Peptide-Labeled Near-Infrared Quantum Dots for Imaging Tumor

Mar 11, 2006 - Molecular Imaging Program at Stanford (MIPS) and Bio-X Program, Department of Radiology, Stanford University School of Medicine, 1201 W...
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NANO LETTERS

Peptide-Labeled Near-Infrared Quantum Dots for Imaging Tumor Vasculature in Living Subjects

2006 Vol. 6, No. 4 669-676

Weibo Cai,† Dong-Woon Shin,‡ Kai Chen,† Olivier Gheysens,† Qizhen Cao,† Shan X. Wang,‡ Sanjiv S. Gambhir,†,§ and Xiaoyuan Chen*,† Molecular Imaging Program at Stanford (MIPS) and Bio-X Program, Department of Radiology, Stanford UniVersity School of Medicine, 1201 Welch Road, Stanford, California 94305, Department of Materials Science and Engineering, Stanford UniVersity, 476 Lomita Mall, Stanford, California 94305, and Department of Bioengineering, Stanford UniVersity School of Medicine, Stanford, California 94305 Received December 5, 2005; Revised Manuscript Received February 18, 2006

ABSTRACT We report the in vivo targeting and imaging of tumor vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled quantum dots (QDs). Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered QD705-RGD intravenously. The tumor fluorescence intensity reached maximum at 6 h postinjection with good contrast. The results reported here open up new perspectives for integrin-targeted near-infrared optical imaging and may aid in cancer detection and management including imaging-guided surgery.

Optical imaging is emerging as a complement to radionuclide imaging methods. The major limitation of light is the limited penetration depth because of high absorption and scattering that occur in biological tissues.1 However, in small animals the required path-length of light is much shorter (a few centimeters), which makes optical imaging more feasible. Near-infrared (NIR, 700-900 nm) fluorescence imaging is expected to have a major impact in biomedical imaging because the absorbance spectra for all biomolecules reach minima in the NIR region, which provides a clear window for in vivo optical imaging.2,3 QDs are nanometer-sized semiconductor crystals that, when surface modified to be water soluble and biocompatible, can be attached to targeting molecules and used as fluorescent probes.4-7 QDs possess many advantages over organic fluorophores, such as sizeand composition-tunable fluorescence emission, large absorption across a wide spectral range, narrow emission spectra, and very high levels of brightness and photostability. Biocompatible QDs have thus been applied for labeling cells (fixed and live) and tissues,8 long-term cell trafficking, multicolor cell imaging,9 fluorescence resonance energy transfer (FRET)-based sensing,10 and sentinel lymph-node mapping.11 Even though NIR QDs have great potential for * Corresponding author. Phone: 650-725-0950. Fax: 650-736-7925. E-mail: [email protected]. † Molecular Imaging Program at Stanford (MIPS) and Bio-X Program, Department of Radiology, Stanford University School of Medicine. ‡ Department of Materials Science and Engineering, Stanford University. § Department of Bioengineering, Stanford University School of Medicine. 10.1021/nl052405t CCC: $33.50 Published on Web 03/11/2006

© 2006 American Chemical Society

imaging in living subjects, there are only very few reports in the literature for in vivo targeting and imaging using QDbased probes. Gao et al. demonstrated the use of ABC triblock copolymer-coated QDs for prostate cancer targeting and imaging in living animals, when conjugated to monoclonal antibody that targets prostate-specific membrane antigen (PSMA).12 In this report, it was estimated that there are about 5-6 antibody residues per QD. Peptides or peptidomimetics are potentially better as targeting ligands than antibodies because tens or even hundreds of peptides can be linked to the surface of one QD and may exhibit stronger binding affinity and better targeting efficacy because of the polyvalency effect.13 The evaluation of tumor targeting by a terminal invasive method does not take full advantage of peptide-labeled QDs.14 To date, no peptide-modified QD has been reported for noninvasive in vivo targeted imaging. Herein we report for the first time the in vivo targeting and imaging of arginine-glycine-aspartic acid (RGD) peptidelabeled QDs for imaging of integrin Rvβ3-positive tumor vasculature in a murine xenograft model. This probe may also have great potential as a universal NIR probe for detecting tumor vasculature in general in living subjects. Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for tumor growth and progression.15,16 Integrin Rvβ3, which binds to RGD-containing components of the interstitial matrix such as vitronectin, fibronectin, and thrombospondin,17 plays a key role in tumor angiogenesis and metastasis. It is significantly upregulated

