Cytotoxicity

DOI: 10.1002/smll.200600218

Evaluation of Quantum Dot CytotoxicityBased on Intracellular Uptake**

Emmanuel Chang, Nadhi Thekkek, William W. Yu,Vicki L. Colvin, and Rebekah Drezek*

Advances in nanomaterials have led to promising candi-dates for many biological applications in research and medi-cine. Their novel physicochemical properties, attributable totheir small size, chemical composition, surface structure, sol-ubility, and shape, have been increasingly utilized in medi-cine for purposes of diagnosis, imaging, and drug delivery.Applications range from the fluorescent tracking of cells[1–3]

and immunostaining assays[4] to magnetic resonance imag-ing.[5] Given the potential for widespread application andcommercialization, nanomaterials will be increasingly uti-ACHTUNGTRENNUNGlized for future biological applications.[6] Quantum dots(QDs) are an example of a nanomaterial that possesses op-tical properties ideal for biological imaging, which makesthem a useful alternative to fluorescent dyes. The organicfluorophores currently used are vulnerable to chemical andmetabolic degradation and are easily photobleached, whichlimits long-term cellular tracking. QDs offer advantages,such as bright photoluminescence, narrow emission, broadUV excitation, and high photostability,[7–9] to help overcomecurrent optical-imaging limitations.

Due to the tremendous focus on developing nanoparti-cles for imaging and therapeutic applications, there hasbeen increasing interest in evaluating the toxicity of nano-materials,[10] in particular, quantum dots. Some studies sug-gest that nanomaterials are not inherently benign and affectbiological systems at the cellular, subcellular, and proteinlevels.[11–15] Akerman et al. demonstrated that some nanopar-ticulates are cleared from the circulation of live mice by themacrophages of the reticuloendothelial system in the liverand spleen.[16] Nevertheless, concerns have surfaced regard-ing the toxicity of QDs, in particular, those nanoparticlesthat are cadmium-containing and thus toxic to both cell cul-

tures and live animals. Previous in vitro labeling experi-ments have demonstrated that QD labeling[17] could occurwithout significant toxicity as long as the QDs are well-coated to be biologically inert.[1, 18,19]

Notwithstanding these results, recent work has demon-strated several significant parameters that identify the toxic-ity of nanoparticles, in particular, QDs. Specifically, the tox-icity of QDs is due to their inherent chemical composition,size, shape, and surface.[14,15,17,20,21] In fact, one of the pri-mary conditions governing cytotoxicity is the degree andstability of the QD surface coating that makes these inor-ganic nanoparticles water-soluble. Several synthesis, storage,and coating strategies have been developed for QDs inorder to ensure stability of these nanoparticles and mini-mize toxicity.[22–27] Derfus et al. examined several parametersof the toxicity of QDs water-solubilized with mercaptoaceticacid.[17] By exposing QDs to prolonged oxidative environ-ments, such as air or photo-oxidation with UV light, theyobserved leaching of Cd2+ from QDs by a blue-shift in theabsorbance and fluorescence spectra due to the decreasingsize of the nanoparticle. Furthermore, they demonstratedthat correct capping and improved surface coating of quan-tum dots can minimize cytotoxicity arising due to air andphoto-oxidation.[17] Hoshino et al. demonstrated that toxicityof QDs is not dependent on the nanocrystal itself but ratheron the surface molecules.[28] Based on this, they suggestedthat it was important to evaluate how surface moleculesaffect the cytotoxicity of QDs. These studies examined howdifferent surface molecules improved the biocompatibilityof QDs. More recently, studies have shown that the surfacemolecules on nanoparticles significantly affect the degree ofnanoparticle uptake into cells by endocytosis.[29–31] In light ofthese new studies, it is important to distinguish whether cel-lular toxicity arises from the interaction of QD surface mol-ecules with cell membranes or from the intracellular uptakeof endocytosed QDs, which is influenced by surface mole-cules on these nanoparticles.

