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July 18, 2012

C 2012 American Chemical Society

Gadolinium-Encapsulating Iron OxideNanoprobe as Activatable NMR/MRIContrast AgentSantimukul Santra,† Samuel D. Jativa,† Charalambos Kaittanis,‡ Guillaume Normand,‡ Jan Grimm,‡ and

J. Manuel Perez†,*

†Nanoscience Technology Center and Chemistry Department, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826, United States,and ‡Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, United States

Magnetic resonance imaging (MRI)has become a powerful techniquein the clinical diagnosis of disease

and in animal imaging.1�4 MRI is capable ofobtaining tomographic images of livingsubjects with high spatial resolution as aresult of perturbation of tissuewater protonsin the presence of an external magneticfield.5�8 MR contrast agents9�11 typicallyenhance contrast for more accurate diag-nosis and most recently to allow for target-ing imaging when a targeting ligand (e.g.,antibody, peptide) is conjugated to the MRcontrast agent.12,13 Among these agents,superparamagnetic nanoparticles14�16 andparamagnetic metal chelates8 are the mostcommonly used contrast agents.17�21 Super-paramagnetic nanoparticles are typicallycomposed of an iron oxide nanoparticle(IONP) surrounded by a polymeric coatingto facilitate increased stability in aqueousmedia.22 They work by shortening the tra-verse relaxation time (T2 and T2*) of sur-rounding water protons, resulting in adecrease of the MR signal (negative con-trast, dark signal) on a T2-weighted MRIsequence.23�28 On the other hand, para-magnetic gadolinium chelates create anincrease in signal intensity on T1-weightedimages (positive contrast, bright signal) byshortening the longitudinal relaxation time(T1) of surrounding water protons.10,17,29�38

The development of an activatable MRimaging agent that reports on a biologicalprocess associated with diseases wouldgreatly advance medical imaging of diseaseat a molecular level.39�41 Activatable T1 orT2 agents, those that result in modulation ofeither the T1 or T2 relaxation time upontarget binding, enzymatic activity, or biolo-gical process associatedwith disease, wouldbe attractive MR imaging agents, resultingin high sensitivity and high signal-to-noise

ratios with low background.20,42�48 Activa-table Gd-based T1 agents have been pre-viously described8,49,50 and include thosedesigned to be biologically activated by anenzyme such as β-galactosidase51,52 andβ-glucoronidase42,44 aswell as thoseactivatedby release of a drug.53,54 However, the lackof sufficient sensitivity and specificity andthe hydrophobic nature of some of thedesigned probes are major problems thathamper their implementation for clinical

* Address correspondence tojmperez@ucf.edu.

Received for review May 30, 2012and accepted July 10, 2012.

Published online10.1021/nn302393e

ABSTRACT Herein we report a

novel gadolinium-encapsulating iron

oxide nanoparticle-based activatable

NMR/MRI nanoprobe. In our design,

Gd-DTPA is encapsulated within the

poly(acrylic acid) (PAA) polymer coat-

ing of a superparamagnetic iron oxide

nanoparticle (IO-PAA), yielding a com-

posite magnetic nanoprobe (IO-PAA-

Gd-DTPA) with quenched longitudinal spin�lattice magnetic relaxation (T1). Upon release of

the Gd-DTPA complex from the nanoprobe's polymeric coating in acidic media, an increase in

the T1 relaxation rate (1/T1) of the composite magnetic nanoprobe was observed, indicating a

dequenching of the nanoprobe with a corresponding increase in the T1-weighted MRI signal.

When a folate-conjugated nanoprobe was incubated in HeLa cells, a cancer cell line

overexpressing folate receptors, an increase in the 1/T1 signal was observed. This result

suggests that, upon receptor-mediated internalization, the composite magnetic nanoprobe

degraded within the cell's lysosome acidic (pH 5.0) environment, resulting in an intracellular

release of Gd-DTPA complex with subsequent T1 activation. In addition, when an anticancer

drug (Taxol) was coencapsulated with the Gd-DTPA within the folate receptor targeting

composite magnetic nanoprobe, the T1 activation of the probe coincided with the rate of drug

release and corresponding cytotoxic effect in cell culture studies. Taken together, these results

suggest that our activatable T1 nanoagent could be of great importance for the detection of

acidic tumors and assessment of drug targeting and release by MRI.

KEYWORDS: activatable MRI imaging . magnetic relaxation . iron oxidenanoprobe . Gd-DTPA complex . theranostic application

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use. Activatable T2 IONP-based agents are less com-mon as it is often difficult to “quench” the strongsuperparamagnetic nature or magnetic moment ofthese nanoparticles.26�29,55 Magnetic relaxationswitches have been developed based on IONPs thatcluster in the presence of a target or enzymatic activityleading to detectable changes in the T2 relaxationtimes.56�59 However, the use of these T2 activatableagents has been difficult to implement in vivo, and ithas so far been limited to their use as nanosensors inmolecular diagnostic applications.57,60

An activatable T1 agent, one that can induce a fasterT1 relaxation, would result in an increase in the T1-weighted MR signal intensity upon target recognitionfor better diagnosis. Such an activatable agent couldbe beneficial in cancer diagnosis if it were designed tobecome activated upon tumor targeting, resulting in abrighter signal. Herein, we report the design, synthesis,and characterization of a new dual-mode MR contrastagent that becomes activated in an acidic environ-ment, resulting in an increase in the T1-weighted signal(brighter contrast). The designed MR agent is com-posed of superparamagnetic iron oxide nanoparticlesthat encapsulate Gd-DTPA chelates within the hydro-phobic pockets of the nanoparticle's poly(acrylic acid)(PAA) coating (IO-PAA-Gd-DTPA). We hypothesizedthat the strong magnetic field induced by the largemagnetic moment of the superparamagnetic ironoxide core will affect the relaxation process of themuch weaker paramagnetic Gd-DTPA, resulting in

quenching of its T1 signal (Scheme 1). The superpara-magnetic iron oxide core is composed of thousands ofiron (Fe2þ and Fe3þ) ions magnetically ordered withinthe nanocrystal in such a way that they collectivelycreate a net magnetic moment larger than that of anysingle paramagnetic Fe2þ/Fe3þ or Gd3þ ion. We thenreasoned that the large magnetic moment of the ironoxide core would create a strong magnetic suscept-ibility in the proximity of the iron oxide core that willaffect the T1 relaxation of the Gd-DTPA encapsulatedwithin the nanoparticle's polymeric coating. We ob-served that the T1 relaxation rate (1/T1) of the Gd(III)-DTPA complex was quenched (off/dark) when the Gd-DTPA complex was encapsulated within the PAA coat-ing of the iron oxide nanoparticle (IO-PAA). Uponrelease of the quenched Gd-DTPA, an increase in theT1 relaxation rate was observed with marginal increasein the T2 relaxation rate (1/T2). This quenching effect isnot observed when the Gd chelate is attached to thesurface of the IONP or when nonmagnetic nanoparti-cles, such as cerium oxide nanoparticles (NC-PAA), areused to encapsulate the Gd-DTPA. Corresponding r1and r2 values for the IO-PAA-Gd-DTPA nanocompositeat different pH values revealed a pH-dependent in-crease in the r1 of the nanocomposite suspension asthe pH decreases, indicating T1 activation at acidic pH.The observed pH-dependent increase in r1 was onlyobserved when Gd-DTPA was encapsulated within thepolymeric coating of the nanoparticle but not when itwas directly attached on the surface of the

Scheme 1. Schematic representation of the acidic pH-mediated activation of the activatable compositemagnetic nanoprobeIO-PAA-Gd-DTPA and corresponding T1MRactivation. In an acidic environment, the quenchedGd-DTPA chelates are releasedfrom the iron oxide's (IO) polymeric coating, becoming activated (ON) and resulting in an enhancement in the T1-weightedMRI image (brighter signal) and no change in the T2-weighted image.

