ARTICLES

Multimodal Silica-Shelled Quantum Dots: Direct Intracellular Delivery,Photosensitization, Toxic, and Microcirculation Effects

Rumiana Bakalova,*,† Zhivko Zhelev,† Ichio Aoki,† Kazuto Masamoto,† Milka Mileva,‡ Takayuki Obata,†

Makoto Higuchi,† Veselina Gadjeva,§ and Iwao Kanno†

Molecular Imaging Center, National Institute of Radiological Sciences (NIRS), 4-9-1 Anagawa, Chiba 263-8555, Japan,Department of Medical Physics and Biophysics, Medical University, Sofia, Bulgaria, and Department of Chemistry andBiochemistry, Medical Faculty, Thracian University, Stara Zagora, Bulgaria. Received November 28, 2007;Revised Manuscript Received March 13, 2008

In the present study, we describe a multimodal QD probe with combined fluorescent and paramagnetic properties,based on silica-shelled single QD micelles with incorporated paramagnetic substances [tris(2,2,6,6-tetramethyl-3,5-heptanedionate)/gadolinium] into the micelle and/or silica coat. The probe was characterized with highphotoluminescence quantum yield and good positive MRI contrast, low cytotoxicity, and easy intracellular deliveryin viable cells. The intravenous administration of the probe in experimental animals did not affect significantlythe physiological parameters and microcirculation (e.g., heart rate, blood pressure, diameter and shape of bloodvessels), which makes it appropriate for tracing of blood circulation and in ViVo multimodal imaging usingfluorescent confocal microscopy, two-photon microscopy, and MRI.

INTRODUCTION

The careful design of the architecture of water-soluble QDsenables the creation of nanobioprobes that are compatible withseveral imaging techniques, so-called multimodal imaging. Thisopens the door to the existing prospect of simultaneous imagingof QD probes by a variety of techniques including opticalimaging, magnetic resonance imaging (MRI), positron emissiontomography (PET), computed tomography (CT), and X-rayimaging.

In particular, by fabricating QD probes that exhibit bothfluorescent and paramagnetic properties, optical imaging andMRI can be used in a complementary fashion. This is a powerfulcombination, as the MRI offers an ability to follow thedistribution of molecules in ViVo or provide an anatomicalreference, whereas the optical imaging can be applied to obtaindetailed information at subcellular levels. The potential applica-tions include monitoring tissue implants and studying the real-time dynamics of tumor metastases and biochemical mediatorsthat might be responsible for many diseases.

The use of a single multimodal probe with two or moreimaging techniques has several advantages. The conventional(nonmultimodal) probes, used in PET, MRI, or optical imaging,have different structures and properties, which determine theirdifferent target affinity and pharmacodynamics in the organism.Often, the target can be detected by several imaging techniquesusing several imaging probes with different properties and

pharmacodynamics, so the results cannot be easily comparedand verified. The researchers and clinicians are faced with thedilemma of which technique to choose and which data to acceptas true. The use of a single multimodal probe with two or moreimaging techniques minimizes the artifacts and enables a precisecomparative analysis of the images, obtained by differenttechniques.

The existing prospect of using QDs as multimodal imagingprobes attracts the interest of researchers in many differentscientific fields, and the number of publications on the topichas increased exponentially in recent years (1-21). However,so far, the results are preliminary and interpretations are stillspeculative, especially in terms of in ViVo deep-tissue imaging.

