This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 4319--4321 4319

Cite this: Chem. Commun.,2013,49, 4319

X-ray excitable luminescent polymer dots doped withan iridium(III) complex†‡

Yasuko Osakada,*ab Guillem Pratx,c Lindsey Hanson,a Paige Elana Solomon,a

Lei Xing*c and Bianxiao Cui*a

In this study, cyclometalated iridium(III) complex-doped polymerdots were synthesized and shown to emit luminescence uponX-ray irradiation, potentially serving as a new probe for molecularimaging during X-ray computed tomography.

X-ray computed tomography (CT) is one of the most widely usedtechniques for medical diagnosis and treatment planning aswell as for small animal imaging research.1 To some extent,contrast agents such as iodinated compounds can be used toincrease the contrast of soft tissue features and providefunctional information such as blood perfusion.2 In addition,metal nanoparticles (NPs), such as gold,3 bismuth4 andtantalum oxide,5 were previously developed as a new generationof absorption contrast agents. These NPs successfully o!er newadvantages, such as contrast enhancement as well as longcirculation time in blood.6 Moreover, surface modification ofthese NPs is possible to target them for specific types of cancer.These NPs are basically designed to increase the contrast viaX-ray absorption. Sensitivity at the molecular level is, however,still limited, because it is sometimes di"cult to discriminateabsorption from targeted probes, tissue and other anatomicalstructures. Recently, we proposed a novel CT imaging metho-dology, called X-ray luminescence computed tomography(XLCT), which o!ers higher molecular sensitivity.7,8 In XLCT,molecular image contrast arises from the conversion ofabsorbed X-rays into luminescent signals, which occursdominantly in injected radioluminescent NPs. As a first proofof concept, we have synthesized lanthanide-doped inorganicnanophosphors, which emit visible light under X-ray

irradiation.9–11 However, inorganic NPs su!er from a numberof drawbacks, such as potential toxicity as well as low biologicalstability. Presently, further instrumental advancement ofimaging as well as development of new probes are necessaryto improve the detection e"ciency of XLCT.

Polymer dots (P-dots) are a novel class of fluorescent organicNPs for biological imaging.12 In particular, it is possible to dopethem with organic or metal compounds to design functionalNPs that have great potential for further biological applica-tions.13,14 On the other hand, some polymers can be used forthe detection of radiation, i.e. plastic scintillators, whichexhibit luminescence upon exposure to ionizing radiation,such as X-rays and g-rays.15 Recently, Campbell and Cronereported that a cyclometalated iridium (Ir(III)) compleximproved the light yield of plastic film scintillators made ofpoly(vinyltoluene) and poly(9-vinylcarbazole) (PVK) upon g-rayirradiation.16 This led us to conceive the possibility of developingPVK P-dots doped with an Ir(III) complex for radioluminescencebio-imaging. In this study, we, for the first time, synthesizednovel cyclometalated Ir(III) complex-doped P-dots and char-acterized their radioluminescence.

Fig. 1 shows our strategy of Ir(III) complex-doped P-dots forradioluminescence imaging. They are designed to contain Ir(III)complexes in PVK P-dots. The PVK polymer functions as thestructural support of the P-dots and possibly as an X-ray

Fig. 1 The design of X-ray induced luminescence of P-dots doped with acyclometalated Ir(III) complex.

a Department of Chemistry, Stanford University, Stanford, CA 94305, USA.E-mail: yosakada@stanford.edu, bcui@stanford.edu; Fax: +1-650-723-4817;Tel: +1-650-723-5177

b PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi,Saitama 332-0012, Japan

c Department of Radiation Oncology, School of Medicine, Stanford University,Stanford, CA 94305, USA. E-mail: lei@stanford.edu

† This article is part of the ‘Emerging Investigators’ themed issue for ChemComm.‡ Electronic supplementary information (ESI) available: Experimental details andFig. S1–S5 and Table S1. See DOI: 10.1039/c2cc37169c

Received 2nd October 2012,Accepted 3rd January 2013

DOI: 10.1039/c2cc37169c

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4320 Chem. Commun., 2013, 49, 4319--4321 This journal is c The Royal Society of Chemistry 2013

absorber as mentioned in the previous film work.16 Polystyrenegraft ethylene oxide functionalized with carboxylic end group(PEG–COOH) is also employed and embedded in P-dots tomake them biocompatible and water soluble.12 Ir(III) complex-doped P-dots were synthesized by coprecipitation methods aspreviously described.14 In brief, PVK and PEG–COOH polymersand the Ir(III) complex were first dissolved in tetrahydrofuran(THF), and the THF solution was then added into water tosynthesize P-dots. The THF was then evaporated to make theaqueous solution of P-dots. We note that the Ir(III) metalcomplex used in this study itself is neutral and not watersoluble. To confirm the formation of NPs, we conductedtransmission electron microscopy (TEM) measurements. Weclearly observed the NPs as shown in Fig. S1 (ESI‡). It is worthnoting that Ir(III) complex-doped P-dots gave better contrastthan the non-doped PVK P-dots under TEM, further supportingthat the metal Ir(III) complex is incorporated into the P-dots.The size of the P-dots estimated by TEM shows a narrowdistribution of P-dots with mean values of 26 nm (non-dopedPVK P-dots) and 35 nm (Ir(III) complex-doped PVK P-dots,500 mg ml!1), respectively (Fig. S1, ESI‡). The hydrodynamicdiameter was also measured (Fig. S1, ESI‡), showing larger sizesthan those estimated by TEM, attributed to the outer PEG layer.