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nanoparticle's polymeric coating. These results confirmthat indeed the superparamagnetic iron oxide nano-crystal acts as amagnetic quencher for the Gd-DTPA T1only when the Gd-DTPA is encapsulated within thenanoparticle's polymeric coating in close proximity tothe superparamagnetic core. Furthermore, when theIO-PAA-Gd-DTPA nanocomposite was conjugatedwithfolic acid, its selective internalization and lysosomallocalization within folate-receptor-positive cells allowfor selective activation due to the lysosome's acidic pH.Finally, when the folate receptor targeting nanocom-posite was used to coencapsulate a cytotoxic drug(Taxol), dual delivery of the drug and T1 imagingactivation was achieved. Therefore, our newly devel-oped activatable nanoprobe (IO-PAA-Gd-DTPA) com-bines features of several important modalities, such as(i) activatable T1-weighted MRI contrast, (ii) T2-weightedMRI contrast, (iii) receptor-targeted internalization,and (iv) delivery of anticancer drug to tumors. Thesefeatures make our magnetic nanoprobe a potentialMR-activatable contrast agent for cancer.

RESULTS AND DISCUSSION

Synthesis and Characterization of Gd-DTPA Composite IronOxide Nanoparticles. Our IO-PAA-Gd-DTPA probe wassynthesized by direct addition of Gd-DTPA during thecourse of the IO-PAA synthesis using a modified ver-sion of our previously published protocol.22 In brief, anaqueous solution of PAA (0.45 mmol) and Gd-DTPA(0.04 mmol) was added and mixed thoroughly beforeaddition of a mixture of iron salts (2.26 mmol ofFeCl3 3 6H2O and 1.61 mmol of FeCl2 3 4H2O in diluteHCl solution) in aqueous ammonium hydroxide solu-tion (0.05 M). The resulting dark-brown suspension ofcomposite IO-PAA-Gd-DTPA nanoprobe was stirred for1 h at room temperature and then centrifuged at4000 rpm for 30 min to get rid of free poly(acrylic acid),not encapsulated Gd-DTPA complex and other un-reacted reagents. Finally, the composite nanoprobesuspension was purified using a magnetic column(Miltenyi Biotech) and washed with phosphate buffersaline (pH 7.4) solution. This in situ encapsulationapproach proved to be effective for the encapsulationof Gd-DTPA as no change in the size and relaxivity ofthe nanoprobes was found over a long period oftime (Supporting Information, Table S1). The encapsu-lation of Gd-DTPA within the nanoprobe was con-firmed by measuring the amount of Gd using ICP-MS(0.289mgGd/mL). The amount of Fe in the IO-PAA-Gd-DTPA preparation was determined as 2.0 mg Fe/mLfor a 0.05:1 Gd/Fe molar ratio. Magnetic relaxationmeasurements at 0.47 T of the composite nano-probes resulted in a Gd concentration based relaxivityof r1 = 50.2(1.8mM�1 s�1 and r2 = 87.3( 2.4mM�1 s�1,and r1 = 43.3( 2.1mM�1 s�1 and r2 = 230( 3mM�1 s�1

based on Fe concentration. These relatively high valuesfor r1 and r2 might be due to the presence of both iron

oxide and Gd(III) within the nanocomposite imagingprobe and are comparable to reported values for highrelaxivity probes.35,40,41 Dynamic light scattering (DLS)studies indicated the presence of a stable and mono-disperse suspension of nanoparticles with a hydrody-namic diameter of D = 79 ( 2 nm. The diameters ofthesemagnetic nanoprobeswere further confirmed byscanning transmittance electron microscopic (STEM)experiments, which show an average diameter of80 nm (Supporting Information, Figure S1). The synthe-sized IO-PAA-Gd-DTPA nanocomposite was found tobe stable in PBS (pH 7.4) and serum, as no binding,clustering, or precipitations of the nanoparticles wereobserved over a long period of time. Similarly, thestability of the composite nanoparticles was furtherconfirmed by observing no significant changes inmagnetic relaxations, as shown in Supporting Informa-tion Table S1. Taken together, these results indicate theeffective encapsulation of Gd-DTPA into the IO-PAApolymeric matrix.

pH-Dependent Activation of the Gd-DTPA Composite MagneticNanoprobes. We first evaluated the magnetic relaxationactivation of the IO-PAA-Gd-DTPA nanoprobes in buf-fered solution within a pH range of 4.0 to 7.4. In theseexperiments, we measured the T1 and T2 of increasingconcentrations of IO-PAA-Gd-DTPA nanoprobes atphysiological (pH 7.4) and acidic (pH 4.0�6.0) bufferedsolutions. T1 and T2 readings were taken upon additionof the magnetic nanoprobes, immediately (0 h) andafter 24 h incubation of the magnetic nanoprobes inthe corresponding buffered solutions at 37 �C. First, weobserved that the T1 relaxation rate (1/T1) of theIO-PAA-Gd-DTPA nanoprobe (0 h, Figure 1A) was similarto that of the control IO-PAA nanoprobe (0 h, Support-ing Information, Figure S2A) at all pH values (pH4.0�7.4). This observation seems to indicate that inthe IO-PAA-Gd-DTPA nanoprobe the 1/T1 of Gd-DTPAwas quenched upon encapsulation in the polymericcoating of IO-PAA. In contrast, we observed a greaterincrease in 1/T1 of the IO-PAA-Gd-DTPA nanoprobewhen incubated in acidic [pH = 4.0 (1), 5.0 (2), and 6.0(b)] buffered solution after 24 h (Figure 1B). However,no changes in 1/T1 were observed either for IO-PAA-Gd-DTPA when incubated at physiological pH over thesame 24 h time period (pH = 7.4, 9, Figure 1B) or forequivalent concentrations of control IO-PAA across thesame pH values (pH 4.0 to 7.4) after 24 h of incubation(Supporting Information, Figure S2B). These resultssuggest that the composite IO-PAA-Gd-DTPA nano-probe gets activated, resulting in high Δ1/T1 numbers(Figure 1C) within 24 h of incubation in the acidicbuffered solutions in contrast to values obtained withthe control IO-PAA probe (Supporting Information,Figure S2C). In another set of experiments, minimalchanges in T2 relaxation rate (Δ1/T2) were observed forboth the composite IO-PAA-Gd-DTPA nanoprobe(Figure 1D�F) and control IO-PAA nanoprobe

