DOI: 10.1021/la903113u 12015Langmuir 2009, 25(20), 12015–12018 Published on Web 09/21/2009

pubs.acs.org/Langmuir

© 2009 American Chemical Society

Hybrid Lanthanide Nanoparticles with Paramagnetic Shell Coated on

Upconversion Fluorescent Nanocrystals

Zhengquan Li,† Yong Zhang,*,†,‡ Borys Shuter,§ and Niagara Muhammad Idris†

†Division of Bioengineering, Faculty of Engineering, ‡Nanoscience and Nanotechnology Initiative, and§Department of Diagnostic Radiology, Yong Loo Lin School of Medicine, National University of Singapore,

7 Engineering Drive 1, Singapore 117574

Received August 20, 2009. Revised Manuscript Received September 14, 2009

Nanoparticles comprising of fluorescent probes and MRI contrast agents are highly desirable for biomedicalapplications due to their ability to be detected at different modes, optically andmagnetically. However, most fluorescentprobes in such nanoparticles synthesized so far are down-conversion phosphors such as organic dyes and quantum dots,which are known to display many intrinsic limitations. Here, we report a core-shell hybrid lanthanide nanoparticleconsisting of an upconverting lanthanide nanocrystal core and a paramagnetic lanthanide complex shell. Thesenanoparticles are uniform in size, stable in water, and show both high MR relaxivities and upconversion fluorescence,which may have the potential to serve as a versatile imaging tool for smart detection or diagnosis in future biomedicalengineering.

Introduction

The recent advances in nanotechnology promise a brightfuture in the use of nanostructured materials for variousbiomedical applications including imaging, diagnosis, andtherapy.1,2 Rational design combined with the use of a cocktailof different functional materials within a single nanoparticlewill support the development of a multifunctional nanoplat-form for enhanced diagnostic and therapeutic effects.3 Inbioimaging, magnetic resonance imaging (MRI) is known toprovide an excellent three-dimensional spatial resolution,while fluorescent imaging has proven to show high screeningsensitivity. Therefore, constructing nanoparticles carryingboth a fluorescent probe and an MRI contrast agent is highlydesirable for in vivo diagnosis of disease and image-guidedsurgery due to their ability to be detected at two differentmodes, optically and magnetically. This duo combination willoffset the limitations of each technique, such as low sensitivityof MRI and limited anatomical background of fluorescentimaging. For such merits, there has been much interest inrecent years in the synthesis of multifunctional nanoparticlescomposing a fluorescent probe and MRI contrast agent. For

example, Gd-dye@MSN nanoparticles, iron oxide/Cy5.5 con-jugates, and quantum dots/Gd-lipid have been developed.4-9

However, most of the multifunctional nanoparticles synthe-sized so far consist of MRI contrast agent and down-conversionphosphors such as organic dyes and quantum dots, which areknown to display some intrinsic limitations.10 For instance, theuse of organic dyes are often associated with a number ofdrawbacks such as poor photostability, broad absorption andemission bands, while the use of semiconductor quantum dotsremained controversial due to their inherent toxicity and chemicalinstability. Besides, the requirement of these down-conversionmaterials to be excited generallywithin theUVorblue light regionhas further compounded the problems in using them includingautofluorescence background, low penetration depth, and photo-damage to the biological specimens being investigated. Near-infrared (NIR)-to-visible upconversion nanomaterials can con-vert NIR light to visible light uponNIR light radiation.11,12 Sincemost biomolecules absorb minimally in the NIR window, theiruse in fluorescent imaging will minimize the extent of autofluor-escence and photodamage induced, as well as enable highpenetration depth of light in tissues.13-16 Furthermore, theseupconverison materials show superior photostability, as thelanthanide ions responsible for their fluorescence emission aredopedwithin the nanocrystal core such that they are well-shieldedfrom the surroundings. On the other hand, although MRIcontrast agents that are both T1-weighted (mainly paramagneticGd3þ complexes) and T2-weighted (generally superparamagneticnanoparticles) have been widely used in clinical applications, thelatter reduce MR signal and sometimes could lead to confusionabout whether the signal comes from bleeding, calcification, or

*Corresponding author. Tel: (þ65) 6516-4871, Fax: (þ65) 6872-3069,E-mail: biezy@nus.edu.sg.(1) Alivisatos, P. Nat. Biotechnol. 2004, 22, 47.(2) Nie, S. M.; Xing, Kim Y.; G., J.; Simons, J. W. Annu. Rev. Biomed. Eng.