Figure 1. Synthesis of QD705-RGD, PEG denotes poly(ethylene glycol) (MW 2000).

in invasive tumor cells of certain cancer types (glioblastoma, melanoma, breast, ovarian, and prostate cancers, and in almost all tumor vasculature) but not in quiescent endothelium and normal tissues.18,19 Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis while integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels. Inhibition of Rvβ3 integrin activity and thereby angiogenesis by monoclonal antibodies,20,21 peptidic and peptidomimetic antagonists,22 and other small molecule integrin Rvβ3 antagonists23-25 has been shown to prevent tumor growth and cause tumor regression in various experimental models. Several integrin Rvβ3 inhibitors are currently in clinical trials as therapeutics for cancer such as MEDI522 (a humanized antibody) and Cilengitide (a cyclic peptide inhibitor of integrin Rvβ3/Rvβ5).22,26,27The ability to noninvasively visualize integrin Rvβ3 expression using QD-based probes will have great potential in cancer imaging in preclinical small animal models and imaging-guided surgery and therapy because the bright QD signal can be detected easily at shallow tissue depth in small animals and during surgery. We and others have previously labeled various cyclic RGD peptides for multimodality imaging of integrin Rvβ3 expression in vivo, such as positron emission tomography,28-34 single photon emission computed tomography,35 and NIR fluorescence.36 Amine-modified QD705 (emission maximum at 705 nm, Quantum Dot Corp., Hayward, CA) was first conjugated to a heterobifunctional cross-linker, 4-maleimidobutyric acid N-hydroxysuccinimide ester, yielding a maleimide-nanocrystal surface (Figure 1). The lysine !-amino group of c(RGDyK) (potent integrin Rvβ3 antagonist) was reacted with S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA), 670

followed by thiol deprotection using hydroxylamine under neutral conditions to yield the thiolated RGD peptide c(RGDy(!-acetylthiol)K), denoted as RGD-SH. The maleimide-functionalized QD705 was allowed to react with RGDSH for 1 h at pH 7.5 and the final conjugate was purified using size-exclusion chromatography (Nap-10 column, GE Healthcare, Piscataway, NJ). The amine-modified QD705 has 80-100 amino groups on the surface of each QD. Because the large excess of the heterobifunctional linker as well as RGD-SH were used for surface modification, we estimated that there are 30-50 RGD peptides per QD, based on the maximum ligand coupling efficiency of 40-50%.12 To visualize the structure of synthesized QD705-RGD conjugate, we performed atomic force microscopy (AFM) on QD705 and QD705-RGD. A silicon wafer of (100) orientation was cleaned with a standard silicon wafer cleaning procedure. QD705-RGD solution was dispensed with a micropipet onto the substrate and rinsed with deionized water. AFM measurement was performed in a tapping mode with a Nanoscope IIIa SPM (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA) using a standard Si tapping-mode tip with a force constant of 40 N/m. The detection frequency was set to the value where the deflection amplitude is 90% of the peak amplitude at the free-air resonance frequency (274.74 kHz) in order to suppress the reduction of the measured height arising from a surface-tip interaction. After image acquisition, the topography of QD705-RGD was analyzed using the offline vendor software. AFM amplitude scans revealed discrete entities with smooth and uniform surface features (Figure 2a-c). Cross-section height analysis of these entities revealed vertical heights of 5-7 nm. The uniform appearance and height values of these entities demonstrated that these Nano Lett., Vol. 6, No. 4, 2006