Perhaps the most attractive for development of multimodalimaging probes (optical and paramagnetic) are silica-shelledQDs. The silica is a bioinert material. The silica shell enablesincorporation of a large amount of contrast substances insidethe silica sphere (e.g., CdSe, CdTe, CdS, InAs, or InP QDs andFe2O3, Fe3O4, CoFe2O4, MnFe2O4, FePt, or CoPt3 paramagneticnanoparticles; fluorescent/paramagnetic heterodimers, such asFe3O4-Au, CoPt-Au, FePt-Ag, Fe3O4-Au-PbSe, γ-Fe2O3-CdS,FePt-CdS, Co/CdS core/shell, Co/CdSe core/shell, CdSe/Zn1-x-MnxS; QDs and paramagnetic fatty acids) (3, 7, 13, 15, 16). Inaddition, paramagnetic substances (e.g., chelators for gado-linium, manganese, etc.) could also be attached on the surfaceof silica-coated QDs (5, 15, 18-20). The high fluorescentcontrast (as a result of high quantum yield of QDs) and thehigh paramagnetic contrast (as a result of incorporation of alarge amount of paramagnetic substances in the small volumeof the silica sphere) enable development of a probe that couldbe detected in ViVo in a single manner, simultaneously usingoptical imaging and MRI. In that case, even single cells andmolecular targets could be visualized.

* Rumiana Bakalova, PhD, Department of Biophysics, MolecularImaging Center, National Institute of Radiological Sciences (NIRS),4-9-1 Anagawa, Chiba 263-8555, Japan. Tel.: +81-43-206-4067. Fax:+81-43-206-3276 . E-mail: bakalova@nirs.go.jp.

† NIRS.‡ Medical University, Sofia, Bulgaria.§ Thracian University.

Bioconjugate Chem. 2008, 19, 1135–1142 1135

10.1021/bc700431c CCC: $40.75 2008 American Chemical SocietyPublished on Web 05/22/2008

The present study accents the biocompatibility of multimodalQD probes based on silica-shelled single QD micelles withincorporated gadolinium complexes into the micelle and/or silicacoat. We tried to clarify the applicability of this probe for bloodvessel tracing in the brain, estimating its effect on the majorphysiological characteristics (such as blood pressure, heart rate,and brain microcirculation) after in ViVo intravenous (i.v.)administration.

EXPERIMENTAL PROCEDURES

Synthesis and Characterization of Multimodal QDs. Twomultimodal QD-based probes were synthesized (Figure 1). Thesynthesis of the first probe (Figure 1A) was performed as itwas described in our previous manuscripts (21). The synthesisof the second probe (Figure 1B) was performed according torefs 22, 23 (with slight modifications) (22, 23) Briefly, thegadolinium complex S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tet-raazacyclododecane tetraacetic acid/gadolinium (DOTA-Bn-NCS/gadolinium) was mixed with amino-functionalized QDs(400:1, mol/mol) in 10 mM PBS (pH 8.0). After 1 h incubationat room temperature, the conjugate was purified using dialysis,as described in our previous manuscript (21). DOTA-Bn-NCS/gadolinium complex was purchased from Macrocyclics Co.(USA). The same molar ratio gadolinium complex/QD ) 400:1was used in the synthesis of the first probe. After purification,the gadolinium concentration in the respective probe wasmeasured using 1H-MRI as described below. The structure andsize of nanoparticles were characterized by TEM and DLS. Theirspectral characteristics were measured using fluorescent spec-troscopy, absorbance spectroscopy, and MRI.

Cell Cultures. HeLa cells were cultured in MinimumEssential Medium (MEM, GIBCO, Invitrogen, USA), supple-mented with 10% inactivated FBS (Sanko, Japan). A-549 cells,derived from human lung cancer, Jurkat and K-562 cell lines,derived from human acute lymphoblastic leukemia or chronicmyeloid leukemia, respectively, were cultured in RPMI-1640medium (GIBCO, Invitrogen, USA), supplemented with 10%

inactivated FBS (Sanko, Japan), 100 U/mL penicillin (GIBCO,Invitrogen, USA), and 100 µg/mL streptomycin (GIBCO,Invitrogen, USA). One day before the treatment with QDs, thecells (1 × 105 cells/mL) were dispensed in 100 µL aliquots in96-well plates and cultured overnight in medium without FBSand antibiotics, in humidified atmosphere (5% CO2, 37 °C).

