Multifunctional polyglycerol-grafted Fe3O4@SiO2 nanoparticles for targeting ovarin cancer cells

Multifunctional polyglycerol-grafted Fe3O4@SiO2 nanoparticles for targeting ovarin cancer cells
Multifunctional polyglycerol-grafted Fe3O4@SiO2 nanoparticles for targeting ovarin cancer cells

Multifunctional polyglycerol-grafted Fe 3O 4@SiO 2nanoparticles for targeting ovarian cancer cells

Liang Wang a ,Koon Gee Neoh a ,*,En-Tang Kang a ,Borys Shuter b

a Department of Chemical and Biomolecular Engineering,National University of Singapore,Kent Ridge,Singapore 119260,Singapore b

Department of Diagnostic Radiology,National University of Singapore,Kent Ridge,Singapore 119260,Singapore

a r t i c l e i n f o

Article history:

Received 2November 2010Accepted 18November 2010

Available online 13December 2010Keywords:Polyglycerol

Iron oxide nanoparticles Silica shell Folic acid ?uorescence MRI

a b s t r a c t

Ligand-mediated magnetic resonance (MR)contrast agents would be highly desirable for cancer diag-nosis.In the present study,nanoparticles of Fe 3O 4core with ?uorescent SiO 2shell were synthesized and grafted with hyperbranched polyglycerol (HPG-grafted Fe 3O 4@SiO 2nanoparticles).These nanoparticles have a hydrodynamic diameter of 47.0?4.0nm,and are very stable in aqueous solution as well as in cell culture medium.Numerous surface hydroxyl groups of these nanoparticles were conjugated with folic acid by a thiol ‘click ’reaction.The successful covalent attachment of folic acid on the nanoparticles was con ?rmed by FTIR and XPS analyses.Both MR imaging and ?uorescence microscopy show signi ?cant preferential uptake of the folic acid-conjugated polyglycerol-grafted Fe 3O 4@SiO 2(FA-HPG-grafted Fe 3O 4@SiO 2)nanoparticles by human ovarian carcinoma cells (SKOV-3)as compared to macrophages and ?broblasts.Such nanoparticles can potentially be used to provide real-time imaging in ovarian cancer resection.

ó2010Elsevier Ltd.All rights reserved.

1.Introduction

Magnetic resonance imaging (MRI)has gained wide acceptance in diagnosis and medical research due to its noninvasive nature and high spatial resolution.MRI images result from the different relaxation times among water protons in the local environment in tissues.Although suf ?cient contrast for distinguishing regions of interest could be achieved without contrast agents,exogenous contrast agents substantially enhance diagnostic utility [1e 5].Contrast agents based on chelated paramagnetic ions,such as gadolinium,and superparamagnetic iron oxide nanoparticles are currently used in clinical practice for bowel contrast,liver and spleen imaging,lymph node imaging,bone marrow imaging,perfusion imaging,and MR angiography [6e 10].In such applica-tions,the accumulation of the contrast agent within the tissue of interest relies on its speci ?c uptake by the targeted cells and its ability to evade endocytosis by macrophages of the mononuclear phagocytic system [11,12].

To increase the local concentration of superparamagnetic iron oxide nanoparticles in tumor tissue,speci ?c targeting molecules,including monoclonal antibodies [13e 15],proteins [16],and peptides [17],have been conjugated on these nanoparticles.However,these targeting agents have their drawbacks.In the case

of antibodies,their relatively large dimension and inherent immunogenicity inhibit the diffusion of the conjugated nano-particles through biological barriers [18,19].Furthermore,the expression of tumor markers,for example,target receptors,on tumor cell surfaces is not a static situation but changes over time depending on the type and amount of antigens present.To avoid such variability,the delivery of magnetic nanoparticles via the nutrient pathways of tumor cells is attractive since this mechanism is directly linked to proliferation.Thus,in principle,it will give rise to increased uptake of the imaging agent and therefore give a greater signal for the more aggressive tumor cells [20,21].Folic acid (FA),a low molecular weight vitamin,whose receptor is over-expressed on the surface of many human tumor cells [22,23],is a key precursor in DNA base synthesis and is thus required for tumor cell proliferation [24].FA has been studied extensively as a targeting agent for cancer cells and applied in systems,such as doxorubicin-loaded liposome,gadolinium complex,and magnetic nanoparticles [12,15,25e 28].When FA is linked via its g -carboxyl group to a drug or imaging agent,its folate receptor binding af ?nity is not signi ?cantly affected,and receptor-mediated endocytosis remains unhindered [29].

In our previous work,we have demonstrated that Fe 3O 4nano-particles grafted with hyperbranched polyglycerol (HPG)can disperse very well in aqueous medium such as water,phosphate buffered saline (PBS),and cell culture medium,and can ef ?ciently evade macrophage uptake [30].In the present study,iron oxide magnetic nanoparticles were ?rst coated with a ?uorescent silica

*Corresponding author.Tel.:t6568742176;fax:t6567791936.E-mail address:chenkg@https://www.360docs.net/doc/b29794385.html,.sg (K.G.

Neoh).Contents lists available at ScienceDirect

Biomaterials

journal h omepage:

https://www.360docs.net/doc/b29794385.html,/locate/biomaterials

0142-9612/$e see front matter ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.biomaterials.2010.11.042

Biomaterials 32(2011)2166e 2173

shell which would allow their visualization by optical means,and then grafted with HPG to attain water dispersibility and macro-phage-evading properties.Subsequently,folic acid was coupled onto the surface of HPG-grafted Fe3O4@SiO2by an effective‘thiol’click reaction to achieve cancer targeting capability.The resulting FA-HPG-grafted Fe3O4@SiO2was characterized by Fourier trans-form infrared spectroscopy(FTIR)and X-ray photoelectron spec-troscopy(XPS)to con?rm the binding of FA on the nanoparticles. Human ovarian carcinoma cells(SKOV-3),3T3?broblasts,and macrophages were chosen as our model cancer cell line,normal cell line,and phagocytic cell line,respectively.In vitro nanoparticle uptake experiments were performed to discern the effects of folic acid conjugation on the nanoparticle internalization by the tar-geted cells.The cells after incubation with the nanoparticles were visualized by confocal microscopy and imaged by MRI.

