOs loading level would have desired properties for MRI. Thecellular uptake of the IOs-PNPs by theMCF-7 breast cancer cells andNIH-3T3 mice fibroblast cells was measured by ICP-MS and visu-alized by TEM. The in vitro cytotoxicity of the IOs-PNPs was alsoinvestigated by MTT assay in close comparison with Resovist�. InvivoMRI investigation found that the IOs-PNPs have high relaxivityand suitable for T2-weighted MRI imaging. Moreover, the IOs-PNPsformulation has advantages over Resovist� for earlier clearance ofthe IOs from the liver that can thus greatly reduce the IOs toxicity tothe organs. The xenograft tumor model MRI indicated the greatpotential of the IOs-PNPs formulation for clinical tumor MRI as wellas for other medical imaging.

Acknowledgement

The authors are thankful for the financial support from the NUSFOE Grant R-279-000-226-112 and NanoCore Grant R-279-000-284-646 as well as A Singapore A*STAR SBIC (Singapore BioimagingConsortium, Agency of Science Technology and Research).

Appendix

Figures with essential color discrimination. Figs. 5, 6 and 7 inthis article are difficult to interpret in black andwhite. The full colorimages can be found in the on-line version, at doi:10.1016/j.biomaterials.2010.03.070.

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