2007, 9, 257.(3) Kim, J.; Piao, Y.; Hyeon, T. Chem. Soc. Rev. 2009, 38, 372.(4) Tsai, C. P.; Hung, Y.; Chou, Y. H.; Huang, D. M.; Hsiao, J. K.; Chang, C.;

Chen, Y. C.; Mou, C. Y. Small 2008, 4, 186.(5) Prinzen, L.; Miserus, R.; Dirksen, A.; Hackeng, T. M.; Deckers, N.; Bitsch,

N. J.; A. Megens, R. T.; Douma, K.; Heemskerk, J. W.; Kooi, M. E.; Frederik,P. M.; Slaaf, D. W.; van Zandvoort, M.; Reutelingsperger, C. P. M. Nano Lett.2007, 7, 93.(6) Mulder, W. J. M.; Koole, R.; Brandwijk, R. J.; Storm, G.; Chin, P. T. K.;

Strijkers, G. J.; Donega, C. D.; Nicolay, K.; Griffioen, A.W.Nano Lett. 2006, 6, 1.(7) Veiseh, O.; Sun, C.; Gunn, J.; Kohler, N.; Gabikian, P.; Lee, D.; Bhattarai,

N.; Ellenbogen, R.; Sze, R.; Hallahan, A.; Olson, J.; Zhang,M.Q.Nano Lett. 2005,5, 1003.(8) Liu, Z. Y.; Yi, G. S.; Zhang, H. T.; Ding, J.; Zhang, Y.W.; Xue, J. M.Chem.

Commun. 2008, 694.(9) Yang, P. P.; Quan, Z. W.; Hou, Z. Y.; Li, C. X.; Kang, X. J.; Cheng, Z. Y.;

Lin, J. Biomaterials 2009, 30, 4786.

(10) Wang, F.; Tan,W. B.; Zhang, Y.; Fan, X. P.; Wang,M. Q.Nanotechnology2006, 17, R1.

(11) Mai, H. X.; Zhang, Y. W.; Si, R.; Yan, Z. G.; Sun, L. D.; You, L. P.; Yan,C. H. J. Am. Chem. Soc. 2006, 128, 6426.

(12) Li, Z. Q.; Zhang, Y. Angew. Chem., Int. Ed. 2006, 45, 7732.(13) Wang, L. Y.; Yan, R. X.; Hao, Z. Y.;Wang, L.; Zeng, J. H.; Bao,H.;Wang,

X.; Peng, Q.; Li, Y. D. Angew. Chem., Int. Ed. 2005, 44, 6054.(14) Chatteriee, D. K.; Rufalhah, A. J.; Zhang, Y. Biomaterials 2008, 29, 937.(15) Jalil, R. A.; Zhang, Y. Biomaterials 2008, 29, 4122.(16) Li, Z. Q.; Zhang, Y.; Jiang, S. Adv. Mater. 2008, 20, 4765.

12016 DOI: 10.1021/la903113u Langmuir 2009, 25(20), 12015–12018

Letter Li et al.

metal deposits.17,18 In view of this, it is thus desirable to use T1-weightedMRI contrast agents to enhance theMRsignal intensity.In order to achieve target-specific MR contrast agents, a feasibleroute is to synthesize nanoparticles with a high payload of Gd3þ

complex. This kind of MR contrast agent can work efficiently atlow concentration.19,20