2.Materials and methods

2.1.Materials

Glycidol purchased from Sigma e Aldrich was dried using4?molecular sieve and distilled before use.1,4-dioxane was purchased from Sigma e Aldrich and dried using sodium before it was used as a solvent for the ring e opening reaction of glycidol. Human ovarian carcinoma cells(SKOV-3),mouse macrophage cells(RAW264.7)and 3T3?broblasts were purchased from ATCC.Folate-free RPMI-1640medium,folate-free Dulbecco’s modi?ed Eagle’s medium(DMEM),fetal bovine serum,L-glutamine, penicillin and3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide were purchased from Sigma e Aldrich.All other solvents and chemicals were purchased from either Fisher Scienti?c or Sigma e Aldrich and used as received.

2.2.Synthesis of HPG-grafted Fe3O4@SiO2

2.2.1.Preparation of magnetic nanoparticles

The magnetic nanoparticles(MNPs)were prepared according to a previously reported method[31].Brie?y,5.4g of iron chloride(FeCl3$6H2O,20m M ol)and18.5g of sodium oleate(60m M ol)were dissolved in a solvent mixture comprising40mL ethanol,30mL distilled water and70mL hexane.The resulting solution was heated to70 C and stirred at that temperature for4h.The upper organic layer was then washed three times with30mL distilled water.Hexane was then evaporated off, resulting in an iron e oleate complex in a waxy solid form.14g(15.6m M ol)of the iron e oleate complex and2.0g of oleic acid(7.0m M ol)were dissolved in20g of 1-octadecene at room temperature.The reaction mixture was heated to320 C at a constant heating rate of3.3 C minà1,and then kept at that temperature for1h. The resulting solution containing the nanocrystals was then cooled to room temperature,and200mL of ethanol was added to the solution to precipitate the nanocrystals.The nanocrystals were separated by centrifugation and then dried under reduced pressure and stored at0e4 C.

2.2.2.Synthesis of Fe3O4@SiO2nanoparticles

Fluorescein isothiocyanate(FITC)was?rst covalently linked to3-aminopropyl triethoxysilane(APTES)by dissolving10mg of FITC in48mg of APTES(1:4molar ratio). Cyclohexane was then added to prepare a10vol%FITC/APTES in cyclohexane solution. This FITC/APTES/cyclohexane solution was stirred for24h in the dark prior to use.

Four milliliters of Igepal CO-520were added to80mL of cyclohexane in a250mL ?ask and stirred at room temperature for5min.2mL of a cyclohexane dispersion of Fe3O4nanocrystals(5mg/mL)were then added to the Igepal CO-520and cyclo-hexane mixture and stirred for an additional5min.750m L of the FITC/APTES/ cyclohexane solution were added dropwise to the nanocrystal dispersion,followed by the dropwise addition of0.65mL of aqueous ammonium hydroxide solution(30% by volume).Since the hydrolysis rate of APTES is?ve times slower than that of tetraethyl orthosilicate(TEOS)[32],the reaction was stirred for24h prior to the TEOS addition to ensure that the APTES bound to the FITC was hydrolyzed and would be incorporated in the SiO2shell.0.30mL of TEOS were added dropwise to the solution and the mixture was stirred for48h.0.1mL of APTES were then added to this system to produce the surface amino groups.The formed Fe3O4@SiO2nano-particles were then puri?ed by extraction:30mL of methanol were added to induce phase separation between cyclohexane-and methanol-rich phases and the meth-anol-rich phase containing the nanocrystals was collected.The solvent was then partially evaporated from the nanoparticle dispersion in a rotary evaporator.Once the dispersion appeared turbid it was removed from the rotary evaporator and centrifuged for15min at8000rpm(8228g).The supernatant was discarded.The nanoparticles were redispersed in10mL ethanol and then centrifuged at8000rpm for15min.The supernatant was again discarded and this washing procedure was repeated?ve times.The Fe3O4@SiO2nanoparticles were stored as a concentrated dispersion in ethanol before further characterization.2.2.3.Synthesis of HPG-grafted Fe3O4@SiO2

60mg of Fe3O4@SiO2nanoparticles were collected by centrifugation from their ethanol dispersion and redispersed in glycidol(5mL).This dispersion was stirred at room temperature in the dark for24h.The resulting Fe3O4@SiO2nanoparticles with hydroxyl surface groups were collected by centrifugation and washed three times with ethanol and then dissolved in1,4-dioxane(10mL)to form a transparent brownish solution.2.0mg of aluminum isopropoxide were added into the solution under nitrogen protection.The mixture solution was heated to80 C and purged with a?ow of nitrogen for2h to remove the resulting isopropanol.The1,4-dioxane solvent was removed under reduced pressure at40 C to produce the nanoparticles with surface initiating active sites.These nanoparticles were redispersed in dry1,4-dioxane(40mL)and heated to80 C.A solution of glycidol(1.0mL)in1,4-dioxane (20mL)was slowly added to this suspension over7h.The system was then main-tained at80 C for24h under nitrogen protection.The resulting suspension was dripped into diethyl ether(200mL)to obtain a black precipitate.The collected precipitate was dispersed in water(20mL)and centrifuged at6000rpm to further remove any agglomerates,and then precipitated in tetrahydrofuran(THF)(30mL)to remove the free HPG polymer.The HPG-grafted nanoparticles were then collected by centrifugation at6000rpm for8min and dried under vacuum to obtain the?nal HPG-grafted Fe3O4@SiO2nanoparticles.