Figure 2. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) demonstrated the excellent monodispersity of QD705RGD. (a) AFM scan (400 × 400 nm2) of QD705-RGD deposited on a silicon wafer. (b and c) Section analysis of three QD705-RGD particles as indicated by the white line. Note that the height variation is due to the fact that the three particles are not fully aligned to the white line. (d) SEM scan of the QD705-RGD conjugate deposited on 5-nm tantalum-coated silicon substrate. The scale bar represents 50 nm.

structures are single QDs. It has been reported the measured height of CdSe nanocrystals using tapping-mode AFM is significantly lower than the particle diameters determined by transmission electron microscopy (TEM),37 which is also observed here. The lateral size of QD705-RGD measured by AFM are about 15-20 nm, which fits well with the expected actual size. AFM scans clearly indicated that our RGD peptide conjugation procedure produced monodisperse single QD705-RGD particles without aggregation. Scanning electron microscopy (SEM) experiments confirmed the excellent monodispersity of QD705-RGD conjugates (Figure 2d). The images were acquired with a field emission gun source (FEI XL30 Sirion) at the acceleration voltage of 10 kV and the beam spot size of 2.3 nm. To determine the integrin Rvβ3 binding affinity of QD705RGD, live and fixed MCF-7 (human breast cancer, integrin Rvβ3 negative), MDA-MB-435 (human breast cancer, medium integrin Rvβ3 expression), and U87MG (human glioblastoma, high integrin Rvβ3 expression) cells were blocked with 0.1% bovine serum albumin (5 min at r.t.), stained with 1 nM QD705, QD705-RGD, or QD705-RGD in the presence of 2 µM c(RGDyK), and examined under the microscope (Carl Zeiss Axiovert 200M, excitation: 420/40 nm, emission 705/40 nm, dichrome 470 nm). The representative brightfield and fluorescence images are shown in Figure 3. It can be seen clearly that QD705-RGD did not bind to integrin-negative cells (MCF-7) because there is minimal fluorescence signal observed, whereas the integrin-positive cells (MDA-MB-435 and U87MG) are clearly visualized. Nano Lett., Vol. 6, No. 4, 2006

All of the staining can be blocked effectively by 2 µM c(RGDyK) (only images of “U87MG + Block” are shown because of spatial limitation; for complete cell-staining results, see Supporting Infornation Figures 1-3). Moreover, the fluorescence intensity correlates well with the integrin Rvβ3 expression level of the cell lines (U87MG > MDAMB-435 . MCF-7). Unconjugated QD705 did not exhibit any significant binding to any of the three cell lines. From the cell-staining experiment it can be concluded that QD705RGD exhibited high affinity integrin Rvβ3-specific binding in vitro. Tissue staining was then performed to confirm the specific binding of QD705-RGD to integrin Rvβ3 ex vivo. The tumor tissues were cryosectioned at -20 °C into slices of 8-µM thickness, fixed in cold acetone for 5 min (-20 °C), blocked with 1:10 goat serum for 30 min, stained with 50 nM QD705 or QD705-RGD, washed three times with phosphate buffered saline (PBS), and examined under a microscope. Figure 4 shows the QD705-RGD and QD705 staining of frozen U87MG tumor tissue. Again, it is obvious that the unmodified QD705 had minimal nonspecific binding, whereas QD705-RGD clearly demonstrated the high integrin expression level of the U87MG tissue. Staining of integrin-negative tumor tissue (MCF-7) gave virtually no fluorescence signal (data not shown). Because both in vitro and ex vivo studies showed that QD705-RGD is able to specifically target integrin Rvβ3, we proceeded to test it in living subjects. Athymic nude mice bearing U87MG tumor (4 weeks post inoculation of 5 × 671

Figure 3. In vitro staining of human breast cancer MCF-7, MDA-MB-435, and human glioblastoma U87MG cells (low, medium, and high integrin Rvβ3 expression, respectively) using 1 nM QD705-RGD (left 3 columns). Staining of U87MG cells with 1 nM QD705 (denoted as “U87MG + QD705”) or 1 nM QD705-RGD in the presence of 2 µM c(RGDyK) (denoted as “U87MG + Block”) are also shown. Filter set: excitation, 420/40 nm; emission, 705/40 nm. Magnification: 400×, 0.5 s exposure. All fluorescence images were acquired under the same condition and displayed under the same scale.