Intracellular Delivery of Multimodal QDs (MRI Measure-ments and Data Analysis). The MRI measurements wereperformed in a 4.7 T magnet (General Electric, CT) or a 7.0 Tmagnet (Kobelco, Japan) interfaced to a Bruker Avance console(Bruker Medical GmbH, Germany). A 30 mm diameter Litzcoil (Doty Scientific Inc., SC, USA) or 70 mm diameter volumecoil (Bruker) was used for measurement of the samples. Thedetails are indicated in the figure captions. The PCR tubes weremounted on a plastic holder for 4.7 T or on a 70 mm diametervolume coil for 7.0 T and set in the center of the coil. Thesample temperature was maintained at room temperature (ap-proximately 22-24 °C). The measurements were performed inthe following order: T1 weighted imaging, using conventionalspin-echo (SE) sequence; multiecho SE imaging for T2 calcu-lations; and inversion-recovery SE imaging for T1 calculations.

Figure 1. Structure of multimodal silica-shelled QD probes. In the firstprobe (A), the gadolinium complex is incorporated into the silica coatand/or hydrophobic micelle around the QD. In the second probe (B),the gadolinium complex is conjugated on the surface of silica sphere.

Figure 2. (A) Transmission electron micrographs of silica-shelled QDmicelles. (B) Retention of gadolinium complex in the silica sphere(probe A) or on the surface of the silica sphere (probe B) afterpurification via dialysis. (C) Fluorescent and paramagnetic character-istics of probes A and B. The probes were purified using 48 h dialysis.Longitudinal relaxation time (T1) and transverse relaxation time (T2)were calculated at equal concentrations of both probes (correspondingto 500 nM QDs). The QD concentration was calculated from theabsorbance spectra of multimodal silica-shelled QDs, using the equationof Yu et al. (38). The MRI measurements were performed in a 4.7 Tmagnet (General Electric, CT) interfaced to a Bruker Avance console(Bruker Medical GmbH, Germany). A 30 mm diameter Litz coil (DotyScientific Inc., SC, USA) was used for measurement of the samples.The sample temperature was maintained at room temperature (∼22-24°C). The measurements were performed in the following order: T1-weighted imaging using conventional spin-echo (SE) sequence;multiecho SE imaging for T2 calculations; and inversion-recovery SEimaging for T1 calculations. For calculation of photoluminescencequantum yield (PL QY), the spectrally integrated emission of particledispersion in different aqueous solutions (distilled water, PBS, or Trisbuffer) was compared with the emission of an ethanol solution ofrhodamine 6G (Fluka) of identical optical density (<0.015) at theexcitation wavelength 365 nm.

1136 Bioconjugate Chem., Vol. 19, No. 6, 2008 Bakalova et al.

T1 weighted imaging: Two dimensional (2D), multislice, T1

weighted images were obtained using a conventional SEsequence with the following parameters: Pulse repetition time(TR) ) 177 or 400 ms, echo time (TE) ) 6.65 ms, matrix size) 64 × 64, slice orientation ) horizontal, field of view (FOV)) 6.4 × 3.2 mm2, slice thickness (ST) ) 3 mm, and numberof acquisitions (NA) ) 1.

Multiecho imaging: 2D multislice multiecho imaging wereperformed to generate T2 maps with the following parameters:TR ) 6000 ms, TE ) 6.65, 13.3, 19.95, 26.6, 33.25, 39.9, 46.55,53.2, 59.85, 66.5, 73.15, 79.8, 86.45, 93.1, 99.75, and 106.4ms, number of echoes ) 16, matrix size ) 64 × 64, sliceorientation ) horizontal, FOV ) 6.4 × 3.2 mm2, ST ) 3 mm,and NA ) 1.

Inversion-recovery: 2D multislice inversion-recovery MRIswere obtained using a spin-echo acquisition for T1 mapcalculation with the following parameters: TR ) 6000 ms, TE

) 6.65 ms, inversion time ) 6.45, 400, 800, 1200, 1600, 2000,3000, 4000 ms, matrix size ) 64 × 64, slice orientation )horizontal, FOV ) 6.4 × 3.2 mm2, ST ) 3 mm, and NA ) 1.