2.3.Synthesis of folic acid modi?ed HPG-grafted Fe3O4@SiO2nanoparticles

2.3.1.Preparation of folate-SH

Folate N-hydroxysuccinimidyl ester(FA-NHS)was prepared according to a procedure reported in the literature[33].In brief,1.0g of folic acid(2.2m M ol)was dissolved in40mL of DMSO and0.5mL of triethylamine.After addition of0.52g of N-hydroxysuccinimide(NHS)(4.5m M ol),and0.50g of dicyclohexylcarbodiimide (DCC)(2.4m M ol),the mixture was stirred in the dark for18h.The mixture was ?ltered to remove the precipitated side-product,dicyclohexylurea,and the resulting clear yellow?ltrate was poured into a large amount of ethyl ether.The crude product (FA-NHS)was collected by?ltration,and then dried at30 C under vacuum for48h. FA-NHS was coupled to2-mercaptoethylamine as follows:FA-NHS was dissolved in 20mL of dry DMSO.2-aminoethanethiol hydrochloride(0.8m M ol,58mg)was added to this solution followed by0.1mL triethylamine.This system was then stirred at room temperature for24h.The resulting yellow solution was precipitated by adding a large amount of ethyl ether.The crude product(folate-SH)was washed with20mL of water three times and dried at30 C under vacuum for48h.

2.3.2.Synthesis of FA-HPG-grafted Fe3O4@SiO2

N-glycinylmaleimide was synthesized from maleic anhydride and glycine by a two-step reaction according to the literature[34].20mg of1,10-carbon-yldiimidazole(CDI)(0.12m M ol)and5mL of tetrahydrofuran were loaded into a dry round-bottom?ask in an ice bath.16mg of N-glycinylmaleimide(0.10m M ol)were added and the clear solution was stirred at room temperature for10h under a nitrogen atmosphere.60mg of HPG-grafted Fe3O4@SiO2were then dispersed into this system.This black suspension was maintained at50 C for2h.The resulting suspension was dripped into diethyl ether(50mL)to obtain a black precipitate.The N-glycinylmaleimide modi?ed HPG-grafted Fe3O4@SiO2nanoparticles were then collected by centrifugation at6000rpm for8min and redispersed in10mL of DMSO. 10mg of folate-SH(0.02m M ol)and20m L of triethylamine were added into the DMSO suspension of the N-glycinylmaleimide modi?ed HPG-grafted Fe3O4@SiO2nano-particles and stirred for24h at room temperature.The FA-HPG-grafted Fe3O4@SiO2 nanoparticles were collected by centrifugation at6000rpm,and washed three times with DMSO followed by three times with diethyl ether,and then dried under vacuum.

2.4.Cytotoxicity assay

The cytotoxicity of the HPG-grafted Fe3O4@SiO2and FA-HPG-grafted Fe3O4@-SiO2nanoparticles was evaluated by determining the viability of SKOV-3,3T3?broblast and mouse macrophage cells(RAW264.7)after incubation in medium containing the nanoparticles at concentrations from0.05to0.2mg/mL.The nano-particles were sterilized with75%ethanol and recovered after drying under vacuum before use.Control experiments were carried out using the complete growth culture media without nanoparticles.Cell viability testing was carried out via the reduction of the MTT reagent(3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide).The MTT assay was performed in a96-well plate following the standard procedure with minor modi?cations.3T3?broblasts were cultured in DMEM sup-plemented with10%fetal bovine serum,1m M L-glutamine,100IU/mL penicillin. These cells were seeded at a density of104cells per well and incubated at37 C for 24h before the medium was replaced with one containing the nanoparticles.The cells were incubated at37 C for another24h in the medium containing the nanoparticles.The culture medium in each well was then removed and90m L of medium and10m L MTT solution(5mg/mL in PBS)were then added to each well. After4h of incubation at37 C,the medium was removed and the formazan crystals were dissolved with100m L of DMSO for15min.The optical absorbance was then measured at560nm on a microplate reader(Tecan GENios).The results were expressed as percentages relative to the optical absorbance obtained in the control experiments.The differences in the results obtained with the nanoparticles and the

L.Wang et al./Biomaterials32(2011)2166e21732167

controls were analyzed statistically using the two-sample t -test.The differences observed between samples were considered signi ?cant for P <0.05.For the SKOV-3and macrophage cells,the MTT procedure was similar as described above for the 3T3?broblasts,except that the culture medium used for SKOV-3and macrophage cells was RPMI-1640medium,supplemented with 10%fetal bovine serum,2m M L -glutamine,100IU/mL penicillin.2.5.Cell uptake experiments

To study the intracellular uptake of HPG-grafted Fe 3O 4@SiO 2and FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles,SKOV-3cells and macrophages were maintained at 37 C and 5%CO 2in folate-free RPMI-1640media supplemented with 10%fetal bovine serum (FBS)and 1%penicillin/streptomycin.3T3?broblasts were cultured in folate-free DMEM media supplemented with 10%FBS and 1%penicillin/strepto-mycin.These cells were seeded at a density of 105cells per well in 24-well culture plates for 24h before the medium was replaced with that containing FA-HPG-grafted Fe 3O 4@SiO 2or HPG-grafted Fe 3O 4@SiO 2nanoparticles at 0.2mg/mL.After incubation at 37 C for 2h,the cells were washed three times with PBS (pH ?7.4)to remove the loosely attached nanoparticles and free nanoparticles in the medium,and then detached with trypsin-EDTA solution.After counting with a hemocytom-eter,the cells were collected by centrifugation,and the cell pellets were dissolved in 37%HCl at 60 C for 4h.The iron concentration was determined using Thermal Jarrell Ash Duo Iris inductively coupled plasma-mass spectrometer (ICP-MS).At each condition,four runs were made and the mean ?SD was

reported.

Fig.1.Synthetic route of folic acid-conjugated HPG-grafted Fe 3O 4@SiO 2?uorescent nanoparticles:(I)overview of the synthetic strategy and (II)chemical synthesis.