Figure 4. Frozen U87MG tumor tissue staining using 50 nM QD705-RGD and QD705. Magnification: 400×. The tissue slices were blocked with 1:10 goat serum for 30 min before staining.

106 cells on the left shoulder, tumor size about 0.5-0.8 cm3) were administered QD705 or QD705-RGD (200 pmol of QD per animal, about 6-10 nmol of RGD peptide) through tail vein injection. The mice were imaged at many time points postinjection (p.i.) using the Maestro in vivo imaging system (CRI, Inc., Woburn, MA; excitation: 575-605 nm, emission: 645 nm long-pass). The Maestro optical system consists of an optical head that includes a liquid crystal 672

tunable filter (LCTF, with a bandwidth of 20 nm and a scanning wavelength range of 500-950 nm) with a customdesigned, spectrally optimized lens system that relays the image to a scientific-grade megapixel CCD. The tunable filter was automatically stepped in 10-nm increments from 650 to 850 nm while the camera captured images at each wavelength with constant 1 s exposure (total acquisition time was about 30 s). The resulting 21 TIFF images (spectral cube, containing a spectrum at every pixel) were loaded into the vendor software (Nuance 1.4.0) and analyzed. Spectral unmixing using a normal mouse as the autofluorescence signal yielded the pseudo-color images of the pure mice autofluorescence and QD signals, which were then quantified using Image J software (http://rsb.info.nih.gov/ij/). The overlayed images are shown in Figure 5a. For the spectra of mice autofluorescence and the QD signal, see Supporting Information Figure 4. As early as 20 min p.i., a fluorescence signal was observed in the tumor. During the next several hours, there was a steady increase in the tumor fluorescence intensity in mice injected with QD705-RGD (Figure 5b). At 6 h p.i., the tumor signal intensity reached its maximum (tumor-to-background ratios were 3.08 ( 1.42, 3.39 ( 1.13, 4.42 ( 1.88, and 2.09 ( 1.17 at 1, 4, 6, and 27 h p.i., respectively, where the background refers to the hind limb, n ) 3). No significant fluorescence signal was observed in the tumor for the QD705 injected mouse (1.12 ( 0.14, 0.78 ( 0.25, 0.84 ( 0.21, and 0.67 ( 0.46 at 1, 4, 6, and 27 h p.i. respectively, n ) 3). Nano Lett., Vol. 6, No. 4, 2006

Figure 5. (a) In vivo NIR fluorescence imaging of U87MG tumor-bearing mice (left shoulder, pointed by white arrows) injected with 200 pmol of QD705-RGD (left) and QD705 (right), respectively. All images were acquired under the same instrumental conditions. The mice autofluorescence is color coded green while the unmixed QD signal is color coded red. Prominent uptake in the liver, bone marrow, and lymph nodes was also visible. (b) Tumor-to-background ratios of mice injected with QD705 or QD705-RGD. The data were represented as mean ( standard deviation (SD). Using one-tailed paired Student’s t-test (n ) 3), “*” denotes where P < 0.05 as compared to the mice injected with QD705. (c) Serum stability of QD705 and QD705-RGD in complete mouse serum over the course of 24 h.