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(Supporting Information, Figure S2D�F) when incu-bated for 24 h in buffered solutions (pH 4.0 to 7.4).These results indicated that the T2 of IO-PAA-Gd-DTPAprobe was not quenched upon encapsulation of theGd-DTPA complex, as hypothesized. Taken together,the above results suggest that the inverse spin�latticemagnetic relaxation (1/T1) of our composite IO-PAA-Gd-DTPA nanoprobes got activated when exposed toacidic environments and could be of potential use asan activatable NMR/MRI imaging agent for the detec-tion of tumors, upon internalization and localization ofthe nanoprobes within lysosomes.

pH-Dependent Studies of a Gd-DTPA Cerium Oxide Nanocom-posite as Control Studies. To confirm that the superpar-amagnetic nature of the iron oxide core is responsiblefor quenching themagnetic relaxation of the Gd-DTPA,we synthesized a PAA-coated cerium oxide nanoparti-cle encapsulating Gd-DTPA (NC-PAA-Gd-DTPA). In thisdesign, we selected a nonmagnetic metal oxide corecomposed of cerium oxide (nanoceria, NC) to replacethe magnetic iron oxide core during our synthesisprotocol. We reasoned that incorporation of Gd-DTPAwithin the polymeric coating of a nonmagnetic nano-particle, such as cerium oxide, will not result in T1quenching or activation upon incubation in acidicmedia. The NC-PAA-Gd-DTPA nanoprobes weresynthesized following a procedure similar to the one

used to synthesize the IO-PAA-Gd-DTPA nanoprobe.Briefly, to a PAA solution in water, Gd-DTPA was addedand mixed thoroughly before addition to a solution ofcerium nitrate in ammonium hydroxide solutions. Thesynthesized NC-PAA-Gd-DTPA composite nanoprobewas purified using the SpectrumLab's Krosflo filtrationsystem. DLS and ICP-MS of the nanoprobe aqueoussuspension indicated the presence of 88 ( 1 nmnanoparticles with a Gd concentration of 0.315 mg/mL(Supporting Information, Scheme S1). These valueswere similar to those obtained for the IO-PAA-Gd-DTPAnanoprobes, suggesting that the size, polymer coatingthickness, and amount of encapsulatedGdwere similarin both preparations. Magnetic relaxation values of theaqueous nanoparticle suspension revealed an r1 = 34.3(2.1mM�1 s�1 and r2 = 60( 5.2mM�1 s�1 (based on Gdconcentration), further confirming the successful en-capsulation of Gd in the nanoparticle's polymeric core.The magnetic relaxation rates 1/T1 and 1/T2 of theNC-PAA-Gd-DTPA nanoprobes indicated no change inΔ1/T1 (Figure 2C) before (0 h, Figure 2A) or after 24 hincubation (Figure 2B) in either physiological (pH 7.4)or acidic (pH 4.0) buffered solutions, indicating nomagnetic relaxation activation at acidic pH. Similarly,no changes in T2 (Δ1/T2, Figure 2D�F) were recorded forthe NC-PAA-Gd-DTPA nanoprobes, and as expected, nochanges in magnetic relaxation rates (1/T1 and 1/T2) were

Figure 1. Assessment of magnetic relaxations of activatable magnetic nanoprobe IO-PAA-Gd-DTPA using a benchtopmagnetic relaxometer (Bruker's Minispec, B = 0.47 T). Inverse spin�lattice (1/T1) and spin�spin (1/T2) magnetic relaxationtimes were measured before and after 24 h of incubation in different PBS solutions (pH 4.0�7.4, 37 �C) and at differentnanoprobe concentrations. (A) Initial 1/T1measurements right after the additionof PBS solutions, (B) 1/T1measurements after24 h of incubation, (C) differential 1/T1 values prior to and after incubation. (D) Initial 1/T2 measurements right after theaddition of PBS solutions, (E) 1/T2 measurements after 24 h of incubation, (F) differential 1/T2 values prior to and afterincubation (means ( SE; SE were within 1�4%, which are too small to be depicted).

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observed in the case of nonmagnetic nanoceria controlprobe NC-PAA61,62 (Supporting Information, Figure S3).Taken together, the above results suggest that the ob-served quenching of the Gd-DTPA T1 relaxation rate (1/T1)only occurred when the Gd-DTPA was encapsulated inclose proximity to a superparamagnetic core (iron oxide)and not when encapsulated within the polymeric coatingof a nonmagnetic core. These data also suggest that theobserved quenching is not due to immobilization of theGd-DTPAwithin a polymermatrix surrounding a nonmag-netic core (ceriumoxide). In another set of experiments, nosignificant pH-dependent activation was observed whenan iron oxide nanocomposite containing Ca-DTPA insteadofGd-DTPAwas used, further indicating that the release ofthe paramagnetic Gd-chelate is responsible for the ob-served activation in acidic pH (Supporting Information,Figure S4). Taking together, these results indicate that theobserved pH-dependent T1 activation observed with ourIO-PAA-Gd-DTPA nanocomposite is due to quenching ofthe T1 relaxativity of the Gd-DPTA chelate when in closeproximity to the ironoxide core and subsequent activationupon release from the nanoparticle's polymeric coating.

Magnetic Relaxations of the Gd-DTPA Surface ConjugatingMagnetic Nanoprobes. In our hypothesis, we stated thatthe close proximity of Gd (a weak paramagnetic ion)within the polymeric matrix of iron oxide nanoparticles(a strong superparamagnetic nanocrystal) affects theT1 relaxation of Gd. If our hypothesis is correct,

conjugation of a Gd-DTPA directly on the nanoparti-cle's surface will not result in quenching of the T1values. In this case, the Gd-DTPA will be further awayfrom the iron oxide core and its T1 relaxationwill be lessaffected by the strong magnetic susceptibility of themagnetic core. To further test this hypothesis, we thensynthesized a IO-PAA-Gd-DTPA magnetic nanoprobewhere the Gd-DTPA was conjugated directly on the IO-PAA surface carboxylic acid groups (Supporting Infor-mation, Scheme S2). Briefly, IO-PAA was first conju-gated with ethylenediamine using the water-solublecarbodiimide chemistry, as previously described.22 Theresulting aminated IO-PAA was then conjugated withGd(III)-chelated 2-(4-isothiocyanatobenzyl)diethylenetri-aminepentaacetic acid (p-SCN-Bn-Gd-DTPA) in basicPBS buffer (pH 8.4). The conjugated magnetic nano-probe was purified using small magnetic columns(Miltenyi Biotech) and washed with PBS (pH 7.4), priorto characterizations and magnetic relaxation measure-ments. The successful conjugation of the functionalGd-DTPA complex was confirmed by performing ICP-MS experiments, and the resulting Gd concentrationwas found to be 0.201 mg/mL. The amount of Fe in thepreparation was determined as 2.0 mg Fe/mL, for a 0.04:1Gd/Fe molar ratio. The magnetic relaxation values of theconjugatednanoprobewere r1=63.4(1.5mM�1 s�1 andr2=92.1(3.8mM�1 s�1 (basedonGdconcentration), andr1 = 49.9 ( 1.3 mM�1 s�1 and r2 = 243 ( 3 mM�1 s�1