L.Wang et al./Biomaterials 32(2011)2166e 2173

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For confocal imaging experiments,SKOV-3cells,3T3?broblasts,and macro-phages were seeded on cover slips for24h.The medium was then aspirated and replaced with a medium containing either the FA-HPG-grafted Fe3O4@SiO2or HPG-grafted Fe3O4@SiO2nanoparticles at a concentration of0.2mg/mL.The cells were cultured for another2h,and then washed with PBS extensively to completely remove loosely attached and free particles in the medium.The cells were then?xed in4%formaldehyde in PBS for15min.Following?xation,the cells were observed under a confocal laser scanning microscope(CLSM)with a FITC?lter(Zeiss LSM510 Meta,Germany).

2.6.Characterization

FTIR spectra were obtained in a transmission mode on a Bio-Rad FTIR spectro-photometer(Model FTS135)under nitrogen atmosphere.Analysis of the chemical composition of oleic acid-stabilized,HPG-grafted Fe3O4@SiO2and FA-HPG-grafted Fe3O4@SiO2nanoparticles was carried out via X-ray photoelectron spectroscopy (XPS)on an AXIS HSi spectrometer(Kratos Analytical Ltd.)using a monochromatized A1K a X-ray source(1486.6eV photons)at a constant dwell time of100ms and a pass energy of40eV.The anode voltage was15kV,and the anode current was10mA.The pressure in the analysis chamber was maintained at6.7?10à6Pa or lower during each measurement.The nanoparticles were mounted on standard sample studs by means of double-sided adhesive tape.The core-level signals were obtained at a photoelectron takeoff angle of90 (with respect to the sample surface).To compensate for surface charging effect,all core-level spectra were referenced to the C 1s hydrocarbon peak at284.6eV.In spectral deconvolution,the line width(full-width at half-maximum)of the Gaussian peaks was maintained constant for all components in a particular spectrum.The peak ratios for various elements were corrected using experimentally determined instrumental sensitivity factors.

Thermogravimetric analysis(TGA)was performed on a TGA2050thermogra-vimetric analyzer(TA Instruments).Samples weighing between5and15mg were heated from30to700 C at a rate of10 C/min in air.Transmission electron microscopy(TEM)images were recorded on a JEOL2010transmission electron microscope at an accelerating voltage of200kV.The TEM specimens were made by placing a drop of the nanoparticle suspension on a carbon-coated copper grid. Measurement of magnetization was carried out with a vibrating sample magne-tometer(VSM)(Model1600,DMS).The hydrodynamic size of the HPG-grafted Fe3O4@SiO2and FA-HPG-grafted Fe3O4@SiO2nanoparticles in water was determined by dynamic light scattering(DLS)using a90Plus particle size analyzer from Broo-khaven Instruments.

MRI experiments were performed at25 C in a clinical magnetic resonance(MR) scanner(Siemens Magnetom;3.0T).HPG-grafted Fe3O4@SiO2nanoparticles were suspended in tubes of water(20mL)at iron concentration of0.04375,0.0875,0.175, 0.35,0.7,1.4m M.The tubes were placed into the MR scanner and a number of MR sequences were run.T2relaxation times were determined from a multi-echo spin-echo sequence(32echoes;repetition time(TR):1600ms;echo times(TE): 15e480ms).T1relaxation times were determined from a saturation recovery experiment using spin-echo images obtained with a number of TRs(7TRs;TR: 100e6400ms;TE:15ms).The relaxation rates for each sample were computed using in-house software(MATLAB V7.1)by non-linear least squares?tting of appropriate exponential functions.Relaxivities were computed using linear regression analysis(Microsoft EXCEL)to correlate relaxation rates and molar Fe concentrations.

For the MRI experiments of cells cultured in medium with HPG-grafted Fe3O4@SiO2and FA-HPG-grafted Fe3O4@SiO2nanoparticles,the cells culture and labeling were as described in Section2.5.The cells were then washed,counted,and resuspended at a cell density of2.0?105,1.0?105,5.0?104,2.5?104,1.25?104, and6.25?103cells/mL in1%agarose solution.The cells were transferred to tubes with a?nal volume of10mL per tube.These tubes of cell-embedded agarose gel were imaged with the clinical MR scanner.Two-dimensional MR imaging using a gradient echo pulse sequence was performed with the following imaging parameters:TE?5e40ms,TR?1600ms,slice thickness?4.0mm,?eld of view?64?64mm, image matrix?256?256.

3.Results and discussion

The synthetic route for HPG-grafted Fe3O4@SiO2nanoparticles is shown in Fig.1.The oleic acid-stabilized MNPs were?rst coated with TEOS,APTES and FITC to form a?uorescent silica shell.The amino groups on the shell were utilized to initiate the surface polymeri-zation of glycidol.Thus,?uorescent core-shell magnetic nano-particles with a hydrophilic HPG surface were obtained.The hydroxyl end groups of HPG were further conjugated with N-glyci-nylmaleimide group by traditional CDI-mediated esteri?cation.Folic acid with a thiol group was then conjugated on the HPG-grafted Fe3O4@SiO2nanoparticles by a‘thiol’click reaction.Since thiols have recently been shown to react under benign conditions with a vast range of chemical species resulting in high yields,their use has been extended to a large number of applications in the chemical,bio-logical,materials and engineering?elds.In particular,these tradi-tional base-catalyzed thiol-Michael reactions between thiol groups and maleimide groups have received increasing attention in the?eld of biomaterials due to their facile and mild reaction conditions [35,36].Compared with other methods,the click chemistry-medi-ated modi?cation of HPG-grafted Fe3O4@SiO2nanoparticles not only improves the reaction ef?ciency but also provides a convenient way to calculate the amount of FA on the nanoparticle surface amount via quantifying the sulfur content by XPS.