First, we characterized the steady state photo-physicalproperties of Ir(III) complex-doped P-dots. Fig. 2a shows thesteady state absorption and emission spectra of P-dots atambient temperature under aerobic conditions. The emissionmaximum for Ir(III) complex-doped P-dots was observed around510 nm. Concomitant to the increase of the doping ratio inP-dots, the intensity of emission from the P-dots linearlyincreased (Fig. 2a and b). Next, we measured the emissionspectra of Ir(III) doped P-dots (500 mg ml!1 in THF solution forthe synthesis) in water and the Ir(III) monomer in toluene underaerobic conditions with multiple concentrations to comparethe relative integrated emission intensity (Fig. 2c). Interest-ingly, the relative integrated emission intensity of the Ir(III)complex-doped P-dots is dramatically enhanced, by 500% withrespect to the monomer of the cyclometalated Ir(III) complex ina toluene solution under aerobic conditions (Fig. 2c). Therelative integrated emission intensity of the Ir(III) monomer isenhanced under N2 (data not shown). On the other hand, therelative quantum yield of luminescence for the Ir(III) complex-doped P-dots is almost the same under aerobic conditions asthat under de-aerated conditions (Fig. S2, ESI‡). To characterizethe luminescence of Ir(III) complex-doped P-dots in detail, theluminescence lifetime (t) was also measured. t for the Ir(III)complex-doped P-dots in water was approximately 3-timeslonger than that of a monomer in a sample in toluene underaerobic conditions (Fig. 2d and e and Table S1, ESI‡). t for theIr(III) complex-doped P-dots was almost the same under aerobicconditions as that under de-aerated conditions. These dataconfirm that the enhanced integrated emission intensity ofIr(III) in P-dots results at least partly from the prevention ofluminescence quenching by oxygen molecules.17

Next, we performed X-ray imaging experiments to examineX-ray induced luminescence. To characterize X-ray luminescencefrom Ir(III) complex-doped P-dots, samples were irradiated with

X-ray (50 kVp and 30 mA), and their luminescence was imagedwith an EM-CCD camera under aerobic conditions.11 The samplewas placed in an eppendorf tube and luminescence was imagedupon X-ray irradiation. The detailed conditions, the photographof the experimental setup and a schematic view of X-ray experi-ments are shown in the experimental section and in Fig. S3(ESI‡), respectively. Fig. 3a shows a representative image of X-rayinduced luminescence. We clearly observed the enhancedluminescence of X-ray irradiated Ir(III) complex-doped P-dots(Fig. 3a #3). Next, we quantified the radioluminescence signalfrom each image. We note that the background signal (approxi-mately 80% of H2O reference) is mainly originated from theeppendorf tube and it was subtracted to estimate the yield ofradioluminescence (Fig. S4, ESI‡). The relative yields of radiolu-minescence are shown in Fig. 3b. The radioluminescence signalfrom Ir(III) doped P-dots was 8-times higher than that fromwater.18,19 The PVK P-dots without doping of Ir(III) had lessluminescence than that of doped P-dots. As control experiments,

Fig. 2 (a) UV-vis absorption spectra, and emission spectra of P-dots excited at400 nm. Samples were doped with the Ir(III) complex (0 (black), 200 (red), 300(green), 400 (blue) mg ml!1 in THF solution for the synthesis). (b) Luminescencedepending on the doping ratio of the Ir(III) complex. The integrated emissionintensity is plotted against the doping amount of Ir(III) complex in P-dots.(c) Magnitude of the integrated emission intensity, excited at 400 nm, againstthe absorbance of the solution for P-dots (triangle) and the Ir(III) monomer intoluene (square) under aerobic conditions. (d, e) Luminescence lifetime measurementsfor (d) P-dots in water under aerobic conditions (black line) and in a de-aerated sample(red line), and (e) the Ir(III) monomer in toluene under aerobic conditions (black line),and in a de-aerated sample (red line). The decay is fit by a single exponential curveshown in green. The represented profiles are obtained from the average of threemeasurements.

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X-ray luminescence images were taken for each component ofP-dots in THF (PVK, Ir(III) monomer, PEG (Fig. S5, ESI‡), all ofthem (Fig. 3a and b, #5) dissolved in THF). We could not observeany significant enhancement of the contrast in THF samples(Fig. 3a and b and Fig. S5, ESI‡). These results clearly confirmthat Ir(III) complex-doped P-dots are excitable by X-rays to inducemore luminescence. To confirm that this emission arises fromthe Ir(III) complex, the emission spectrum was measured using amonochromator (Fig. 3c). The emission maximum of Ir(III)complex-doped P-dots upon X-ray irradiation was around530 nm, which is only slightly red-shifted from that of the steadystate measurement as shown in Fig. 2a. This result suggests thatthe emission upon X-ray irradiation arises from the Ir(III)complex.