Ideally, a multimodal nanoparticle composed of a strongupconversion probe and numerous active paramagnetic centers,and having a water-dispersible and easy-to-conjugate surface,may offer tremendous potential in biomedical research andclinical applications. Herein, we report a hybrid lanthanidenanoparticle consisting of an upconverting lanthanide nanocrys-tal core and a paramagnetic lanthanide complex shell, as illu-strated in Figure 1A. The core material is made up of hexagonallanthanide-doped NaYF4 nanocrystals, which have been provento be the best upconversion materials developed so far.21 Anefficient T1-weighted MRI contrast agent, Si-DTTA-Gd3þ, wasthen uniformly coated on the NaYF4 nanocrystals with a highpayload of Gd3þ ions. Rational combination of the lanthanidenanocrystals and lanthanide complexes brings many advantages:(1) the surface of the core nanocrystals is transformed fromhydrophobic to hydrophilic without disturbing their originalsurface state and intrinsic fluorescent intensity; (2) a high payloadof Gd3þ complex in one nanoparticle can significantly improveboth r1 and r2 MR relaxivities, which not only makes thenanoparticles excellent T1-weighted contrast agents but alsoconfers good T2-weighted functionality; (3) a silica surface

decorated with abundant carboxylic groups (from Si-DTTA) ismore soluble and stable in solution and easily conjugated tovarious biomolecules for targetting.Wehypothesize that this kindof core-shell structured multifunctional nanoparticle may serveas a versatile imaging tool to correlate preoperative diagnosticimages with intraoperative pathology for future biomedicalapplications.

Experimental Section

Synthesis of NaYF4@Si-DTTA Nanoparticles. High-quality upconversionNaYF4 nanocrystals were synthesized usinga user-friendly protocol that we previously established.22 Asilanized complex, 3-aminopropyl(trimethoxysilyl)diethylenetri-amine tetraacetic acid (Si-DTTA), was prepared by graftingcarboxylic groups on 3-(trimethoxysilylpropyl)diethylene tria-mine (Supporting Information).19 In a typical synthesis ofNaYF4@Si-DTTA nanoparticles, a mixing solution of 2 mLTri-100, 2mL1-hexanol, 15mLcyclohexane, and0.3mLaqueousammonia (10 wt %) and 2 mL of NaYF4 nanocrystal solution(0.02 M in cyclohexane) were vigorously stirred for 20 min andformed a transparent microemulsion solution. Then, 20 uL oftetraethyl orthosilicate (TEOS)was added into themicroemulsionsolution, and a thin silica layer on NaYF4 nanocrystal wasinitially deposited in 24 h. After that, 100 uL aqueous solutionof Si-DTTA (0.1M) and 20 uLTEOSwere added into the stirringsolution every 2 h four times. The Si-DTTA complex wascovalently bonded within the silica shell through cross-linkingof the silanized group of Si-DTTA and tetraethyl orthosilicate(TEOS) during hydrolysis under basic solution. After continuousstirringof the solutionat roomtemperature for anadditional 48h,NaYF4@Si-DTTA nanoparticles could be precipitated from themicroemulsion solution by adding ethanol. The collected nano-particles were washed with water/ethanol three times. Successfulcoating of Si-DTTA on the nanoparticles could be confirmed byFourier transform infrared (FTIR) spectra (Supporting Informa-tion Figure S1).

Loading Gd3þ Ions to Nanoparticles. In a typical process,aliquots of the prepared NaYF4@Si-DTTA nanoparticles weredispersed in a 10 mL of 0.05M Tris-HCl buffer solution (pH=7.4) with a concentration of 0.12 mM (on Si-DTTA basis).Different volumes (0-1 mL) of 1 mM GdCl3 aqueous solutionwere then added dropwise inside the buffer solutionwhile stirring.After incubation for 12 h, the nanoparticles were centrifugeddown and redispersed in water at least three times. The nanopar-ticles were finally dispersed in DI water and formed a shallow,white transparent solution.