Successful tumor imaging demonstrated the specific in vivo targeting of integrin Rvβ3 using QD705-RGD. QD705 and QD705-RGD were incubated in complete mouse serum at 37 °C to evaluate the fluorescence intensity change as a function of time. As can be seen in Figure 5c, a significant drop in fluorescence intensity was observed for both conjugates. After 24 h of incubation in mouse serum, the fluorescence intensity was 73.6% and 66.4% of the initial fluorescence for QD705 and QD705-RGD, respectively. This phenomenon may be partially responsible for the decrease in tumor contrast at late time points and may also limit the longitudinal studies where QDs need to be monitored over weeks or even longer. At 6 h p.i. where the best tumor contrast was observed, mice injected with QD705 or QD705-RGD were sacrificed and the tumors were harvested and imaged immediately. The IVIS200TM system (Xenogen Corp., Alameda, CA) was used for ex vivo imaging because of its higher sensitivity. As can be seen in Figure 6a, the QD signal is clearly visible in the U87MG tumor of the mouse injected with QD705-RGD, whereas virtually no signal was seen in the tumor of the mouse injected with QD705. The fluorescence signal intensity of ex vivo tumor imaging is a true reflection of the QDs retained inside the tumor due to the lack of autofluorescence. Because the ex vivo tumor fluorescence signal appeared to Nano Lett., Vol. 6, No. 4, 2006

Figure 6. (a) Ex vivo U87MG tumor fluorescence images of QD705-RGD (left) and QD705 (right) injected mice at 6 h p.i. (b) Microscopic images of frozen tumor slices stained for CD31 (green). The QD signal is shown in red and pointed by white arrows. Magnification: 200×. All fluorescence images were acquired under the same condition and displayed under the same scale.

be heterogeneous, we reasoned that the fluorescence signal might come mainly from the tumor vasculature because these QD conjugates are about 15-20 nm in diameter, which may 673

not extravasate very well. To testify this, the tumor tissue was frozen in optimal cutting temperature (OCT) medium, cryosectioned at -20 °C into 5-µm slices, and immunostained for CD31 to allow visualizing of the tumor vasculature (Figure 6b). Indeed, the microscope images confirmed the presence of QD in the tumor tissue of the QD705-RGDinjected mouse but not in the QD705-injected mouse. Furthermore, the majority of the fluorescence signal (pointed by white arrows) colocalizes with the CD31 staining, indicating that QD705-RGD did not extravasate, consistent with previous reports in the literature.14 For in vivo imaging, QDs must have adequate stability in biological fluids and minimal nonspecific binding, must retain their fluorescence for a sufficiently long time, and must emit in the NIR range. It was reported recently that antibody conjugated polymer-encapsulated QD probes allow for cancer targeting and imaging in vivo.12 However, the use of orange/ red emitting QDs in the visible range is not optimal for in vivo imaging. Akerman et al. first reported the use of peptidecoated ZnS-capped CdSe QDs for the targeting of lung, blood vessels, or lymphatic vessels in tumors after intravenous injection by means of microscopic imaging of tissue slices.14 They also demonstrated that PEG coating of the QD could prevent nonselective accumulation in the reticuloendothelial system (RES, part of the body’s immune system, concentrated mostly in the liver, spleen, lymph nodes and bone marrow38). However, no in vivo imaging was achieved. In this study, we report the use of peptide-labeled NIR QD for in vitro cell labeling, ex vivo tumor tissue staining, as well as in vivo tumor vasculature imaging. The aminemodified QD705 used in this study are built with a 2000 MW PEG spacer covalently attached to the QD705 surface. We found that the PEG linker gave much improved stability compared to the corresponding carboxyl-modified QD705, which does not possess a PEG linker (our unpublished data). No aggregation was observed after the peptide conjugation as well as over the experimental time frame of this study, likely because of the increased hydrophilicity and higher stability resulted from the PEG-coating. For both QD705 and QD705-RGD, accumulation of the injected QDs is rapid and high in liver, spleen, bone marrow, and lymph nodes, indicating that the mononuclear phagocytes of the RES are involved in the clearance of some of the circulating QDs in the mice. The QD signal in the liver can be observed as early as 10 min p.i., and ex vivo fluorescence images of the liver reveals that the liver uptake of the QD705-RGD injected mice was slightly lower than that of the QD705 injected mice, although the difference was marginal and not statistically different (data not shown). It cannot be ruled out that the overall charge of the QD705-RGD may also play a role. QD705 is highly cationic under physiological conditions because there are about 80-100 primary amino groups per QD. For QD705-RGD, about half of the amino groups are converted to maleimide; therefore the overall charges are much less, which may lead to improved in vivo pharmacokinetics. As for the excitation wavelength for the in vivo imaging, QDs can be excited at any wavelength shorter than their emission wavelength and the shorter the excitation 674