Quantitative T1 maps were calculated by a nonlinear least-squares fitting using inversion-recovery MRIs. In addition,quantitative T2 maps were calculated by a nonlinear least-squaresfitting using multiecho imaging. All calculations and analyseswere performed using MRVision image analysis software(version 1.5.8, MRVision Co., MA) on Solaris (Sun Microsys-tems, Inc., CA) and Macintosh (Mac OS X, version 10.4, AppleComputer, CA). All data are presented as mean ( SD.

Intracellular Delivery of Multimodal QDs (FluorescentConfocal Microscopy). 10 µL of QD probe (50 pmol) wasincubated with 90 µL cancer cells (containing 1 × 105 cells) inmedium, within 30 min or 2 h, in a humidified atmosphere.The cell suspension was washed twice by PBS and analyzedby fluorescence confocal microscopy for intracellular delivery

Figure 3. (A) Fluorescent confocal images of the intracellular delivery of multimodal silica-shelled QD probe in living cultured cells. The cells,dispensed in 96-well glass plates, were incubated with the probe (approximately 500 nM of QDs) within 30 min or 2 h in humidified atmosphere(5% CO2, 37 °C), in the dark. After incubation, the cells were washed three times with PBS and subjected to fluorescent confocal microscopy. (B)Fluorescent confocal images of intracellular delivery of the QD probe in living cells in the absence and in the presence of cytochalasin (5 µg/mL).The cells were incubated with the probe within 2 h in humidified atmosphere (in the dark), washed three times with PBS, and subjected to fluorescenceconfocal microscopy. In both cases, the images were obtained using an Olympus FV1000 microscope (excitation wavelength, 488 nm; emissionfilters, 535 nm for lung cancer cells, 615 nm for HeLa cells; differential interference contrast; section scanning).

Multimodal Silica-Shelled QDs Bioconjugate Chem., Vol. 19, No. 6, 2008 1137

of QDs. An Olympus FV1000 microscope was used in theanalysis (excitation wavelength, 488 nm; emission filters, 535or 615 nm; differential interference contrast; section scanning).

Photosensitization. The cells (90 µL, 2 × 105 cells/mL) wereincubated with QDs (50 pmol in 10 µL) in medium,within 24 h,in humidified atmosphere (5% CO2, 37 °C). The cells werewashed twice with fresh medium and subjected to irradiationwith laser or UV mercury lamp.

The laser irradiation of the cell suspension was carried outcontinuously on a confocal microscope for 30 min, changingthe plane of irradiation in the well area (96-well plate). Thecells were washed twice with PBS, and cell viability wasdetected using flow cytometry.

The UV irradiation was carried out under the experimentalscheme mentioned below, to ensure irradiation with low-intensity UV light without significant heating of the sample andside effects: mercury lampf UV-filter (UTF-34U, Sigma-Koki)(to restrict the light under 340 nm)f quartz platformf plateswith cell suspension. The samples were placed in a fixed positionrelative to the camera to ensure equal spatial distribution of theenergy during irradiation. The irradiation was carried out withthe following regime: 10 min irradiation followed by 10 minbreak, repeated for a total duration of 30 min. Time-dependentmonitoring of the level and spatial distribution of the energyduring UV irradiation was carried out and was found to berelatively constant during the experiment. The temperature ofthe sample was also monitored over time and was found to varyin the interval 22-28 °C. After irradiation, the cells were washedtwice by PBS, and the cell viability was detected by flowcytometry.

In both cases, the control samples contained QD nontreatedcells.