Characterization. Size distributions of nanoparticles weremeasured by dynamic light scattering (DLS), which was per-formed on a Malvern Zetasizer Nano ZS. Transmission electronmicroscopy (TEM) images were recorded on a JEOL 2010F TEMoperating at 200 kV. The TEM samples were prepared by drop-ping an ethanol solution of nanoparticles on a carbon-film coatedcopper grid. The accurate concentration of Gd3þ ions was deter-mined using a conductively coupled plasma-atomic emissionspectrometry (ICP-AES). Fluorescence spectra were acquired ona SpetroPro 2150i fluorescence spectrometer equipped with acommercial 980 nm NIR laser.

Relaxivity data were determined from vials containing nano-particles with Gd concentrations of 0 mM, 0.05 mM, 0.1 mM,0.2 mM, 0.4 mM, and 0.8 mM. Images were acquired using a 3 Tclinical magnetic resonance imaging scanner (Siemens Trio,Erlangen, Germany). A series of single echo (echo time (TE) =8ms) spin-echo imageswere acquiredatnine repetition times (TR:25-6400 ms) to obtain T1 relaxation times. T2 relaxation timeswere obtained from a multiecho spin-echo sequence (TR: 3000ms) which acquired images at 32 echo times (TE: 8-256 ms).

Figure 1. (A) Schematic illustration of single NaYF4@Si-DTTA-Gd3þ nanoparticles. (B) DLS data of NaYF4 nanoparticles before(dashed line) and after Si-DTTA-Gd3þ coating (solid line). (C,D)TEM images of NaYF4@Si-DTTA-Gd3þ nanoparticles at differ-ent magnification.

(17) Na,H. B.; Lee, J. H.; An,K. J.; Park, Y. I.; Park,M.; Lee, I. S.; Nam,D.H.;Kim, S. T.; Kim, S. H.; Kim, S. W.; Lim, K. H.; Kim, K. S.; Kim, S. O.; Hyeon, T.Angew. Chem., Int. Ed. 2007, 46, 5397.(18) Bulte, J. W. M.; Kraitchman, D. L. NMR Biomed. 2004, 17, 484.(19) Rieter, W. J.; Kim, J. S.; Taylor, K. M. L.; An, H. Y.; Lin, W. L.; Tarrant,

T.; Lin, W. B. Angew. Chem., Int. Ed. 2007, 46, 3680.(20) Taylor, K. M. L.; Kim, J. S.; Rieter, W. J.; An, H.; Lin, W. L.; Lin, W. B.

J. Am. Chem. Soc. 2008, 130, 2154.(21) Kramer, K. W.; Biner, D.; Frei, G.; Gudel, H. U.; Hehlen, M. P.; Luthi,

S. R. Chem. Mater. 2004, 16, 1244. (22) Li, Z. Q.; Zhang, Y. Nanotechnology 2008, 19, 345606.

DOI: 10.1021/la903113u 12017Langmuir 2009, 25(20), 12015–12018

Li et al. Letter

MRsignal values for each vialwere obtained from the images usingin-house software (MATLAB v 7.1:MathWorks,Natick,MA).T1

values were obtained by fitting an increasing exponential function(saturation recovery equation) to signal-TR plot. T2 values wereobtained by fitting a decreasing monoexponential function tosignal-TE plots. r1 and r2 were calculated as the slope of linearregression fits of inverse relaxation times (relaxation rates) plottedagainst Gd3þ concentration.

Results and Discussion

Size Distribution andMorphology of Nanoparticles.DLSdata of NaYF4 nanocrystals before and after Si-DTTA coating isshown in Figure 1B. The symmetric and regular curve displayedinfers that these nanocrystals are uniform in size both before andafter Si-DTTAcoating.Anobvious size increase from24 to 54nmsuggests a uniform shell has been coated. Themorphologies of thepreparedNaYF4@Si-DTTAnanoparticles were studied byTEMand are shown in Figure 1C,D, which further confirmed thesuccessful preparation of uniform core-shell structuredNaYF4@Si-DTTA nanoparticles. The diameter of the corenanocrystals alone is around 19 nm, while the whole nanoparticleextends to about 47 nm in diameter. It should be noted that thediameter reading derived from DLS data is slightly larger thanthat shown in TEM images due to the different measuringmechanism behind these two methods.Gd3þ Loading and MR Properties of Nanoparticles.