During endocytosis of nanoparticles, the vesicles encap-sulating the nanoparticles are called endosomes. These en-dosomes are then trafficked to various cellular compart-ments. Nanoparticulates that cannot be utilized by the cellare trafficked to acidic (�pH5) and oxidative environmentsof lysosomes and peroxisomes for degradation.[32] Since thisintracellular environment has been shown to degrade cer-tain nanoparticles such as QDs,[17] it would be reasonable toexpect that for nanoparticles that degrade into toxic compo-nents, intracellular nanoparticle concentration affects cell vi-ability. Therefore, the improved biocompatibility that cer-tain surface coatings provide could arise from decreased cel-lular uptake of these nanoparticles for cellular degradation.

So far, all previous studies have only examined the cyto-toxicity of QDs based on extracellular nanoparticle expo-sure concentrations. Since size and surface coatings havebeen demonstrated to influence the intracellular uptake ofnanoparticles by endocytosis into cells,[14, 20,29,30,33] we hy-pothesize that the degree of nanoparticle uptake into cells(intracellular exposure to nanoparticles) is a significantfactor in determining inherent nanoparticle toxicity insteadof only evaluating extracellular exposure concentrations.

[*] E. Chang, N. Thekkek, Prof. R. DrezekRice UniversityDepartment of Bioengineering MS-142PO Box 1892, Houston, TX 77251-1892 (USA)Fax: (+1)713-348-5877E-mail: drezek@rice.edu

Dr. W.W. Yu, Prof. V. L. ColvinRice UniversityDepartment of Chemistry MS-60PO Box 1892, Houston, TX 77251-1892 (USA)

[**] The authors would like to thank Diego Diaz and Christie Sayesfor assistance with the use of the equipment. This work is sup-ported in part by the Nanoscale Science and Engineering Initia-tive of the National Science Foundation under NSF AwardNumber EEC-0118007.

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Therefore, in this study, we characterize the cytotoxicity ofvarious surface-coated CdSe/CdS (core/shell) QDs based onboth extracellular and intracellular nanoparticle exposurelevels in cells and provide evidence that the number ofnanoparticles taken up into cells (intracellular exposure)plays a significant role in determining cell toxicity. Whileprevious work has studied the cytotoxicity of extracellularexposure to CdSe,[17,21] CdSe/ZnS,[17,21] and CdTe QDs,[20]

here we examine CdSe/CdS-based QDs. This is the first re-ported cytotoxicity study to demonstrate the significance ofevaluating intracellular levels of nanoparticles, in particular,with nanoparticles that potentially degrade to release toxicmaterials in low-pH and highly oxidative environmentsfrom cellular degradative mechanisms. We correlate cellulartoxicity with the intracellular exposure of different surface-modified QDs and demonstrate that the improved biocom-patibility of surface coatings arises due to the decreased cel-lular uptake of QDs via endocytosis. By understanding theparameters that govern the toxicity of nanoparticles to cells,progress can be made to improve synthesis, processing, andsurface-coating strategies, thus minimizing the potential fornanoparticle toxicity in biological applications.

The QD surface coatings used were nonPEG-substituted(bare), or were 750 and 6000 weight-average molecularweight (MW) PEG substituted, to evaluate varying levels oftoxicity for different surface-modified QDs (PEG: poly(eth-ylene glycol). Figure 1 illustrates that QD-surface modifica-

tion does play a significant role in cell cytotoxicity as previ-ously observed. In addition, increasing cytotoxicity withhigher QD concentration for all three surface-modifiedQDs was observed, with nonPEG-substituted QDs exhibit-ing the highest cell toxicity. PEG substitution of QDs result-ed in decreased cell death, as reported in previous stud-ies.[17]

Several groups have implied that the cytotoxicity ofnanoparticles is based on a surface interaction with the cellmembrane, which leads to cell toxicity. However, since vari-ous surface coatings dramatically affect the degree of cellendocytosis of nanoparticles, it is difficult to assess whether

or not nanoparticle cytotoxicity is due to cell-membrane–nanoparticle-surface interactions or the intracellular break-down of endocytosed nanoparticles. To elucidate whether ornot intracellular breakdown of nanoparticles plays the keyrole in governing cell toxicity instead of cell-membrane–sur-face interactions, cells exposed to QDs were incubated at4 8C to inhibit cell endocytosis of the QDs.[34, 35] We observeda significant decrease in the intracellular uptake of QDs at4 8C using methods previously described[29] (Figure 2). One