Figure 2. Assessment of magnetic relaxations of composite nanoceria NC-PAA-Gd-DTPA using a benchtop magneticrelaxometer (Bruker's Minispec, B = 0.47 T). Inverse spin�lattice (1/T1) and spin�spin (1/T2) magnetic relaxation times weremeasured before and after 24 h of incubation in different PBS solutions (pH 4.0 and 7.4, 37 �C) and at different nanoprobeconcentrations. (A) Initial 1/T1 measurements right after the addition of PBS solutions, (B) 1/T1 measurements after 24 h ofincubation, and (C) differential 1/T1 values prior to and after incubation. (D) Initial 1/T2 measurements right after the additionof PBS solutions, (E) 1/T2 measurements after 24 h of incubation, and (F) differential 1/T2 values prior to and after incubation(means ( SE; SE were within 1�4%, which are too small to be depicted).

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(based on Fe concentration). The T1 and T2 relaxation rates(1/T1 and 1/T2) of the nanoprobe were measured in asimilar fashion as described earlier and results com-pared to those of the control IO-PAA nanoprobe.Results showed no change in Δ1/T1 (Figure 3C) before(0 h, Figure 3A) or after (24 h, Figure 3B) incubating invarious buffered solutions andwere found to be similarto that of the control IO-PAA probe with no magneticactivation (Supporting Information, Figure S2). Simi-larly, no changes in spin�spin relaxations (Δ1/T2,Figure 3D�F) were observed after the 24 h of treat-ment. Overall, the above results indicate the encap-sulation of the Gd-DTPA within the polymeric coatingand close proximity to the iron oxide core responsiblefor the Gd relaxation quenching, which was thenactivated upon release.

Meanwhile, the r1 and r2 relaxation values based onGd concentrations of the IO-PAA-Gd-DTPA nano-probes indicate a significant pH-dependent increasein the r1 of the nanoprobes when the Gd is encapsu-lated within the polymeric coating of the iron oxidenanoparticles (Table 1). Results show that, by decreas-ing the pH of the solution to a mildly acidic condition(pH 6.0), a significant percent increase of 44% in theGd-based r1 is observed. This value contrasts with asmall increase of 5% observed when the Gd is con-jugated on the nanoparticle surface, further indicatingthat indeed encapsulation within the nanoparticle's

polymeric matrix is essential for the observed T1 acti-vation. The observed increase in r1 is larger at higherpH, observing a 68% increasing at pH5.0, the typical pHwithin lysosomes. Even though pH-dependent percentchanges in r2 are also observed in the Gd-encapsulatednanocomposite, they are not as large as the valuesobtained with r1. Taken together, these results confirmthe T1 activation of the IO-PAA-Gd-DTPA nanoprobesupon decreases in pH, particularly within the range (pH6�5) observed within lysosomes.

MRI-Based T1-Weighted Activation of the Composite IO-PAA-Gd-DTPA Nanoprobe. Next, we investigated if the ob-served pH-dependent increases in r1 of the IO-PAA-Gd-DTPA nanoprobe results in increases in theT1-weighted signal by MRI, leading to an increase inthe brightness of the image. For these experiments, theT1- and T2-weighted MR images (MRI, B = 4.7 T) ofnanoprobe solutions at pH 5.0 were acquired immedi-ately (Figure 4A1) and after a 24 h incubation(Figure 4A2) in the pH 5.0 buffer. An increase inthe T1-weighted MR signals was observed as theconcentration of the activatable IO-PAA-Gd-DTPAnanoprobes increased (from 0.06 to 2.4 μM of Gd),resulting in an increase in the signal of the correspondingMR images (Figure 4A2). The observed signal increaseafter a 24 h incubation in the pH 5.0 buffer correspondedto an increase in the (1/T1) relaxation rate (2, Figure 4C).As expected, a minimal increase in T2-weighted MR

Figure 3. Assessment of magnetic relaxations of Gd-DTPA surface conjugating IO-PAA magnetic nanoprobes, using abenchtop magnetic relaxometer (Bruker's Minispec, B = 0.47 T). Inverse spin�lattice (1/T1) and spin�spin (1/T2) magneticrelaxation times were measured before and after 24 h of incubation in different PBS solutions (pH 4.0�7.4, 37 �C) and atdifferent nanoprobe concentrations. (A) Initial 1/T1 measurements right after the addition of PBS solutions, (B) 1/T1measurements after 24 h of incubation, and (C) differential 1/T1 values prior to and after incubation. (D) Initial 1/T2measurements right after the addition of PBS solutions, (E) 1/T2 measurements after 24 h of incubation, and (F) differential1/T2 values prior to and after incubation (means ( SE; SE were within 1�4%, which are too small to be depicted).

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signals (T2 map, Figure 4B) or corresponding inversespin�spin magnetic relaxations (1/T2, Figure 4D) wasobserved from the IO-PAA-Gd-DTPA nanoprobes dueto the absence of any T2 activation. However, in thiscase, the MR signals were found to be decreased sincethe iron concentrations increased with the rising nano-probe concentrations. The calculated r1 and r2 values at4.7 T for the IO-PAA-Gd-DTPA nanoprobe before andafter a 24 h incubation at pH 5.0 also show an increasein r1 values (24.8 ( 1.2 vs 45.2 ( 1.9 mM�1 s�1) for apercent increase in r1 of 87%. Meanwhile, a modestincrease in r2 was observed (75.4 ( 2.3 vs 91.5 (3.1 mM�1 s�1) for a percent increase of only 21%. Inanother set of experiments, no change in the MRsignals (both T1 and T2 map) or corresponding mag-netic relaxations were observed due to the absence ofany magnetic activation from our control IO-PAAprobe (Supporting Information, Figure S5). Takentogether, the above results confirm that our activa-table IO-PAA-Gd-DTPA nanoprobes get activated atacidic pH, and activation was indicated by the in-crease in the T1-weighted MRI signal. These resultsalso suggest the potential diagnostic applications of

our novel NMR/MRI activatable composite iron oxidenanoprobes.