wavelength, the stronger the absorbance and fluorescence.4,7 However, excitation at longer wavelengths should improve visualization and resolution at increased tissue depth, although at some cost in efficiency of excitation. Different excitation wavelengths were compared and we found that the 590/30 nm excitation gave the best result and required the shortest imaging time. Excitation at shorter wavelengths such as 525/50 nm reduced the fluorescence signal dramatically because of the poorer tissue penetration of the green light although QDs have much stronger absorbance at this wavelength than at 590 nm. As for the optimal dose and toxicity of these QD conjugates, we found that injection of 100 pmol QD705-RGD did not give significant tumor contrast; higher doses were not tested. No toxicity was noticed in vitro, and no abnormal behavior of the mice was observed in all of the in vivo imaging experiments. To the best of our knowledge, this is the first successful demonstration of peptide-labeled QD for in vivo tumor vasculature imaging. Much can be done in future studies to further improve the in vivo targeting and imaging of QDbased probes. We have reported that dimeric and tetrameric RGD peptides exhibited better in vivo targeting efficacy and pharmacokinetics because of the polyvalency effect.32,33 Therefore, it might be worth exploring the conjugation of dimeric or tetrameric RGD peptides to QD. We are currently in the process of investigating whether the polyvalency of RGD peptide ligands can give an extra boost to the already polyvalent QD705-RGD conjugates and lead to improve targeting efficacy and better in vivo imaging properties. Multiple ligands can be attached to QD to target multiple receptors on the tumor vasculature as well as on the tumor cells. For example, half of the amino group can be modified with RGD peptides, while the other half can be modified with ligands targeting VEGFR-2 (Flk-1/KDR) on the tumor vasculature. For in vivo application, reducing QD uptake by the RES and increasing circulation lifetime will be critical. Although the tumor-to-background ratio was satisfactory in this study, the uptake of QD705 and QD705-RGD by the RES is quite obvious. It has been reported that long-chain methoxy-PEG (MW 5000)-coated QDs have significantly prolonged circulation half-lives compared to poly(acrylic acid) or short-chain methoxy-PEG-coated QDs.39 Whether incorporation of a long PEG linker into the QD705-RGD conjugate will reduce the nonspecific uptake in the liver, spleen, and bone marrows without compromising integrin Rvβ3 specific tumor uptake and retention remains to be determined. One needs to bear in mind that long PEGmodified QDs may prolong the circulation time and reduce the tumor washout rate but they may also reduce the probability of QD-based probes to extravasate and target tumor cells because of the substantially increased particle sizes. Moreover, a longer PEG linker may also hamper the binding of RGD peptide to integrin Rvβ3 because of the steric hindrance. The optimal PEG chain length as well as the use of other hydrophilic or amphiphilic linkers needs to be finetuned on a case-by-case basis. Reducing the size of QDbased probes is also necessary to achieve better tissue penetration and tumor targeting. Direct replacement of the Nano Lett., Vol. 6, No. 4, 2006