3.1.Size and stability of the HPG-grafted Fe3O4@SiO2and FA-HPG-grafted Fe3O4@SiO2nanoparticles

The typical TEM micrographs of oleic acid-stabilized MNPs and HPG-grafted Fe3O4@SiO2nanoparticles are shown in Fig.2.The oleic acid-stabilized nanoparticles are essentially monodisperse and the size deduced from the TEM image(Fig.2a)is around14nm which is close to the mean hydrodynamic diameter of17nm.The TEM image of HPG-grafted Fe3O4@SiO2nanoparticles(Fig.2b) shows w30nm spherical particles with a distinct core-shell structure.The thickness of the silica shell is about8nm,and the Fe3O4core is around13nm.ICP results show that the Fe3O4content in the HPG-grafted Fe3O4@SiO2nanoparticles is7.6%by weight.The hydrodynamic diameter of the HPG-grafted Fe3O4@SiO2nano-particles was46nm.The hydrodynamic diameter is larger than the size calculated from the TEM image since the polymer shell is not visible in the latter.After FA conjugation,the mean hydrodynamic diameter of FA-HPG-grafted Fe3O4@SiO2nanoparticles increased slightly to54nm.From the TEM and DLS results,it can be concluded that the HPG-grafted Fe3O4@SiO2and FA-HPG-grafted Fe3O4@SiO2nanoparticles can disperse well in water and exist mostly as single

particles.

Fig.2.TEM images of(a)oleic acid-stabilized MNPs and(b)HPG-grafted Fe3O4@SiO2nanoparticles.

L.Wang et al./Biomaterials32(2011)2166e21732169

The stability of HPG-grafted Fe 3O 4@SiO 2and FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles in water was assessed by DLS.The hydrodynamic diameters of these two types of nanoparticles were measured by DLS after 0,7,14,and 30days in water.From Fig.3,it can be seen that there was no signi ?cant change in the diameter over the 30day period which con ?rms the high level of stability of these nanoparticles in water.Furthermore,these nanoparticles can be stored at 4 C for months,and the dispersibility and re-dispersibility remain very good after the storage period.3.2.XPS and FTIR characterization of HPG-grafted Fe 3O 4@SiO 2nanoparticles

The success of the covalent conjugation of FA on HPG-grafted Fe 3O 4@SiO 2nanoparticles can be ascertained by comparing the XPS spectra after various stages of surface modi ?cation.Fig.4shows the XPS wide-scan spectra of oleic acid-stabilized MNPs,HPG-grafted Fe 3O 4@SiO 2and FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles.In the wide-scan spectrum of oleic acid-stabilized MNPs (Fig.4a),the

predominant components are C 1s (285eV),O 1s (530eV),and Fe (710eV).The N 1s peak component is not discernible in this spec-trum.For HPG-grafted Fe 3O 4@SiO 2nanoparticles,the appearance of the N 1s signal at a binding energy of 400eV,the Si 2p signal at 100eV,and the Si 2s signal at 150eV is consistent with the presence of APTES coating (Fig.4b).After HPG grafting,the O 1s/C 1s intensity ratio increased signi ?cantly,indicating that surface poly-merization has resulted in increased O content on the nanoparticle surface,consistent with the chemical structure of HPG.In the case of FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles (Fig.4c),the appearance of the S 2p (168eV)signal indicates that FA has been successfully immobilized onto the HPG-grafted Fe 3O 4@SiO 2nanoparticles.Analysis of the S 2p and O 1s core-level spectra indicates that the mole ratio of FA to the hydroxyl groups of HPG is approximately 1:40.

The nanoparticles were further characterized by FTIR to con ?rm the chemical binding of FA to the surface of the nanoparticles.FTIR spectra of HPG-grafted Fe 3O 4@SiO 2nanoparticles,FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles and FA are shown in Fig.5.The presence of HPG on the nanoparticle surface was con ?rmed by the absor-bance peaks for -OH stretch at 3400cm -1and for C e O e C ether stretch at 1080cm -1.The spectra of HPG-grafted Fe 3O 4@SiO 2nanoparticles and FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles also display strong bands at around 2912cm -1corresponding to the CH 2stretching vibrations.The FTIR spectrum of the FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles display peaks at 1610and 1410cm -1corresponding to the aromatic ring stretch of the pteridine ring and p -amino benzoic acid moieties of FA.Signi ?cant peak broadening at 1610cm -1is also attributable to the amide and ester linkage formed between folic acid and the hydroxyl groups of HPG.

3.3.Magnetic properties

It is important to characterize the magnetic properties of the HPG-grafted Fe 3O 4@SiO 2nanoparticles before their use as a contrast agent for MRI.Fig.6shows that the VSM curves of both oleic acid-stabilized and HPG-grafted Fe 3O 4@SiO 2nanoparticles rapidly approach a saturation magnetization of 89and 80emu/g Fe res-pectively,compared to the bulk value of 90emu/g Fe [39].The size of the nanoparticles and the absence of hysteresis in their magnetic pro ?les suggest that these nanoparticles are

superparamagnetic.

Fig.3.Stability of HPG-grafted Fe 3O 4@SiO 2and FA-HPG-grafted Fe 3O 4@SiO 2nano-particles in water as assessed by

DLS.

Fig. 4.XPS wide-scan spectra of (a)oleic acid-stabilized MNPs,(b)HPG-grafted Fe 3O 4@SiO 2and (c)FA-HPG-grafted Fe 3O 4@SiO 2

nanoparticles.Fig.5.FTIR spectra of (a)HPG-grafted Fe 3O 4@SiO 2nanoparticles,(b)FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles and (c)folic acid.