In Vitro Activation of the Composite IO-PAA-Gd-DTPA Nano-probe. To evaluate the potential biomedical applica-tions of the activatable IO-PAA-Gd-DTPA nanoprobes,we assessed their magnetic activations using culturedcells. We hypothesized that, upon receptor-mediatedendocytosis, the nanoparticles will localize in acidiclysosomes, therefore becoming activated as the en-capsulated Gd-DTPA complex gets released at lowerpH. For these experiments, the magnetic nanoprobeswere functionalizedwith folic acid, following publishedprotocols,22,63 in order to assess their targeted imagingcapabilities toward folate receptor (FR)-expressing can-cer cells. We hypothesized that, upon internalizationinto FR-expressing cancer cells, T1 activation of thecomposite IO-PAA-Gd-DTPA-Fol nanoprobe would betriggered by the lysosomal acidic environment (pH5.0), resulting in in vitro activation of the MRI signals.In these experiments, we used a FR-positive humancervical carcinoma cell line (HeLa cells, 10 000 cells/well), and as negative control, we used H9c2 cardio-myocyte (10 000 cells/well) that do not express FR. Cells

TABLE 1. Magnetic Relaxation Values at 0.47 T of the Nanocomposite Based on Gd Concentrations at Different pH

nanoprobe pH r1 (mM�1 s�1) r2 (mM

�1 s�1) % change r1 % change r2

IO-PAA-Gd-DTPA (encapsulated) [Gd] = 0.289 mg/mL 7.4 50.2 ( 1.8 87.3 ( 2.46.0 72.5 ( 1.3 98.2 ( 3.2 44 125.0 84.3 ( 1.2 111.6 ( 2.8 68 284.0 97.0 ( 2.5 118.5 ( 3.4 93 38

IO-PAA-Gd-DTPA (surface) [Gd] = 0.201 mg/mL 7.4 63.4 ( 1.5 92.1 ( 3.86.0 66.3 ( 2.2 95.2 ( 1.2 5 35.0 68.1 ( 1.4 97.4 ( 2.1 7 64.0 69.3 ( 1.3 98.5 ( 1.8 9 7

Figure 4. Magnetic resonance imaging (MRI) studies measuring the magnetic activations (T1 and T2 map) of our activatablemagnetic IO-PAA-Gd-DTPA nanoprobes in PBS at pH 5.0. (A) T1-weighted MRI images of increasing Gd concentrations(0.06�2.4μM)of IO-PAA-Gd-DTPAnanoprobes prior to (1) and after 24 hof incubation (2) at 37 �C. (B) T2-weightedMRI imagesof increasingFe concentrations (0.3�11.5mM) of IO-PAA-Gd-DTPAnanoprobesprior to (1) andafter 24hof incubation (2) at 37 �C.(C) Corresponding 1/T1 relaxation rates prior to (b) and after (2) 24 h of incubation. (D) Corresponding 1/T2 relaxation rate prior to(b) and after (2) 24 h of incubation (means ( SE; SE were within 1�4%, which are too small to be depicted).

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were incubated with the nanoprobes (100 μL, 28 mM)at different time points, trypsinized, centrifuged, andresuspended in PBS (pH 7.4) before measuring T1 andT2 of the nanoparticle cell suspension. As hypothe-sized, compared to the control IO-PAA-Fol nanoprobes,significant activation in inverse spin�lattice magneticrelaxations (1/T1) was observed in HeLa cells incubatedwith the activatable IO-PAA-Gd-DTPA-Fol nanoprobes(b, Figure 5A). No significant changes in 1/T2 wereobserved from HeLa cells incubated with either of theprobes (Figure 5B). These results further supported thein vitro activatable MR imaging capability of the com-posite nanoprobes, whereas the control probe's (9, IO-PAA-Fol) magnetic relaxation remained unchangedafter the FR-mediated internalizations. In contrast, nosignificant changes in magnetic relaxations (both 1/T1and 1/T2) were observed from H9c2 cells (FR-negative)incubated with either one of the nanoprobes, suggest-ing the lack of any receptor-mediated internalizationsof our magnetic nanoprobes (Figure 5C,D). Further-more, control experiments using HeLa cells preincu-bated with excess folic acid (0.2 mg/mL) in theculturing media resulted in abrogation of the pH-dependent 1/T1 activation (Supporting Information,Figure S6). Taken together, our results confirm thatthe FR-mediated internalizations and lysosomal acidicpH-assisted release of the encapsulating Gd-DTPAcomplex were responsible for the enhanced MR signalfrom our composite IO-PAA-Gd-DTPA-Fol nanoprobe.

These results also indicate that the activatable MRimaging capability of our composite nanoprobe couldpotentially be useful in the detection and treatment ofcancer in clinical settings.

pH-Dependent Dual Release of the Gd-DTPA Complex andTaxol. Previously, we have reported the encapsulationand release of a cytotoxic drug (Taxol) from the poly-meric coating of iron oxide nanoparticles as a thera-nostic nanoagent for the potential treatment ofcancer.22 We then hypothesized whether we couldencapsulate both Taxol and Gd-DTPA within the samenanoparticle, creating a dual therapeutic and activata-ble magnetic nanoprobe. The IO-PAA-Gd-DTPA nano-probes were then used to encapsulate Taxol aspreviously described using the solvent diffusionmethod.22,63 Briefly, to a suspension of IO-PAA-Gd-DTPA nanoprobes (2.5 mL, 28 mmol) in PBS, thedimethyl sulfoxide (DMSO) solution of Taxol (10 μL,0.5 μg/μL) was added dropwise at room temperature.The resulting purified IO-PAA-Gd-DTPA-Taxol nanopar-ticles were characterized by measuring their size usingDLS (D = 84 ( 2 nm), taxol encapsulation efficiency(EE) = 52 ( 2.4% using HPLC (λabs = 227 nm), andcalculating the Gd concentration (0.215 mg/mL) byperforming ICP-MS experiments. To evaluate the dualrelease of Taxol and Gd, the IO-PAA-Gd-DTPA-Taxolnanoprobes were incubated in a pH 5.0 PBS solu-tion, and the rate of release of the drug and Gd wasassessed using a dynamic dialysis technique. Briefly,

Figure 5. Intracellular magnetic activations of our folate-decorated activatable IO-PAA-Gd-DTPA-Fol nanoprobe (b, 100 μL,28mM) and the control IO-PAA-Fol nanoprobe (9, 100 μL, 28mM) using FR-expressing HeLa cells (A,B) and FR-negative H9c2cells (C,D). Significant activation in inverse spin�lattice magnetic relaxations (1/T1) was observed from HeLa cells incubatedwith the activatable IO-PAA-Gd-DTPA-Fol nanoprobes (b,A). As expected, no significant changes in 1/T2 were observed fromHeLa cells due to absence of any T2 activations (B). Neither 1/T1 (C) nor 1/T2 (D) activationswere observed fromH9c2 cells dueto lack of any receptor-mediated internalizations.