would expect that if nanoparticle cytotoxicity is mediatedby cell-membrane–surface interactions with nanoparticles,the pattern of cytotoxicity observed at 4 8C would be similarto that at 37 8C. The values may have decreased due toslowed biochemical reactions at 4 8C. Interestingly, when in-cubating QDs with cells at 4 8C, no difference in cytotoxicitywas observed for various surface-modified QDs (Figure 3).However, minimal toxicity does exist at increased concen-tration, which suggests that the interaction of nanoparticleswith cell membranes does induce some toxicity. This obser-

Figure 1. Cytotoxicity of bare (*) and PEG-substituted (&: 750 Mw;� : 6000 Mw) surface-modified QDs versus QD-exposure concentra-tion, incubated at 37 8C. Cells were exposed to QDs for 4 h prior tolive/dead assay for cytotoxicity analysis. Cytotoxicity increased withincreasing extracellular QD concentration (sample size n=4).

Figure 2. Decreased intracellular uptake of QDs by endocytosis, incu-bated for 4 h at 4 8C (black columns) compared to 37 8C (hashed col-umns). Cellular-endocytosis mechanisms were inhibited when incu-bated at 4 8C. Intracellular QD levels were quantified based on QDphotoluminescence with background subtraction of cellular auto-fluorescence. The QD concentration in the incubation solution was25 nm (n=3).

Figure 3. Cytotoxicity of bare (^)and PEG-substituted (&: 750 Mw;� : 6000 Mw) surface-modified QDs versus. QD-exposure concentra-tion, after incubation at 4 8C. Similar cytotoxicity profiles for differentsurface-modified QDs were present at 4 8C, whereas significant differ-ences existed when incubated at 37 8C (compare with Figure 1). Cellswere exposed to QDs for 4 h prior to live/dead assay. Cytotoxicityincreased with increasing extracellular QD concentration (n=4).

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vation strongly suggests that endocytosis of nanoparticles,which is inhibited at 4 8C, has a significant impact on overallcell toxicity, most likely arising from the intracellular break-down of nanoparticles in lysosomes. The QDs were exposedto acidic (pH5) and highly oxidative environments (10 mm

H2O2) to simulate the biological degradation within lyso-somes and peroxisomes; a blue-shift was observed, whichdemonstrates QD degradation, in agreement with the find-ings of Derfus et al.[17] (Figure 4). This also provides a rea-sonable explanation as to why Lovric et al. observed thatpretreatment of cells with antioxidants significantly reducedQD-induced cell death.[20]

The cytotoxicity of surface-modified QDs, examined forextracellular exposure at different concentrations, wasfound to increase in the following order: 6000-Mw <

750-Mw!bare QDs. Previous studies have also shown thatcellular QD uptake by endocytosis increases in the follow-ing order: 6000-Mw<750Mw!bare QDs.[29] Since we foundthat endocytosis of QDs significantly alters cytotoxicity andstudies have demonstrated how QD surface coatings signifi-cantly affect the endocytic uptake of cells, we examined thecytotoxicity of QDs based on intracellular exposure for vari-ous surface-modified QDs. These results are shown in Fig-ures 5 and 6 and illustrate the correlation between intracel-lular levels of QDs and cell toxicity. Accounting for surface-

coating effects on the cellular uptake of QDs, the averagenumber of QDs taken up per cell was the same for differentsurface-modified QDs, obtained by incubation at appropri-ate concentrations. The results show that the exposure con-centrations that led to equivalent QD uptake in cells for dif-ferent surface-modified QDs resulted in no statistical differ-ence in cytotoxicity for bare, 750-Mw, or 6000-Mw QDs(Figure 6). Interestingly, the cytotoxicities at 25, 50, 75, 100,and 150 attomol of intracellular QDs per cell were similarfor all three surface-modified QDs, despite extracellular-tox-icity studies revealing different cytotoxicities for each sur-face-modified QD. Studies based solely on extracellular-ex-posure concentrations would have concluded that the 6000-Mw QDs were the least cytotoxic; however, intracellular-ex-posure studies demonstrated that all surface-modified QDsare equally cytotoxic when intracellular-exposure levels arecompared.