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Fig.7shows the inverse relaxation times,1/T 1and 1/T 2,as a function of the iron molar concentration [Fe],for the HPG-grafted Fe 3O 4@SiO 2nanoparticles in water.The inverse relaxation times were found to be linearly correlated with the iron concentration,according to the following equations [40]:

1=T 1?1=T 1e?Fe ?0Ttr 1?Fe (1)1=T 2?1=T 2e?Fe ?0Ttr 2?Fe

(2)

where r 1and r 2are the longitudinal and transverse relaxivities,respectively.The intercepts,1/T 1([Fe]?0)and 1/T 2([Fe]?0),are the proton inverse relaxation times in aqueous solutions without nanoparticles.The r 1value per millimole Fe of the HPG-grafted Fe 3O 4@SiO 2was found to be 2.41?0.03m M -1s à1which is much smaller than r 1of a commercial carboxydextran-coated USPIO (SHU 555C from Bayer Schering Pharma AG,7.3m M -1s à1)[41].On the other hand,the r 2value per millimole Fe of the HPG-grafted Fe 3O 4@SiO 2is 58.6?1.8m M -1s à1,which is close to the r 2value of

the carboxydextran-coated USPIO (SHU 555C from Bayer Schering Pharma AG,57m M -1s à1)[41].It is well known that the relaxivity ratio,r 2/r 1,is an important parameter to estimate the ef ?ciency of T 2-contrast agents.In this work,r 2/r 1was calculated to be 24.3,which is much larger than that of the carboxydextran-coated USPIO nanoparticle (7.8)[41].

3.4.Cytotoxicity and cellular uptake

For the ultimate use of the modi ?ed iron oxide nanoparticles as targeted contrast agent for MRI,it is critical that these nano-particles after coating with silica and HPG retain their low toxicity properties.SKOV-3,RAW macrophages and 3T3?broblasts were incubated with HPG-grafted Fe 3O 4@SiO 2nanoparticles at concen-trations of 0.05,0.10and 0.20mg/mL for 24h.Fig.8shows that for all three types of cells there is no correlation between the nano-particle concentration and the cell viabilities,and in all cases,

the

Fig.7.Relaxation rates (1/T 1and 1/T 2,s à1

)in a 3T magnetic ?eld at 25 C

as a function of iron concentration (mM)of HPG-grafted Fe 3O 4@SiO 2nanoparticles in

water.

Fig.8.Viability of SKOV-3,macrophage,and 3T3?broblast cells incubated with HPG-grafted Fe 3O 4@SiO 2nanoparticles at different concentrations for 24h.Control exper-iments were carried out without

nanoparticles.

Fig.6.Field dependent magnetization at 25 C for (a)oleic acid-stabilized MNPs and (b)HPG-grafted Fe 3O 4@SiO 2

nanoparticles.

Fig.9.Uptake of HPG-grafted Fe 3O 4@SiO 2and FA-HPG-grafted Fe 3O 4@SiO 2nano-particles by SKOV-3,macrophage and 3T3?broblast cells after an incubation time of 2h.

L.Wang et al./Biomaterials 32(2011)2166e 21732171

viabilities were above 95%and not signi ?cantly different from that in the control experiment (P >0.05).Similar results were obtained with the FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles.Thus,it can be concluded that both FA-HPG-grafted Fe 3O 4@SiO 2and HPG-grafted Fe 3O 4@SiO 2nanoparticles have very low or no toxicity towards these three types of cells.

To evaluate the targeting speci ?city of the FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles for tumor cells,the uptake of these nanoparticles by SKOV-3cells was compared to the uptake by macrophages and 3T3?broblasts.The folate receptor is generally over-expressed in non-mucinous human ovarian carcinomas.SKOV-3is known to be a folate receptor over-expressed cell line [23,37,38]and was chosen as the cancer cell model for our study.The macrophages and 3T3?broblasts act as the phagocytic and normal cell model,respectively.It can be observed from Fig.9that the uptake of the HPG-grafted Fe 3O 4@SiO 2nanoparticles by these three cell lines were all less than 2pg/cell,indicating that the HPG coating successfully reduces phagocytosis by macrophage as well as SKOV-3and 3T3?broblast cells.In the case of the FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles,these three cell lines show signi ?cantly different extents of uptake.The SKOV-3cells have the highest iron uptake of 22pg/cell while the lowest is 3pg/cell for 3T3?broblasts.The functionalization of the nanoparticles with FA results in an order of magnitude increase in their uptake by the SKOV-3cells,and the uptake is about four times that by the macrophages.These results indicate that for the FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles,the HPG coating inhibits phagocytosis by the macrophages while FA enables the nanoparticles to rapidly bind to the folate receptors expressed on the surface of SKOV-3cells and subsequently be internalized by receptor-mediated endocy-tosis.On the other hand,nanoparticles without FA enter the cancer cells by the non-speci ?c binding/penetration process.Fig.10shows the MR images of various concentrations of SKOV-3and macrophage cells suspended in a low-melting agarose after culturing in medium containing 0.2mg/mL FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles.Differentiation between the SKOV-3and macrophage cells can be detected from the images at a cell density at 5.0?104cells/mL (Fig.10d).When the cell density is increased to 1.0?105cells/mL,the difference in image contrast is very clear (Fig.10e).Since each image slice was 4.0mm in thickness,it is estimated that the slice shown in Fig.10e contained approximately 1.6?105cells.On the other hand,with HPG-grafted Fe 3O 4@SiO 2nanoparticles,even when the cell density is increased to 2.0?105cells/mL,there is only a slight difference between the images of SKOV-3and macrophage cells (Fig.10g).

To ascertain that the FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles were indeed internalized by the cells rather than being bound to cellular membranes,SKOV-3cells,macrophages,and 3T3?bro-blasts incubated with FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles were examined by confocal ?uorescence microscopy.Fig.11shows images from the middle section of these three types of cells.Fluorescence can be observed from the images of SKOV-3cells (Fig.11a)and macrophages (Fig.11b)but the former is much stronger.The dark nuclei of the SKOV-3and macrophage cells (magni ?ed in the inset of Fig.11b)con ?rm that the sections shown are in the middle of the cells and the nanoparticles were indeed internalized and accumulated uniformly within the cytoplasm.But for the 3T3?broblasts,no obvious ?uorescence can be observed (Fig.11c)indicating a lack of signi ?cant uptake via the folate receptor-mediated pathway.The very strong ?uorescence observed from the SKOV-3cells enables them to be differentiated from the macrophages.These results con ?rm that the uptake of FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles is more favorable for SKOV-3than

macrophages.