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the IO-PAA-Gd-DTPA-Taxol nanoprobes (50 μL, 28mM)were taken in a small dialysis cup (MWCO6�8 kDa) andincubated in PBS (pH 5.0) solution at 37 �C. The rate ofrelease of Taxol and Gd was monitored by collectingaliquots from the outside reservoir buffer and measur-ing the amount of released taxol via HPLC experiment(λabs = 227 nm) and Gd bymeasuring the increase in T1relaxation rate with time. Results showed a time-de-pendent increase in the amount of Taxol (2, Figure 6A)and Gd-DTPA (1, Figure 6B) released upon incubationat pH 5.0. These results suggest that indeed the acid-mediated degradation and/or swelling of the PAAcoatings results in the simultaneous release of bothTaxol and Gd. Interestingly, a slower rate of Gd-DTPArelease from the nanoprobe is observed in contrast toTaxol, and this could be due to a possible higher extentof hydrogen bonding between Gd-DTPA and thecarboxylic groups within the polymeric coating inter-nal cavities surrounding the iron oxide core. In contrast,when similar experiments were performed at physio-logical pH (PBS, pH 7.4, 37 �C), no significant release ofTaxol (9, Figure 6A) or Gd was observed (b, Figure 6B).Similarly, no significant release of Taxol or increase inmagnetic relaxations (1/T1) was observed when the IO-PAA-Gd-DTPA-Taxol nanoprobe was incubated in serumat 37 �C (Supporting Information, Figure S7). These find-ings indicate that the IO-PAA-Gd-DTPA-Taxol nanoprobe isstable at neutral pH and physiological conditions, onlyreleasing its cargo (Taxol andGd) inanacidic environment.

In Vitro Cytotoxicity of Taxol-Encapsulating Activatable IO-PAA-Gd-DTPA Nanoprobes. Finally, we examined the differentialin vitro cytotoxicity of the functionalized magneticnanoprobes (35 μL, 28 mM in PBS, pH 7.4) using FR-expressing human cervical cancer cells (HeLa, 2500 cells/well) and FR-negative cardiomyocyte cell lines (H9c2,2500 cells/well). Results confirmed a time-dependentdecrease in the number of viable HeLa cells whenincubated with folate-decorated IO-PAA-Gd-DTPA-Taxol

nanoprobes 5 (Figure 7A), showing more than 90%reduction in cell viability after 24 h of incubation. How-ever, the folate-decorated IO-PAA-Gd-DTPA nanoprobesshowed nominal toxicity (4) and comparablewith the IO-PAA-Fol (1) lacking the Gd-DTPA complex, as publishedearlier.22 As expected, nominal cytotoxicity was observedwhen HeLa cells were incubated with the IO-PAA-Gd-DTPA (2) and IO-PAA-Gd-DTPA-Taxol (3), due to theabsenceof any receptor-mediated internalizations. Theseresults suggest that the cytotoxicity of the nanoprobeswas not affected by the encapsulation of Gd due to thepresence of PAA polymer coatings. In addition, no sig-nificant reduction in cell viability was observed whenH9c2 cells, which do not overexpress FR, were in-cubated with all of the functional magnetic nanoprobes(Figure 7B), suggesting biocompatibility and potentialapplications of our nanoprobes for the targeted imagingand treatment of cancers. Taken together, the aboveresults suggest that our folate-decorated activatableIONP-PAA-Gd-DTPA-Taxol nanoprobes can potentiallybe used to detect tumors using MR imaging, whiletargeting and delivering a chemotherapeutic agent(Taxol) via folate receptors.

CONCLUSION

In summary, we report an activatable Gd-DTPA-encapsulating iron oxide NMR/MRI nanoprobe, withquenched longitudinal (spin�lattice) magnetic relaxa-tion (T1) of the encapsulated Gd-DTPA (low 1/T1) by theiron oxide nanoparticles. In our design, the T1 relaxa-tion of the Gd-DTPA complex becomes activated(dequenched), resulting in higher 1/T1 values andenhanced T1-weighted MRI contrast, upon acid-mediated degradation and release of the T1 agent.Our results clearly indicated that the magnetic relaxa-tion of the Gd-DTPA chelate (T1 agent) is quenched as aresult of such encapsulation, whereas the transverse(spin�spin) magnetic relaxation (T2) of iron oxide was

Figure 6. Rate of release of Taxol and Gd-DTPA at 37 �C. (A) HPLC experiment (λabs = 227 nm) indicated the time-dependentrelease of Taxol from the activatable IO-PAA-Gd-DTPA nanoprobes (50 μL, 28 mM) when incubated at pH 5.0 (2) solution. Nosignificant release of Taxol was observed (9) when incubated in PBS at pH 7.4. (B) Observed increase rate of Taxol release wasaccompanied by a gradual increase in the inverse spin�lattice magnetic relaxation (1/T1) recorded using magneticrelaxometer (1, B = 0.47 T, pH 5.0). As expected, nominal increase in the inverse spin�lattice magnetic relaxation (b, 1/T1)was observed when incubated in PBS at pH 7.4.

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minimally affected. Our results also demonstrated thatthe folate-receptor-mediated internalization and thesubsequent lysosomal localization induced an intracel-lular release of Gd-DTPA complex, resulting in anenhanced 1/T1 signal. In addition, when a Taxol-encapsulating activatable magnetic nanoprobe wasused, the intracellular drug's release was monitoredby the observed changes in 1/T1. The presence of folateon the activatable magnetic nanoprobe guarantees aselective activation and release of the drug only infolate-receptor-positive cells, minimizing toxicity tohealthy cells. In contrast, no T1 activation is observed

in theGd-DTPA surface-conjugated IONPs or Gd-DTPA-encapsulating nonmagnetic NC-PAA, confirming thatquenching was due to the close residence of the Gd-DTPA to the superparamagnetic iron oxide (IO) coreand not due to the presence of any nonmagneticmetallic core (cerium oxide) or polymeric (PAA) coat-ings. Overall, the pH dependence and targeted activa-tion upon cellular internalization of our designed IO-PAA-Gd-DTPA nanoprobe are important attributes ofthis novel platform technology that can be furtherdeveloped into others MR-activatable and theranosticnanoprobes.