The results here agree with published studies[17,20,21] thatindicate that various surface modifications can improve thebiocompatibility of QDs. Interestingly, the results of thiswork strongly suggest that improved biocompatibility arisesfrom the minimized intracellular uptake of QDs by endocy-tosis. This uptake is influenced significantly by surface-coat-ing modifications. While QDs demonstrate minimal cytotox-icity at extracellular concentrations typical of most molecu-lar-imaging experiments (5–20 nm), high intracellular levelsof QDs do exhibit significant cytotoxicity. Therefore, as newnanoparticles that possess useful physical and chemicalproperties are developed, understanding the parametersthat control biocompatibility, such as composition, intracel-lular uptake, and degradation, is important for designingnanoparticles used in biological applications. In addition,since cell types have varying degrees of hardiness and resis-tance to cytotoxic environments, each biological applicationneeds to be individually evaluated.[36]

In conclusion, quantum dots have generated tremendousinterest in terms of biological applications; however, con-cern has also been raised over toxicity due to their chemicalcomposition. For that reason, this study suggests that, for in

Figure 4. Degradation of QDs after exposure to an acidic and oxida-tive environment at room temperature in the dark. Legend: no degra-dationc; 4-ha, and 24-hg, degradation. A) QD degradationdemonstrated by a blue-shift in the first quantum-confinement peakabsorbance (in water). B) The decrease in QD size due to degrada-tion is consistent with a slight blue-shift of fluorescence peak aswell as loss in the quantum yield. A blue-shift of �1.5 nm wasobserved after a 4-h exposure to pH 5.0 and 10 mm H2O2 at roomtemperature.

Figure 5. Images of the live/dead cytotoxicity stain on SK-BR-3 cells.Cells were exposed to yield the same number of intracellular QDs(50 attomolcell�1) for (left to right) nonPEG-substituted and 750- and6000-Mw PEG-substituted QDs.

Figure 6. Cytotoxicity evaluation based on the same number of intra-cellular QDs for nonPEG-substituted, 750- and 6000-Mw PEG-substi-tuted QDs (black, dark gray, and light gray, respectively) on SK-BR-3cells (n=4). QDs were uptaken into cells by endocytosis during a4-h incubation, and intracellular levels were quantified by photolumi-nescence measurements. Cytotoxicity was determined using a com-mercial live/dead assay.

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vitro studies, the cytotoxicity of QDs correlates with the in-tracellular levels of QDs rather than extracellular levels andthis toxicity most likely arises from the breakdown of endo-cytosed QDs by cellular degradative mechanisms. Therefore,it has been demonstrated that the improved biocompatibil-ity of QDs due to PEG-substitution is a result of the mini-mized endocytic uptake of QDs. This observed differencebetween intracellular and extracellular trends is importantin evaluating the toxicity of other nanomaterials that poten-tially degrade within cells. By demonstrating how nanomate-rial surface coatings can influence cell toxicity, these find-ings serve to suggest an additional parameter to be consid-ered in the design and evaluation of biocompatible nanopar-ticles for future biological applications.

Experimental Section

Synthesis of water-soluble quantum dots: The CdSe/CdS(core/shell) QDs were synthesized as previously de-scribed.[29,37–40] The target emission wavelength of the QDs wasselected to be >600 nm in order to minimize spectral overlapwith cellular autofluorescence. An amphiphilic polymer, poly(ma-leic anhydride-alt-1-octadecene) (PMAO, number-average molec-ular weight Mn=30000–50000, Aldrich) was used as thephase-transfer reagent to transfer QDs into water.[40] NH2-termi-nated poly(ethylene glycol) methyl esters (NH2-PEGs, Mw=750and 6000, bonded to PMAO through the reaction between NH2and anhydride groups) were used to increase the water solubilityand stability of the QDs. The above-mentioned synthesized mon-odispersed QDs (purified and dispersed in chloroform) and theamphiphilic polymer (PMAO or PMAO-PEGs) were mixed inchloroform and stirred overnight (room temperature). Pure waterwas then added to the chloroform solution of the complexeswith a 1:1 volume ratio; chloroform was then gradually removedby rotary evaporation at room temperature, resulting in clear,colorful, water-soluble QDs. Ultracentrifugation (Beckman CoulterOptima L-80XP) was applied to concentrate and purify (i.e.,remove excess amphiphilic polymers) the water-soluble QDs(typically at 300000 g for 2 h).