Cytotoxicity Test (Flow Cytometry). The cells (2 × 105

cells/mL) were incubated with QDs (500 nM; λem ) 525nmsgreen fluorescence) in medium, within 24 h, in a humidifiedatmosphere (5% CO2, 37 °C), and after that washed twice byPBS. The dead cells were labeled by propidium iodide (PI),and cell viability was analyzed by flow cytometry (flowcytometer Beckman Coulter-Epics XL). The flow cytometer wasoperated in accordance with the manufacturer’s recommenda-tions after fine adjustments for optimization. The forward- andsite-scatter parameters were adjusted to accommodate theinclusion of both live and dead cells within the acquisition datawith quadrant markers drawn to distinguish PI labeled dead cells(red fluorescence) from PI nonlabeled viable cells. The controlsample contained QD nontreated cells. The viability of QDtreated cells was calculated as the percentage from viability ofnontreated (control) cells. No cells were excluded from theanalysis and ∼5000 cells were counted for each sample. Thedata were collected and analyzed using XL System II software.

Pharmacodynamics. Mice (ICR, ∼20-25 g) were injectedintravenously through the tail vain with 50 pmol (in 100 µL, asingle injection) multimodal silica-shelled QDs. Serial sacrificeswere carried out at the sixth and 24th hour after the injection.Several organs (brain, spleen, liver, lung, kidneys) were isolated,homogenized, and analyzed for QD amount, using NMRspectroscopy.

Two-Photon Imaging of Brain Circulation in ViWo. Theanimal (Sprague-Dawley rats, n ) 4, ∼340 g) was anesthetizedwith isoflurane (4% for induction, 2% for surgery, and 1.4%during the measurements). The tracheal intubation was per-formed, and the femoral vein was catheterized for drugadministration. The skull over the somatosensory cortex (3 mm× 3 mm) was removed, and the dura was kept intact. The bodytemperature was maintained (37 °C), and the systemic arterialblood pressure was periodically monitored from the tail artery.The heartbeat and end tidal gas levels were continuously

monitored. To visualize brain microcirculation, 5% FITC-Dextran(70 kDa) was intravenously (i.v.) injected (0.3 mL/h), andexcited with 900 nm pulse-laser (Maitai HP, Spectral Physics)under a microscope (SP5, Leica) equipped with a 10× objectivelens (0.30 numerical aperture). The vessel diameter was thendetermined by measuring the width of the vessel image projectedacross the 15 z-images (up to ∼0.3 mm from the cortical surface)in each vessel segment. All measured parameters were consid-ered as controls under the conditions mentioned above. Afterthe stoppage of FITC-Dextran infusion, FITC-Dextran waspartially washed from the brain circulation by short i.v. infusionof saline solution. Then, QD was injected (single injection, 0.3mL of 0.5 µM QD concentration) into the rat, and all parameterswere measured as described above.

RESULTS AND DISCUSSION

Structure and Characterization of Multimodal Silica-Shelled QDs. Our multimodal QD probe was composed ofsingle QD micelles incorporated in small silica spheres (15-20nm in diameter) and amphiphilic gadolinium complex [tris(2,2,6,6-tetramethyl-3,5-heptanedionate)/gadolinium, trivial name Re-solve Al-Gd] incorporated into the micelle part and/or silicacoat (Figure 1A). For comparison, we used a second probe,consisting of QDs conjugated with DOTA-Bn-NCS/gadolinium(Figure 1B).

In the first probe, the micelle around the QD allows anincorporation of strongly hydrophobic paramagnetic substancesin the silica sphere. The hydrophobic micelle isolates QDnanocrystal from the polar molecules, penetrating from theenvironment in the silica sphere, and maintains the fluorescentproperties. At the same time, the paramagnetic molecules,incorporated in the silica coat or micelle, are accessible to thewater, which is beneficial for 1H MRI contrast agents and, inparticular, for gadolinium ions. The surface of the silica spherewas amino-functionalized and ready for bioconjugation. Atacidic pH (3.0), the probe (A) had a surface potential of +31mV (detected by zeta-potential titration), which decreased to-37 mV at pH 10.0. At physiological pH (7.4), the zeta potentialwas +6.9 mV.