MATERIALS AND METHODS

Materials. Iron salts, including ferrous(II) chloride tetrahydrate(FeCl2 3 4H2O) and ferric(III) chloride hexahydrate (FeCl3 3 6H2O),gadolinium(III) chloride hexahydrate (GdCl3 3 6H2O), cerium(III)nitrate hexahydrate (CeNO3 3 6H2O), diethylenetriaminepentaaceticacid (DTPA), ammonium hydroxide, hydrochloric acid, sodiumhydroxide, chloropropryl amine, sodium azide, copper(I) iodide,ethylenediamine (EDA), folic acid, N,N-dimethylformamide(DMF), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide (MTT), N-hydroxysuccinimide(NHS), 2-(N-morpholino) ethanesulfonic acid (MES), poly(acrylicacid) (PAA) and other chemicals, were purchased from Sigma-Aldrich. 2-(4-Isothiocyanatobenzyl)diethylenetriaminepentaaceticacid [p-SCN-Bn-DTPA] was purchased from Macrocyclics. EDC[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride]was obtained from Pierce Biotechnology. The human cervicalcarcinoma (HeLa) and cardiomyocyte (H9c2) cell lines wereobtained from ATCC. Magnetic columns (LS Column) were

Figure 7. In vitro cytotoxicity of Taxol-encapsulating activatable IO-PAA-Gd-DTPA nanoprobes. Time-dependent in vitroMTTassays for the determination of cytotoxicity of the functional magnetic nanoprobes (1�5, 35 μL, 28 mM in PBS, pH 7.4). HeLacells (A) and H9c2 cells (B) treated with the functional magnetic nanoprobes. Folate-conjugated (1), Gd-DTPA-encapsulating(2), Gd-DTPA, and taxol-encapsulating (3) magnetic nanoprobes showed biocompatibility with nominal toxicity in both thecell lines. The Gd-DTPA-encapsulating folate-conjugated magnetic nanoprobes (4) showed more than 15% reduction in cellviability, whereas Gd-DTPA- and Taxol-encapsulating folate-conjugated magnetic nanoprobes (5) showed more than 90%reduction in cell viabilitywhen treatedwithHeLa cells (A) andnotwithH9c2 cells (B), confirming the folate-receptor-mediatedinternalizations and ability for targeted therapy. Average values of four measurements are depicted (standard errors.

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purchased from Miltenyi Biotech for the purification of mag-netic nanoprobes usingQuadroMACS separators. Dialysismem-branes were obtained from Spectrum Laboratories. Nitrogen-purged DI water was used in all syntheses.

Synthesis of the Gd-DTPA Complexes. Chelation of the rare-earthelement gadolinium (Gd) with diethylenetriaminepentaaceticacid (DTPA) or with functional DTPA, p-SCN-Bn-DTPA [2-(4-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid], re-sults in a strongly paramagnetic, stable complex that iswell-tolerated in animals. These complexes were synthesizedfollowing the reported literature method.64,65 Briefly, a solutionof GdCl3 3 6H2O (4.49 g, 0.0121 mol) in H2O (10 mL) was addeddropwise to a solution of DTPA (5.0 g, 0.0127 mol) or p-SCN-Bn-DTPA (0.0127mol) in H2O (30mL) containing 2 N NaOH (5.0 mL)solution. The pH of the final reaction mixture was maintained at6.8 by constant addition of 2 N NaOH solution. The reaction wascontinued at 80 �C for 12 h before concentrated to 20 mL. Theobserved white crystals were dissolved in a minimum amountof water before precipitating in ethanol. The precipitate wasfiltered and dried under vacuum to obtain the Gd(III) complex asa white solid (yield: 86%).

Synthesis of the Gd-DTPA-Encapsulating Composite Iron Oxide Nano-probes (IO-PAA-Gd-DTPA). For the synthesis of Gd-DTPA-encapsulat-ing composite nanoprobe (IO-PAA-Gd-DTPA), we used a novelwater-based, in situ encapsulation approach for the successfulencapsulation of the Gd-DTPA complex. In this approach, threedifferent solutions were prepared: an iron salt solution [0.61 g ofFeCl3 3 6H2Oand0.32gof FeCl2 3 4H2O indiluteHCl solution (100μLof 12 N HCl in 2.0 mL H2O)], an alkaline solution [1.8 mL of 30%NH4OH solution in 15 mL of N2 purged DI water], and a para-magnetic stabilizing solution [800 mg of PAA and 20 mg ofGd-DTPA complex in 5 mL of DI water]. To synthesize thecomposite IO-PAA-Gd-DTPA nanoprobe, the iron salt solutionwas added to the alkaline solution under vigorous stirring.The resulting dark suspension of iron oxide nanoparticles wasstirred for 10 s before addition of the paramagnetic stabiliz-ing solution and stirred for 1 h. The resulting suspension ofthe composite IO-PAA-Gd-DTPA nanoprobe was then centri-fuged at 4000 rpm for 30 min to get rid of free poly(acrylicacid), Gd-DTPA complex, and other unreacted reagents.Finally, the composite IO-PAA-Gd-DTPA nanoprobe suspen-sion was purified using magnetic columns and washed withphosphate buffer saline (pH 7.4) solution. The iron concen-tration and magnetic relaxation of the PAA-IONPs was de-termined as previously reported.22 The successful coating ofthe IONPs with PAA was confirmed by the presence of anegative zeta-potential (ζ = �41 mV) and the characteristicacid carbonyl band on the FT-IR spectroscopic analysis of thenanoparticles (Supporting Information, Figure S1).

Synthesis of the Theranostic Cargo-Encapsulating Composite Activata-ble Magnetic Nanoprobes. Taxol was encapsulated in the PAApolymer coating of the magnetic nanoprobe, following thepreviously reported solvent diffusion method.22,66 Briefly, to asuspension of IO-PAA-Gd-DTPA nanoprobes (2.5 mL, 28 mmol)in PBS, a dimethyl sulfoxide (DMSO) solution of Taxol (10 μL,0.5 μg/μL) was added dropwise at room temperature withcontinuous stirring at 1000 rpm. The Taxol-encapsulating nano-probes (IO-PAA-Gd-DTPA-Taxol) were purified using amagneticcolumn (Miltenyi Biotech) and then dialyzed (using 6�8KMWCO dialysis bag) three times against deionized water andfinally against phosphate buffered saline solution. The resultingIO-PAA-Gd-DTPA-Taxol nanoparticles were characterized bymeasuring their size using DLS (D = 84 ( 2 nm), the Taxolencapsulation efficiency (EE) = 52 ( 2.4% using HPLC (λabs =227 nm).

Synthesis of Folate-Decorated Magnetic Nanoprobes: Click Chemistry.To synthesize folate-decorated functional IO-PAA nanoprobes,the surface carboxylic acid groups of the nanoprobes werealkynated using propargylamine as a reagent, and the water-based carbodiimide chemistry was followed as previouslyreported.22 The resulting alkynated IO-PAA nanoprobes werepurified using magnetic columns. The highly specific “click”chemistry was used to conjugate an azide-functionalized folicacid with the purified alkynated IO-PAA, as described in thepreviously reported methods.22,67 Briefly, the alkynated IO-PAA

(4.0� 10�3mmol) in bicarbonate buffer (pH 8.5) was taken to anEppendorf tube containing a catalytic amount of CuI (5.0 �10�10 mmol) in 250 μL of bicarbonate buffer (pH 8.5) andvortexed. To the resulting solution, the azide-functionalizedfolic acid22,63 (8.0 � 10�2 mmol) in DMSO was added and thereaction was incubated at room temperature for 12 h. Thesynthesized folate-decorated IO-PAA was purified using themagnetic column and finally washed using PBS solution (pH7.4). The folate-decorate IO-PAA was stored in refrigerator forfurther characterization.