The resulting synthesized QDs were carboxylate terminated(from reactions of amino acid or water with anhydride groups)with no PEG coating (nonPEG-substituted or bare QDs), a 750-Mw

PEG coating, or a 6000-Mw PEG coating. The final water-solubleQDs were free of Cd2+ ions in solution and had a peak emissionmeasured at 620 nm. QD concentration was determined by ab-sorbance using previously determined extinction coefficients.[41]

Transmission electron microscopy (TEM) showed the QDs to bemonodispersed (<10%) elongated rodlike particles, with shortand long axes of 6 and 9 nm, respectively. Quantum-yield (QY)measurements with rhodamine 640 resulted in a QY of 30% forbare QDs, and 43 and 53% for 750- and 6000-Mw PEG-substitut-ed QDs, respectively. The QDs demonstrated high photostabilitywith negligible photobleaching or shift in emission wavelengtheven after one hour of continuous 100-W long-wave ultraviolet ir-radiation or exposure to an intracellular physiologic pH range of5–8. In addition, the hydrodynamic diameter (HD) of the QDswas also determined by size-exclusion chromatography using ahydroxylated polymethacrylate-based gel and an Agilent high-

performance liquid chromatograph (1100 series) equipped witha diode array and UV/Vis and differential refractive-index detec-tors.[42,43] The results were a HD of 25 nm for bare QDs, and 30and 37 nm for 750- and 6000-Mw PEG-substituted QDs, respec-tively.

Cell culture: The human breast-cancer cell line SK-BR-3 wasordered from American Type Culture Collection (ATCC, Manassas,VA). Cells were plated at a seeding density of 70000 cellscm�2

(22500 cellswell�1) onto 96-well culture plates in McCoy’s 5Amedium with 10% (v/v) fetal bovine serum (Invitrogen Corp.,Carlsbad, CA) for 18 h (37 8C, 5% CO2) to allow cells to attach.The cells underwent a 4-h exposure to the corresponding QD in-cubation concentration that would yield the same intracellularuptake of QDs in SK-BR-3 cells for various surface-coated QDs.

Quantum-dot exposure: QDs were added to the full-growthmedium of confluent SK-BR-3 cells to yield final QD concentra-tions of 10, 25, 50, 100, and 150 nm. Three different surface-modified QDs were examined: bare, and 750- and 6000-Mw PEG-substituted QDs. Cells were incubated for 4 h at both 37 8C and4 8C in darkness to observe the effects of QD cytotoxicity.

Viability assay: The cells were gently washed with phos-phate-buffered saline (PBS) to avoid losing any detached cells,and stained using the live/dead reagent [4 mm ethidium homodi-mer-1 (EthD-1) and 2 mm calcein AM] (Molecular Probes, Eugene,OR). This assay has been utilized to quantitate apoptotic celldeath and cell-mediated cytotoxicity. It identifies live versusdead cells on the basis of membrane integrity and esterase ac-tivity. EthD-1 is excluded by the intact plasma membrane of livecells. Wells in which SK-BR-3 cells were cultured without expo-sure to QDs served as the positive (live) control. For the negative(dead) control, SK-BR-3 cells were exposed to a 70% (v/v) meth-anol solution for 30 min prior to addition of the live/dead re-agent. Due to the time-sensitive response of the assay, eachsample group has a live and dead control that was simulta-ACHTUNGTRENNUNGneously dosed with live/dead reagent using an 8-well multichan-nel pipettor to minimize sample variability. After a 45-min incu-bation at room temperature, the cytotoxicity was quantified byrecording fluorescence via a plate reader (SpectraMax M2 Micro-plate Reader, Molecular Devices Corp., Sunnyvale, CA) set to494/517 nm (excitation/emission) for calcein AM (live cells) and528/617 nm for EthD-1 (dead cells). Background subtractionwas performed to remove any autofluorescence from the cellsand QDs. After measurement, the fluorescence of each samplewell was normalized to that obtained from the positive and neg-ative controls. After quantification, representative wells wereimaged via fluorescence microscopy with a 10� objective lens(Zeiss 510 Meta with appropriate filter settings).