Both probes consist of a single QD per one silica sphere(Figure 2A), which allows normalization of their concentrationsto the QD concentration in moles and facilitates the dose-dependent analyses in life science research. Both probes stronglyretain about 25-30% of the gadolinium complex added initiallyto the reaction mixture (Figure 2B). The concentration of thegadolinium complex decreased during purification by dialysisup to 10 h. After this time, there were no changes (Figure 2B),which was an indication for a retention of the complex in thesilica coat or on the surface of the silica sphere.

The close distance between QDs and paramagnetic substances(especially paramagnetic nanoparticles) results in a decrease oftheir fluorescent and paramagnetic characteristics in comparisonwith the isolated materials (13, 15). In the present study, wecompared the spectral properties of both multimodal probes,described above (Figure 2C). In probe A, gadolinium ions arein close proximity to QD (Figure 1A), and in probe B,gadolinium ions are far from QD (Figure 1B). The fluorescencequantum yield (QY) of both probes was ∼35-50% in aqueoussolution (depending on QD size). It was ∼1.4 times lower thanthe QY of QDs in organic solvent and the same as the QY ofsilica-shelled QDs without gadolinium. The incorporation ofgadolinium ions into the silica sphere also did not affect theprofile of the absorbance and fluorescent spectra of silica-shelledQDs, as well as their paramagnetic characteristics. Afterpurification, T1 (longitudinal relaxation time) and T2 (transverserelaxation time) of probe (A) were shorter than T1 and T2 ofprobe (B) (Figure 2C). We calculated that the numbers of

1138 Bioconjugate Chem., Vol. 19, No. 6, 2008 Bakalova et al.

chelating molecules attached on or incorporated in one silicasphere were approximately same (Gd/QD ∼ 18:1 and ∼17.6:1,

mol/mol, for probe A and probe B, respectively). This indicatesthat the incorporation of gadolinium in close proximity to QD

Figure 4. T1- and T2-weighted images and quantitative values of samples, containing probe only (A2-A4), cells only (B2-B4), or cells incubatedwith probe (C2-C4). Incubation conditions: Lung cancer cells (2 × 107) were dispensed in plates, containing 20 mL cultured medium. The probewas added to the plates in different concentrations (1 µM, 0.5 µM, or 0.1 µM QDs, respectively). The incubation was carried out during 2 h inhumidified atmosphere (5% CO2, 37 °C). The cells were collected by centrifugation, washed three times by PBS (containing 1% Trypsin), andfinally resuspended in 200 µL PBS. All samples were treated in the same manner. The concentration of the probe in moles was approximate. It wascalculated from the absorbance spectra of QD incorporated into the silica spheres, using the equation of Yu et al. (38). The MRI measurementswere performed in a 7.0 T magnet (Kobelco, Japan) interfaced to a Bruker Avance console (Bruker Medical GmbH, Germany). A 70 mm diametervolume coil (Bruker) was used for measurement of the samples. The sample temperature was maintained at room temperature (∼22-24 °C). Themeasurements were performed as follows: T1-weighted imaging using conventional spin-echo (SE) sequence and inversion-recovery SE imagingfor T1 calculations. The multiecho SE imaging was measured for T2-weighted imaging and T2 calculations.

Multimodal Silica-Shelled QDs Bioconjugate Chem., Vol. 19, No. 6, 2008 1139

does not significantly affect its paramagnetic properties, presum-ably because of the isolation of both materials from thehydrophobic micelle.

All experiments on viable cells and animals were performedusing probe A only.

Direct Intracellular Delivery of Multimodal Silica-ShelledQDs. Figure 3A represents fluorescent images of HeLa and lungcancer cells, labeled with probe A. The amino-functionalized(and positively charged) probe possessed an easy and compara-tively fast intracellular delivery without transfection techniques.It was found that the probe penetrates through the cytoplasmicmembrane via an endocytotic mechanism. The cytochalasin (aninhibitor of endocytosis) suppressed its intracellular delivery(Figure 3B). The probe was localized into the cytoplasm,uniformly distributed. A concentration of the probe in closeproximity to the nuclear membrane was detected, whichindicates an interaction with some intracellular compartments.No fluorescence was detected in the nucleus within 4 hincubation.