According to the hard and soft acids and bases (HSAB) theory,lanthanide ions are hard acids while carboxylic groups are hardbases, so that Gd3þ and DTTA can form stable complexesespecially when the ligands are multidentate.19,20 After metalliza-tion with Gd3þ, the Si-DTTA complex can irreversibly bind toGd3þ ions within the nanoparticle and prevent Gd3þ ions frombeing released out into the solution. Figure 2A gives the Gdconcentration determined by ICP-AES for samples having differ-ent amounts ofGd3þ added intoNaYF4@Si-DTTAnanoparticles

solution (1 mM, on Si-DTTA basis). The ICP-AES data show aclose correlation with the actual amount of Gd3þ added to thenanoparticles, with the former showing a slightly lower value dueto the inevitable loss of nanoparticles during the centrifugation andredispersion process. The release profiles of Gd3þ in the NaYF4@Si-DTTA-Gd3þ nanoparticles were also evaluated over a period of7 days during which no obvious leakage of Gd3þ was found in thesolution (Supporting Information Figure S2). In the controlexperiments, it was found that Gd3þ ions could not be efficientlyattached to pure NaYF4@SiO2 nanoparticles without DTTAcoating. The Gd3þ ions were released into the solution, asdemonstrated by ICP measurement, suggesting that DTTA playsa very important role in the attachment.

The MR relaxivities of the NaYF4@Si-DTTA-Gd3þ nanopar-ticles are shown in Figure 2B. The nanoparticles have a long-itudinal relaxivity (r1) of 20.1mM-1 s-1 and a transverse relaxivity(r2) of 55.0 mM-1 s-1 on a per millimolar Gd3þ-ion basis. Theserelaxivity values are higher than those of the pure Si-DTTA-Gd3þ

complex (r1 = 6.8 mM-1 s-1 and r2 = 7.0 mM-1 s-1), mostprobably a result of the slower tumbling rate of complex in thenanoparticulate form.19,20 If calculated on a per millimolar parti-cles basis, the nanoparticles could reach a much higher value of r1and r2, since at least the 104 Si-DTTA-Gd3þ complex has beencoated within a single nanoparticle, suggesting that these nano-particles may function as an efficient MRI contrast agent due totheir large payloads of active paramagnetic centers. Figure 2C,Dshow the T1-weighted and T2-weighted images, respectively, ofnanoparticles dispersed in deionized water at different concentra-tions. MR signal intensity is increased along with increasingconcentration of the nanoparticles from 0.05 to 0.8 mM (onGd3þ-ions basis) in the T1-weighted mode, while signals graduallybecame weaker with increasing concentration of nanoparticles inthe T2-weighted mode. These results support that the view that thenanoparticles may be a useful contrast agent for both T1- and T2-weighted MR imaging.UpconversionPropertiesandPhotostabilityofNanoparticles.

The upconversion fluorescence spectra of pure NaYF4:Yb,Er/Tmnanocrystals and NaYF4:Yb,Er/Tm@Si-DTTA nanoparticles,before and after Gd3þ labeling, are shown in Figure 3A and B.The Yb and Er co-doped NaYF4 nanocrystals exhibit two greenpeaks (521 and 539 nm) and a red peak (651 nm), which are ascribedto the energy transitions from 2H11,

4S3/2, and4F9/2 to

4I15/2 of Er3þ

ions, respectively. Two blue emissions (450 and 479 nm) weredisplayed in the Yb and Tm co-doped NaYF4 nanocrystals, owingto the transitionof 1D2 to

3F4 and1G4 to

3H6ofTm3þ ions.23 Inboth

Figure 2. (A)PlotofGd3þ loading amount versusGd3þ bonded innanoparticles (measured by ICP-AES). (B) r1 and r2 relaxivities ofNaYF4@Si-DTTA-Gd3þ nanoparticles. (C) T1-weighted (TR =200ms,TE=8ms). (D)T2-weighted (TR=3000ms,TE=88ms)MR images of aqueous NaYF4@Si-DTTA-Gd3þ nanoparticlesolution with different Gd3þ concentrations. (The “no Gd3þ”sample is a 0.8 mM solution of unlabeled NaYF4@Si-DTTAnanoparticles.)