Intracellular QD quantification : The QD solution was removedand the cells were washed twice with full-growth media toremove any extracellular QDs. The cells were trypsinized and re-suspended in chilled PBS. The cell concentration was deter-mined by standard hemocytometry. The cells were then trans-ACHTUNGTRENNUNGferred into a quartz cuvette (Starna Cells, Atascadero, CA) at aconcentration of 1.5�106 cellsmL�1 for photoluminescence mea-ACHTUNGTRENNUNGsurements. Prior measurements demonstrated that SK-BR-3 cellsuspensions containing concentrations of fewer than 6�106 cellsmL�1 were in the instrument’s linear range of photolu-minescence for the measurement conditions. The luminescenceper cell was calculated by dividing the measured luminescence

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by the number of cells in the excitation volume of the cuvette.All measurements were performed at room temperature with a500-mL quartz cuvette (Starna Cells Inc., Atascadero, CA) on aHoriba Jobin Yvon SPEX FL3-22 fluorimeter (Edison, NJ) with dualexcitation and emission monochromators. QDs were excited at awavelength of 360 nm and photoluminescence was measuredfrom 500–700 nm. Excitation/emission bandpass slits and theintegration time were set on the fluorimeter to 5 nm/5 nm and150 ms, respectively. All values were normalized over time to arhodamine 6G standard to avoid any artifacts that could arisefrom possible lamp fluctuations. Control samples (SK-BR-3 cellswithout QDs) were used to subtract any cellular autofluores-cence. The area under the curve from 600–640 nm was calculat-ed for QD luminescence for each sample. Reference-QD calibra-tion standards in PBS were made at various concentrations(250 pm, 500 pm, 1 nm, and 2 nm) obtained by serial dilution.Changes to PBS composition resulted in no significant change inQD luminescence. All standard measurements fitted a linear re-gression curve of R2=0.98 or better for use as calibration inten-sity curves. Due to small differences in the QY of each QDsample, calibration curves were made for bare, and 750 and6000 Mw PEG-substituted QDs in order to determine the meannumber of QDs per cell. This is reported because photolumines-cence measurements were taken from a cell suspension of 1.5�106 cellsmL�1.

QD intracellular correlation study : A second study comparedQD cytotoxicity with the degree of QD uptake in the SK-BR-3cells. Continuing previously published work on the intracellularuptake of different surface-coated QDs,[29] the following equa-tions governing endocytic uptake of quantum dots in SK-BR-3cells were used to determine the appropriate QD concentrationfor incubation solutions to attain the desired intracellular levelof QDs: bare QDs, y=5.3676 x; 750-Mw QDs, y=1.1696 x, and6000-Mw QDs, y=0.8929 x, where y represents the averagenumber of QDs per cell (attomolcell�1) taken up during endocy-tosis, and x represents the QD incubation concentration (nm)after 4 h. The intracellular number of quantum dots per cell wasmeasured and verified using previously reported methods.[29]

The cytotoxicity of surface-modified QDs (bare, and 750- and6000-Mw PEG-substituted) on SK-BR-3 cells was examined; thecells contained a mean intracellular number of QDs of 25, 50,75, 100, and 150 attomolcell�1, formed by incubation at the cor-responding QD concentration (bare QDs: 4.7, 9.3, 14, 19, and28 nm; 750-Mw QDs: 21, 43, 64, 85, and 128 nm; 6000-Mw

QDs: 28, 56, 84, 112, and 168 nm). After a 4-h incubation withthe QDs, the media in each well were aspirated and the cellswere rinsed gently with PBS prior to cytotoxicity assay.

Keywords:biocompatible materials · cytotoxicity · endocytosis ·intracellular exposure · quantum dots

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Received: May 3, 2006Published online on October 9, 2006

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