The facilitated permeability of silica-shelled QDs allows theirapplication in the detection of intracellular targets (antigens,membrane compartments, subcellular organelles, etc.) in viablecells without transfection techniques. Currently, QDs are appliedpredominantly for labeling of cell surface antigens in Vitro andin ViVo or for extra- and intracellular labeling of fixed cells.Their application for labeling of intracellular components inviable cells requires transfection techniques for penetrationthrough plasmatic membrane. The already published dataindicate that QDs could be delivered in viable mammalian cellsvia four different mechanisms: nonspecific endocytosis/pinocy-tosis facilitated by polycationic transfectants (as lipofectamine);microinjection; electroporation; and peptide-induced transport(24-28). It is widely accepted that the transfection techniquesmaintain the risk of artifacts, some of them are toxic, and theyhave to be replaced with direct approaches for intracellulardelivery of QDs. We observed that an appropriate polymercoating of QDs (such as positively charged silica) could facilitatetheir direct intracellular delivery and random distribution intothe cells (29), which was also reported by other authors (30).

If the cells were washed several times with phosphate-buffered saline, concentrated, and subjected to MRI, a high MRIcontrast was observed on the T1- and T2-weighted images(Figures 4). The T1 and T2 values allow calculation of theapproximated absolute amount of multimodal QDs deliveredinto the cells, using two independent calibration curves. Thecalibration curve represents the relationship between the pureprobe in known concentrations and T1 or T2 values, respectively.It was found that approximately 58-75% of the probe wasdelivered into the cells and/or bound on the cell surface.

Cytotoxicity, Photosensitization, and Pharmacodynamics.All experiments with cultured cells were provided in mediumwithout FBS. The cell viability of A459, Jurkat, K562, and HeLacells was ∼100% after 24 h incubation with QDs in 500 nMconcentration (Table 1). The nanoparticles (500 nM) were also

nontoxic under continuous UV or laser irradiation of the cellsuspension after preliminary incubation with QDs for 24 h. Thecell viability did not change within 30 min of continuous laserirradiation of QD-treated and QD-nontreated (control) cells.Under UV irradiation, the viability of QD-treated and QD-nontreated (control) cells decreased slightly (∼10%) in com-parison with nonirradiated cells, as a result of nonspecific effectsof UV light on cell viability (Table 1).

Several groups have demonstrated photosensitization of livingcells using CdSe or CdTe core QDs (31-35). However, theovercoating (inorganic or organic) usually suppresses or elimi-nates the photosensitization effect (31, 32, 35).

Regarding cytotoxicity of nanoparticles, it is necessary to notethat this parameter is usually clarified on cancer cells that areinherently resistant to many external agents. The data on normalcells are scanty. Currently, there are insufficient data on themetabolism of QDs in live organisms, and it is not clear whatwill happen if the nanoparticles are accumulated in differentorgans for a long time. Their excretion through kidneys avoidingany long-term accumulation and metabolism in the liver, bonemarrow, or other organs seems to be the best possibility. Choiand colleagues have demonstrated that a hydrodynamic diameterof <5.5 nm of organic-coated QDs results in rapid and efficientrenal filtration and urinary excretion from the body. Increasingthe diameter by >15 nm usually prevents renal excretion andincreases the blood half-life by about 300 times and the wholebody half-life by about 700 times (36). The authors also havereported that the purely cationic charge is associated with anincrease in hydrodynamic diameter of the QDs after incubationwith serum.

Our pharmacodynamic study shows that nonPEGylated silica-shelled QDs persist in the blood circulation within an hour afteri.v. injection in mice (half-life ∼20 min). Six hours after theinjection, a comparatively large amount of QDs were detectedin spleen, a very low amount in liver and kidney, and none inlung and brain (data are not shown). A similar result wasobtained 24 h after the injection. Recently, Yang and colleaguesreported that QDs (commercial Qtracker 705) are accumulatedpredominantly in spleen, liver, and kidney after i.v. injectionin mice, and they do not metabolize within 28 h (36).