Here, we successfully designed and synthesized oxygen-independent luminescent Ir(III) complex-doped P-dots.17 Asmentioned in the earlier film work, Ir(III) complexes are amongthe most promising compounds for plastic scintillators due tothe high electron density of Ir(III) (atomic number of 77).16

Although the specific mechanism of X-ray induced emission isstill unclear, we speculate the following two mechanisms:direct and indirect excitation of the Ir(III) complex. With directexcitation, the Ir(III) complex is excited via X-ray irradiation,

thus emitting visible light. In the indirect mechanism, wespeculate that the excitons formed in P-dots recombinetogether to generate a triplet excited state of polymers, andthen, energy is transferred to the Ir(III) complex to emit light.Kinetic measurements will prove the detailed mechanisms tounderstand and improve X-ray induced luminescencee"ciency.20

In this study, we synthesized and characterized novel Ir(III)complex-doped P-dots. The Ir(III) complex-doped P-dots in waterare 5-times brighter than that of monomer cyclometalated Ir(III)in organic solvent under aerobic conditions. More importantly,under X-ray irradiation, we observed luminescence from Ir(III)complex-doped P-dots. Taken together, the present datademonstrate that Ir(III) complex-doped P-dots have a potentialuse in XLCT imaging. Future work will be directed towardimproving the photo-physical properties, as well as using P-dotsfor in vivo study, in particular, combining them with anatomicalCT imaging to improve XLCT.

We thank Prof. Eric Kool and Dr Armando Ricardo Hernandezfor providing the apparatus for lifetime decay measurements. Thiswork was supported by JST PRESTO program to YO.

Notes and references1 R. Weissleder, Nat. Rev. Cancer, 2002, 2, 11–19.2 S.-B. Yu and A. D. Watson, Chem. Rev., 1999, 99, 2353–2377.3 R. Popovtzer, A. Agrawal, N. A. Kotov, A. Popovtzer, J. Balter,

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W. K. Moon, S. H. Choi and T. Hyeon, J. Am. Chem. Soc., 2011, 133,5508–5515.

6 F. M. Kievit and M. Zhang, Adv. Mater., 2011, 23, H217–H247.7 G. Pratx, C. M. Carpenter, C. Sun, R. P. Rao and L. Xing, Opt. Lett.,

2010, 35, 3345–3347.8 A. Ale, V. Ermolayev, E. Herzog, C. Cohrs, M. Hrabe de Angelis and

V. Ntziachristos, Nat. Methods, 2012, 9, 615–620.9 I. N. Stanton, J. A. Ayres and M. J. Therien, Dalton Trans., 2012, 41,

11576–11578.10 B. Henke, S. Schweizer, J. A. Johnson and D. T. Keane, J. Synchrotron

Radiat., 2007, 14, 252–256.11 C. Sun, G. Pratx, M. Carpenter Colin, H. Liu, Z. Cheng, S. Gambhir

Sanjiv and L. Xing, Adv. Mater., 2011, 23, H195–H199.12 C. Wu, T. Schneider, M. Zeigler, J. Yu, P. G. Schiro, D. R. Burnham,

J. D. McNeill and D. T. Chiu, J. Am. Chem. Soc., 2010, 132,15410–15417.

13 C. Wu, Y. Jin, T. Schneider, D. R. Burnham, P. B. Smith andD. T. Chiu, Angew. Chem., Int. Ed., 2010, 49, 9436–9440.

14 Y. Osakada, L. Hanson and B. Cui, Chem. Commun., 2012, 48,3285–3287.

15 G. F. Knoll, Radiation Detection and Measurement, Third edn, 1999.16 I. H. Campbell and B. K. Crone, Appl. Phys. Lett., 2007, 90, 012117.17 S. Zanarini, E. Rampazzo, S. Bonacchi, R. Juris, M. Marcaccio,

M. Montalti, F. Paolucci and L. Prodi, J. Am. Chem. Soc., 2009,131, 14208–14209.

18 M. D. Tarasov, S. L. El’yash, V. F. Goncharova, O. N. Petrushin,Y. A. Savel’ev, M. Y. Tarakanov and Y. S. Shigaev, Instrum. Exp. Tech.,2007, 50, 761–763.

19 T. I. Quickenden and H. S. S. Que, Radiat. Res., 1971, 46, 28–35.20 H. Odaka, T. Miura, K. Hatanaka, S. Wiebel and H. Fukumura,

J. Phys. Chem. C, 2009, 113, 11969–11974.

Fig. 3 (a) Representative image of X-ray induced luminescence (top) and brightfield image (bottom). 1; water, 2; non-doped P-dots, 3; Ir(III) complex-dopedP-dots (500 mg ml!1), 4; THF, 5; Ir(III) and polymers dissolved in THF (the sameamount as P-dots, 500 mg ml!1). (b) The relative radio-luminescence yield ofsamples shown in (a). (c) Luminescence spectrum of P-dots upon X-ray irradiation.

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