Fig.10.MR images of phantoms containing SKOV-3and macrophage cells at TE ?40ms after incubation with FA-HPG-grafted Fe 3O 4@SiO 2at a cell density of (a)6.25?103,(b)1.25?104,(c)2.5?104,(d)5.0?104,(e)1.0?105and (f)2.0?105cells/mL.(g)shows the corresponding phantoms containing cells (at 2.0?105cells/mL)after incubation with HPG-grafted Fe 3O 4@SiO 2

nanoparticles.

Fig.11.Confocal laser scanning microscopy images of (a)SKOV-3,(b)macrophages,and (c)3T3?broblasts after culturing in medium containing 0.2mg/mL FA-HPG-grafted Fe 3O 4@SiO 2nanoparticles for 2h.Scale bar:100m m.

L.Wang et al./Biomaterials 32(2011)2166e 2173

2172

4.Conclusion

A multifunctional nano-platform comprising folic acid-conju-gated?uorescent HPG-grafted Fe3O4@SiO2nanoparticles has been created for targeting ovarian cancer cells.The HPG coating confers macrophage-evading properties while folic acid provides the folate receptor targeting capability.The uptake of FA-HPG-grafted Fe3O4@SiO2nanoparticles by SKOV-3cells is about four and seven times that by macrophages and3T3?broblasts,respectively.These nanoparticles possess a high T2relaxivity and r2/r1ratio of58.6m M-1sà1and24.3,respectively,indicating their suitability as an MRI T2-contrast agent.Concomitantly,by virtue of the?uorescent group,detection of the targeted cells by optical means is also possible.MTT assays with SKOV-3,macrophages and3T3?bro-blasts indicate that these nanoparticles are not cytotoxic.With these favorable properties,the FA-HPG-grafted Fe3O4@SiO2nano-particles have good potential for targeted diagnostic imaging with MRI and real-time intraoperative visualization of tumor margins.

Acknowledgment

This work was?nancially supported by the National Medical Research Council of Singapore Grant NMRC/EDG/028/2008. Appendix

Figures with essential color discrimination.Figs.1and11in this article are dif?cult to interpret in black and white.The full color images can be found in the online version,at doi:10.1016/j. biomaterials.2010.11.042.

References

[1]Sun C,Sze R,Zhang MQ.Folic acid-PEG conjugated superparamagnetic

nanoparticles for targeted cellular uptake and detection by MRI.J Biomed Mater Res A2006;78A:550e7.

[2]Artemov D.Molecular magnetic resonance imaging with targeted contrast

agents.J Cell Biochem2003;90:518e24.

[3]Bulte JWM,Kraitchman DL.Iron oxide MR contrast agents for molecular and

cellular imaging.Nmr Biomed2004;17:484e99.

[4]Sunderland CJ,Steiert M,Talmadge JE,Derfus AM,Barry SE.Targeted nano-

particles for detecting and treating cancer.Drug Dev Res2006;67:70e93. [5]Luciani N,Wilhelm C,Gazeau F.The role of cell-released microvesicles in the

intercellular transfer of magnetic nanoparticles in the monocyte/macrophage system.Biomaterials2010;31:7061e9.

[6]Frank H,Weissleder R,Brady TJ.Enhancement of MR-angiography with iron-

oxide-Preliminary studies in whole-blood phantom and in animals.

Am J Roentgenol1994;162:209e13.

[7]Weinstein JS,Varallyay CG,Dosa E,Gahramanov S,Hamilton B,Rooney WD,

et al.Superparamagnetic iron oxide nanoparticles:diagnostic magnetic resonance imaging and potential therapeutic applications in neurooncology and central nervous system in?ammatory pathologies,a review.J Cereb Blood Flow Metab2010;30:15e35.

[8]Choi H,Choi SR,Zhou R,Kung HF,Chen IW.Iron oxide nanoparticles as

magnetic resonance contrast agent for tumor imaging via folate receptor-targeted delivery.Acad Radiol2004;11:996e1004.

[9]Lin SP,Brown JJ.MR contrast agents:Physical and pharmacologic basics.

J Magn Reson Imaging2007;25:884e99.

[10]Wang YXJ,Hussain SM,Krestin GP.Superparamagnetic iron oxide contrast

agents:physicochemical characteristics and applications in MR imaging.Eur Radiol2001;11:2319e31.

[11]Veiseh O,Sun C,Gunn J,Kohler N,Gabikian P,Lee D,et al.Optical and MRI

multifunctional nanoprobe for targeting gliomas.Nano Lett2005;5:1003e8.

[12]Landmark KJ,DiMaggio S,Ward J,Kelly CV,Vogt S,Hong S,et al.Synthesis,

characterization,and in vitro testing of superparamagnetic iron oxide nano-particles targeted using folic acid-conjugated dendrimers.ACS Nano 2008;2:773e83.

[13]Okuhata Y.Delivery of diagnostic agents for magnetic resonance imaging.Adv

Drug Deliv Rev1999;37:121e37.[14]Baio G,Fabbi M,Salvi S,de Totero D,Truini M,Ferrini S,et al.Two-step in vivo

tumor targeting by biotin-conjugated antibodies and superparamagnetic nanoparticles assessed by magnetic resonance imaging at1.5T.Mol Imaging Biol2010;12:305e15.

[15]Scheinberg DA,Villa CH,Escorcia FE,McDevitt MR.Conscripts of the in?nite

armada:systemic cancer therapy using nanomaterials.Nat Rev Clin Oncol 2010;7:266e76.