Effects on Physiological Characteristics and Brain Cir-culation. The intravenous application of the probe in rat didnot affect significantly the physiological parameters, e.g., hartrate, blood pressure, and microcirculation (Figure 5). Themultimodal QDs (300 µL of 1 µM stock solution) were injectedinto the femoral vain. Ten minutes after the injection, the heartrate increased slightly with subsequent normalization to thebaseline level within 40 min. The blood pressure was stablewithin 20 min after the injection and decreased about 10% after80 min (Table 2). The brain vasculature was visualized in thesomatosensory cortex using two-photon microscopy, and thefluorescence was detected up to a 200 µm depth (Figure 5).The probe did not affect the diameter and structure of the bloodvessels, which makes it an appropriate tracer for brain vascu-lature in the study of brain phenomena, e.g., visualization ofneuron/astrocyte/blood vessel structure and investigation ofneurovascular coupling.

In conclusion, the described strategy of the design ofmultimodal QDs allows the development of probes with highfluorescent and paramagnetic contrast characteristics and com-paratively soft effects on cell homeostasis and animal physiol-ogy. The described QD probe is appropriate for in Vitro and inViVo tracing of cells and blood vessels, simultaneously usingfluorescent microscopy and MRI. After conjugation with ap-propriate ligands, the probe could be applied for target-selectivemolecular imaging in Vitro and in ViVo. The easy intracellular

Table 1. Cytotoxicity and Photosensitization of MultimodalSilica-Shelled QDs

treatment HeLa cellsa A549 cellsa Jurkat cellsa K562 cellsa

+ 500 nM QDs 98 +/- 6 95 +/- 9 97 +/- 7 101 +/- 5+ Laser irradiation 96 +/- 8 97 +/- 9 98 +/- 5 98 +/- 8+ 500 nM QDs 95 +/- 4 98 +/- 10 96 +/- 6 96 +/- 8+ Laser irradiation+ UV irradiation 87 +/- 4 89 +/- 5 91 +/- 5 89 +/- 6+ 500 nM QDs 85 +/- 7 84 +/- 8 88 +/- 3 87 +/- 6+ UV irradiation

a Cell viability (% from control). The control samples containnontreated cells. The results are mean +/- SD from four independentexperiments.

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delivery of these silica-shelled QDs allows avoidance ofconventional transfectants that are inherently toxic.

ACKNOWLEDGMENT

The technical assistance of Misao Yoneyama and JeffKershaw (NIRS, Chiba, Japan) in MRI measurements and dataanalysis is gratefully acknowledged. The animal experimentswere approved by the Animal Care and Use Committee ofNIRS-Chiba.

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Figure 5. Two-photon images of brain circulation before and after i.v. injection of multimodal silica-shelled QDs in rat. In the images: (A)FITC-Dextran (0.5 mL of 5 µM concentration) was injected in ∼340 g rat, and the image was obtained after 20 min at λex ) 900 nm and λem )525 nm. (B) FITC-Dextran was washed from the brain vasculature by i.v. infusion of saline solution, and the image was obtained at λex ) 900 nmand λem ) 525 nm. (C) QDs (0.3 mL of 1 µM QD concentration) were injected in the same rat. The image was obtained after 20 min at λex ) 900nm and λem ) 560 nm. Representative images from three independent experiments are shown in the figure.

Table 2. Physiological Characteristics before and After i.v. Injectionof Silica-Shelled QDs in Experimental Animals under Anesthesiaa

parameter before ∼20 min after i.v. injection

blood pressure, (mmHg) 95.3 +/- 7.8 93.5 +/- 16.2heart rate, (BPM) 304 +/- 62 348 +/- 43blood vessel diameterarteries, (µm) 26 +/- 8 constantveins, (µm) 43 +/- 20 constant

a n ) 3 (n ) number of rats in the group); QD concentration ) 80pmol (in 300 L).

Multimodal Silica-Shelled QDs Bioconjugate Chem., Vol. 19, No. 6, 2008 1141

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