Synthesis of the Gd-DTPA-Encapsulating Composite Nanoceria (NC-PAA-Gd-DTPA). For the synthesis of Gd-DTPA-encapsulating compo-site nanoceria, we have modified our previously reportedstepwise method61,68 and followed the in situ encapsulationapproach. In this approach, 1 M cerium(III) nitrate (2.17 g in5.0 mL of water) solution was added to 30.0 mL of ammoniumhydroxide solution (30%w/v) under continuous stirring at roomtemperature. Then, after 45 s of stirring, an aqueous mixturecontaining the PAA polymer and Gd-DTPA complex (800 mg ofPAA and 20 mg of Gd-DTPA in 5 mL of water) was added andallowed to stir for 3 h at room temperature. The preparationwasthen centrifuged at 4000 rpm for two 30 min cycles to settledown any debris and large agglomerates. The supernatantsolution was then purified from free PAA, Gd-DTPA complex,or other chemicals and concentrated using SpectrumLab'sKrosFlo filtration system.

Synthesis of the Gd-DTPA Surface-Conjugating Magnetic Nanoprobes(IO-PAA-Gd-DTPA-Surface). The poly(acrylic acid)-coated iron oxidenanoparticles (IO-PAA) were synthesized using our previouslyreported alkaline precipitation method.22 Briefly, an Fe3þ/Fe2þ

solution in water was rapidly mixed with an ammonium hydro-xide solution for 30 s, prior to addition of the PAA polymersolution in water. The synthesized IO-PAA was purified usingmagnetic columns to remove any unreacted reagents, andphosphate buffered saline (PBS, pH 7.4) was used as runningsolvent. To incorporate amine groups to the nanoparticles,ethylenediamine was used as an aminating agent and thewater-based carbodiimide chemistry (using EDC and NHSreagents) was followed, as previously reported.22,62 The suc-cessful amination of the IO-PAAnanoparticles was confirmed bymeasuring their overall positive surface charge (ζ = þ15 mV)using Malvern's Zetasizer. To synthesize the Gd-DTPA surface-conjugating IO-PAA nanoprobe, the aminated IO-PAA wasreacted with the isothiocyanate group of the p-SCN-Bn-DTPAchelated with GdCl3 3 6H2O salt. In a typical reaction, the iso-thiocyanate functional Gd-DTPA chelate (p-SCN-Bn-Gd-DTPA,25 mmol) was added to the aminated IO-PAA nanoprobe(1 mmol) in the presence of basic phosphate buffered saline(PBS, pH8.4) and incubated overnight at room temperature. Theresulting Gd-DTPA surface-conjugating IO-PAA nanoprobe waspurified using small magnetic columns (Miltenyi Biotech) andwashed with phosphate buffered saline (PBS, pH 7.4), prior tocharacterizations and magnetic relaxation measurements.

Measurement of the Hydrodynamic Diameter and Surface Zeta-Poten-tial of the Functional IO-PAA. The size and dispersity of the synthe-sized composite IO-PAA was measured using dynamic lightscattering (DLS) PDDLS/CoolBatch 40T instrument with Preci-sion Deconvolve 32 software. The overall surface charges (zeta-potential) of this functional IO-PAA were measured using aZetasizer Nano ZS from Malvern Instruments. These experi-ments were performed by placing 10 μL of the compositemagnetic nanoprobes in 990 μL of distilled water. All measure-ments were performed in triplicate.

Measurement of Magnetic Relaxations. Magnetic relaxationmea-surements were conducted with a compact magnetic relaxo-meter (0.47 T mq20, Bruker), by taking composite magneticnanoprobes with various concentrations. Magnetic resonanceimaging (MRI) of the magnetic phantoms was achieved usingthe MRI/MRS facility utilizing a 4.7 T 33 cm bore magnetimaging/spectroscopy system (MSKCC, New York). All measure-ments were performed in triplicate.

HPLC Experiment. HPLC experiments were carried out using aPerkinElmer's Series 200 instrument to study drug releasekinetics. In a typical experiment, upon addition of acidic PBSsolution (pH 5.0) to the Taxol-encapsulating IO-PAA-Gd-DTPA

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(50 μL, 28 mM), the rate of release of encapsulating Taxol wasmonitored in a timely manner at 37 �C using HPLC (λabs =227 nm) chromatography.

Cell Cultures. The human cervical cancer (HeLa) and cardio-myocyte (H9c2) cells were obtained from ATCC andmaintainedin accordance to the supplier's protocols. Briefly, the cervicalcancer cells were grown in a 5% FBS-containing DMEMmediumsupplemented with L-glutamine, streptomycin, amphotericin B,and sodium bicarbonate. The H9c2 cells were propagated in a10% FBS-containing MEM medium containing penicillin, strep-tomycin, and bovine insulin (0.01mg/mL). Cells were grown in ahumidified incubator at 37 �C under 5% CO2 atmosphere.

In Vitro Magnetic Activations of the Composite Nanoprobes. Thehuman cells (HeLa and H9c2, 10 000 cells/well) were incubatedwith the folate-decorated activatable IO-PAA-Gd-DTPA-Fol na-noprobe and the control IO-PAA-Fol nanoprobe (100μL, 28mM)at different incubation times. The cells were then trypsinizedand centrifuged. The resulting cell pellet was suspended inphosphate buffer saline (PBS, pH 7.4), and magnetic relaxationsof these solutions weremeasured using the benchtopmagneticrelaxometer (B = 0.47 T mq 20) from Bruker. All measurementswere performed in triplicate.

Cytotoxicity Assay. H9c2 and HeLa cells (2500 cells/well) wereseeded in 96-well plates and incubated with the correspondingcomposite IO-PAA nanoprobes (35 μL, 28 mM in PBS pH 7.4) at37 �C. After the specific time incubation, each well was washedthree times with 1� PBS and treatedwith 30 μL ofMTT (2 μg/μL)for 2 h. The resulting formazan crystals were dissolved in acidicisopropyl alcohol (0.1 N HCl), and the absorbance was recordedat 570 and 750 nm (background), using a Synergy μQuantmicrotiter plate reader (Biotek). Experiments were performedin triplicate.

Conflict of Interest: The authors declare no competingfinancial interest.

Acknowledgment. This work was supported in part by NIHGrant GM084331 and UCF-NSTC Start Up Fund, all to J.M.P. Thisstudywas also partly supported by NIH Grant P30 CA008748-44S5 and MSKCC Gerstner Young Investigator Award (to J.G.). Wethank Dr. S. M. Hussain from the US Air Force's Wright-PattersonAir Force Base, Ohio, USA, for assistance with ICP-MSexperiments.

Supporting Information Available: Detailed physical charac-terizations of the magnetic probes including dynamic lightscattering (DLS), scanning transmittance electron microscopy(STEM), FT-IR, zeta-potential, stability of the nanoprobes atdifferent conditions, MRI-based magnetic relaxations, encapsu-lating drug release, and cytotoxicity studies. This material isavailable free of charge via the Internet at http://pubs.acs.org.

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