[16]Kresse M,Wagner S,Pfefferer D,Lawaczeck R,Elste V,Semmler W.Targeting

of ultrasmall superparamagnetic iron oxide(USPIO)particles to tumor cells in vivo by using transferrin receptor pathways.Magn Reson Med 1998;40:236e42.

[17]Wunderbaldinger P,Josephson L,Weissleder R.Tat peptide directs enhanced

clearance and hepatic permeability of magnetic nanoparticles.Bioconjug Chem2002;13:264e8.

[18]McNeil SE.Nanotechnology for the biologist.J Leukoc Biol2005;78:585e94.

[19]Jain RK.Transport of molecules in the tumor interstitium-A review.Cancer

Res1987;47:3039e51.

[20]Chen DY,Jiang MJ,Li NJ,Gu HW,Xu QF,Ge JF,et al.Modi?cation of magnetic

silica/iron oxide nanocomposites with?uorescent polymethacrylic acid for cancer targeting and drug delivery.J Mater Chem2010;20:6422e9.

[21]Arote RB,Hwang SK,Lim HT,Kim TH,Jere D,Jiang HL,et al.The therapeutic

ef?ciency of FP-PEA/TAM67gene complexes via folate receptor-mediated endocytosis in a xenograft mice model.Biomaterials2010;31:2435e45. [22]Weitman SD,Lark RH,Coney LR,Fort DW,Frasca V,Zurawski VR,et al.

Distribution of the folate receptor Gp38in normal and malignant-cell lines and tissues.Cancer Res1992;52:3396e401.

[23]Ross JF,Chaudhuri PK,Ratnam M.Differential regulation of folate receptor

isoforms in normal and malignant-tissues in-vivo and in established cell-lines-Physiological and clinical implications.Cancer-Am Cancer Soc1994;73: 2432e43.

[24]James SJ,Miller BJ,Mcgarrity LJ,Morris SM.The effect of folic-acid and/or

methionine de?ciency on deoxyribonucleotide pools and cell-cycle distribu-tion in mitogen-stimulated rat lymphocytes.Cell Prolif1994;27:395e406. [25]Pradhan P,Giri J,Rieken F,Koch C,Mykhaylyk O,Doblinger M,et al.Targeted

temperature sensitive magnetic liposomes for thermo-chemotherapy.

J Control Release2010;142:108e21.

[26]Liu YT,Li K,Pan J,Liu B,Feng SS.Folic acid conjugated nanoparticles of mixed

lipid monolayer shell and biodegradable polymer core for targeted delivery of Docetaxel.Biomaterials2010;31:330e8.

[27]Sudimack J,Lee RJ.Targeted drug delivery via the folate receptor.Adv Drug

Deliv Rev2000;41:147e62.

[28]Park EK,Lee SB,Lee YM.Preparation and characterization of methoxy poly

(ethylene glycol)/poly(epsilon-caprolactone)amphiphilic block copolymeric nanospheres for tumor-speci?c folate-mediated targeting of anticancer drugs.

Biomaterials2005;26:1053e61.

[29]Reddy JA,Low PS.Folate-mediated targeting of therapeutic and imaging

agents to cancers.Crit Rev Ther Drug1998;15:587e627.

[30]Wang L,Neoh KG,Kang ET,Shuter B,Wang SC.Superparamagnetic hyper-

branched polyglycerol-grafted Fe3O4nanoparticles as a novel magnetic reso-nance imaging contrast agent:an in vitro assessment.Adv Funct Mater 2009;19:2615e22.

[31]Park J,An KJ,Hwang YS,Park JG,Noh HJ,Kim JY,et al.Ultra-large-scale syntheses

of monodisperse nanocrystals.Nat Mater2004;3:891e5.

[32]Wang L,Tan WH.Multicolor FRET silica nanoparticles by single wavelength

excitation.Nano Lett2006;6:84e8.

[33]Guo WJ,Lee RJ.Receptor-targeted gene delivery via folate-conjugated poly-

ethylenimine.AAPS PharmSci1999;1:19.

[34]Zeng YX,Bei FL,Wang C,Yang XJ,Lu LD,Wang X.Synthesis and quantum

chemical calculation of N-(4-sulfophenyl)maleimide and its polymer.Acta Chim Sinica2006;64:1079e84.

[35]Hoyle CE,Lowe AB,Bowman CN.Thiol-click chemistry:a multifaceted toolbox

for small molecule and polymer synthesis.Chem Soc Rev2010;39:1355e87.

[36]Hong V,Kislukhin AA,Finn MG.Thiol-selective?uorogenic probes for labeling

and release.J Am Chem Soc2009;131:9986e94.

[37]Corona G,Giannini F,Fabris M,Toffoli G,Boiocchi M.Role of folate receptor

and reduced folate carrier in the transport of5-methyltetrahydrofolic acid in human ovarian carcinoma cells.Int J Cancer1998;75:125e33.

[38]Miotti S,Bagnoli M,Ottone F,Tomassetti A,Colnaghi MI,Canevari S.Simul-

taneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines.J Cell Biochem1997;65:479e91.

[39]Yu SS,Wan JQ,Yu XG,Chen KZ.Preparation and characterization of hydro-

phobic magnetite microspheres by a simple solvothermal method.J Phys Chem Solids2010;71:412e5.

[40]Gillis P,Koenig SH.Transverse relaxation of solvent protons induced by

magnetized spheres-Application to ferritin,erythrocytes,and magnetite.

Magn Reson Med1987;5:323e45.

[41]Rohrer M,Bauer H,Mintorovitch J,Requardt M,Weinmann https://www.360docs.net/doc/b29794385.html,parison of

magnetic properties of MRI contrast media solutions at different magnetic ?eld strengths.Invest Radiol2005;40:715e24.

L.Wang et al./Biomaterials32(2011)2166e21732173

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