Figure 3. Upconversion fluorescence spectra of (A) NaYF4:Yb,Er and (B) NaYF4:Yb,Tm nanocrystals in cyclohexane (dashedline); after Si-DTTA coating and dispersed in water (dotted line);and after Gd3þ labeling and dispersed in water (solid line).

(23) Suyver, J. F.; Aebischer, A.; Biner, D.; Gerner, P.; Grimm, J.; Heer, S.;Kramer, K. W.; Reinhard, C.; Gudel, H. U. Opt. Mater. 2005, 27, 1111.

12018 DOI: 10.1021/la903113u Langmuir 2009, 25(20), 12015–12018

Letter Li et al.

types of nanocrystals, the dopant Yb3þ acted as an NIR absorberand efficiently transferred energy to the emitter Er3þ or Tm3þ ion.After modification with a 14 nm Si-DTTA shell, the fluorescentintensity of these nanocrystals showed a slight decrease. This isreasonable, since the additional silica layer is able to scatter bothincident NIR light and emissive visible lights, implying that lessexcitation light can now reach the core nanocrystal and less emissionlight can penetrate out as well. No obvious fluorescence change wasobserved when the Gd3þ ion was labeled into the NaYF4:Yb,Er/Tm@Si-DTTA nanoparticles. Fluorescence stability of the NaYF4:Yb,Er@Si-DTTA-Gd3þ nanoparticles was also studied over aperiod of 7 days (Supporting Information Figure S3) and showeda relatively stable fluorescence of the nanoparticles in water.

Despite the slight fluorescence decrease that resulted from lightscattering, silica coating remained a favorable route to conferwater-solubility to these hydrophobic upconversion nanocrystalsbesides having the advantage of being able to preserve the inherentfluorescence of the nanoparticles. Indeed, other surface modifica-tion methods that we have tried including ligand exchange withpoly(acrylic acid) (PAA) or oxidation of the surfactant (oleic acid)have caused a dramatic decrease in the fluorescence intensity ofthese nanocrystals.24,25 For example, the green peak of modifiedNaYF4:Yb,Er nanocrystals in water decreased to nearly 1/8th and

1/11th of the unmodified nanocrystals in cychohexane after beingsurface-modified by ligand exchange with PAA and oxidation ofoleic acid, respectively. These phenomena presumably result fromthe fact that silica coating does not change the original surfacestate of the nanocrystals and thus reduce its possibility to inducesurface defects on the nanocrystals as compared to other surfacemodification methods.

Conclusion

In summary, we have developed a core-shell hybrid lantha-nide nanoparticle consisting of upconversion luminescent coreand a paramagnetic shell. Uniform Si-DTTA coating on theupconversion NaYF4 nanocrystals resulted in good water solu-bility and fluorescence stability. The paramagnetic shell with alarge payload of Gd3þ complex showed high r1 and r2 relaxivity,and could be used as an efficient contrast agent in dual modes.These multifunctional nanoparticles may have the potential toserve as versatile imaging tools for smart detection or diagnosis infuture biomedical engineering.

Acknowledgment. We acknowledge financial support fromSingapore A*STAR BMRC (grant number R-397-000-062-305).

Supporting Information Available: Experimental detail,FTIR spectra, release profile of Gd3þ, and photostability ofnanoparticles. Thismaterial is available free of charge via theInternet at http://pubs.acs.org.

(24) Naccache, R.; Vetrone, F.; Mahalingam, V.; Cuccia, L. A.; Capobianco,J. A. Chem. Mater. 2009, 21, 717.(25) Chen, Z. G.; Chen, H. L.; Hu, H.; Yu, M. X.; Li, F. Y.; Zhang, Q.; Zhou,

Z. G.; Yi, T.; Huang, C. H. J. Am. Chem. Soc. 2008, 130, 3023.