TOPO coating on the QD by presynthesized HS-PEG-RGD may give a QD-RGD conjugate of much smaller size.40 In addition, more RGD peptides will be available per QD, which may lead to much better in vivo targeting and imaging. Mixtures of HS-PEG-RGD and HS-PEG at different ratios can also be investigated. QDs that emit at even longer wavelengths in the NIR region, for example, 800-850 nm, will be better for in vivo imaging especially in human studies.3 However, the technology for manufacturing such QDs is still immature compared to the CdSe- or CdTe-based QDs.7 Multimodality imaging of QD-based probes can also be developed. For example, QD705-RGD can be further labeled with positron emitting isotope 124I through the D-Tyr residue so that optical imaging and PET imaging (no depth limit in tissue penetration41) can be performed on the same animal. Dual modality imaging can compensate for some of the disadvantages of optical imaging alone, such as poor tissue penetration and lack of quantification. Other positron emitting isotopes may also be incorporated through different strategies (e.g., various radiometals can be labeled through chelation). Recently, we reported the use of peptide-dye conjugate Cy5.5-c(RGDyK) (denoted as Cy5.5-RGD) for in vivo imaging of subcutaneous U87MG xenograft at different doses (0.1, 0.5, and 3 nmol).36 Cy5.5 has maximum emission at 694 nm, similar to QD705. The intermediate dose (0.5 nmol) produced better tumor contrast than the high dose (3 nmol) and low dose (0.1 nmol) during 30 min to 24 h p.i., because of partial self-inhibition of receptor-specific tumor uptake at high dose and the presence of significant amount of background fluorescence at low dose, respectively. Comparing these two studies, we found that QD705-RGD delineates the tumor better than Cy5.5-RGD, likely for several reasons. First, much stronger absorbance and higher quantum yield of the QD gave a better signal at lower levels of fluorescent probes administered; Second, the polyvalency effect of the RGD ligands yielded better in vivo targeting efficacy and imaging properties. However, the uptake of QD705-RGD in other nontargeting organs is much higher than Cy5.5-RGD. Because of its much smaller size, Cy5.5-RGD exhibited more rapid tumor localization than QD705-RGD and it was also able to bind to integrin Rvβ3 on both tumor vasculature and tumor cells as evidenced by the homogeneous fluorescence images of the tumor ex vivo, whereas the QDs can only bind to vascular integrin Rvβ3. Overall, the major advantages of QD705-RGD over Cy5.5-RGD are better photostability (much less photo bleaching for QD705-RGD both in vitro and in vivo), much stronger signal intensity, and the possibility of multiplexing, which is the goal of future studies. In clinical settings, optical imaging is relevant for tissues close to the surface of the skin, tissues accessible by endoscopy, and intraoperative visualization. NIR fluorescence imaging using QD705-RGD may assist in differentiating the integrin Rvβ3-positive neoplastic lesions and normal tissue during surgery. Soon, multicolor QDs for multitarget imaging in preclinical studies can also add tremendously to our understanding of tumor biology and tumor susceptibility to therapy. Multiple wavelength QDs in the NIR range will Nano Lett., Vol. 6, No. 4, 2006

allow for multiplex imaging of deeper tissues, greatly extending potential human applications. A key question is whether QDs can be used directly in patients. Although we did not observe any toxicity in this study, complete cellular toxicity studies and in vivo toxicology need to be carried out before any application in humans. In conclusion, for the first time we have demonstrated that RGD peptide-labeled quantum dot QD705-RGD can specifically target integrin Rvβ3 in vitro, ex vivo, and in living mice. Because the majority of tumor vasculature overexpresses integrin Rvβ3 during angiogenesis, QD705-RGD has great potential as a universal NIR probe for detecting tumor vasculature in vivo in general for most cancer types. Further testing of QD705-RGD is currently under way in our laboratory. The results reported here open up new perspectives for integrin-targeted NIR optical imaging and will have great potential in cancer diagnosis and management as well as imaging-guided surgery and therapy. Acknowledgment. This work was supported, in part, by National Cancer Institute (NCI) Grant R21 CA102123, National Institute of Biomedical Imaging and Bioengineering (NIBIB) Grant R21 EB001785, Department of Defense (DOD) Breast Cancer Research Program (BCRP) Concept Award DAMD17-03-1-0752, DOD BCRP IDEA Award W81XWH-04-1-0697, DOD Ovarian Cancer Research Program (OCRP) Award OC050120, DOD Prostate Cancer Research Program (PCRP) New Investigator Award (NIA) DAMD1717-03-1-0143, American Lung Association California (ALAC), the Society of Nuclear Medicine Education and Research Foundation, National Cancer Institute (NCI) Small Animal Imaging Resource Program (SAIRP) Grant R24 CA93862, NCI In Vivo Cellular Molecular Imaging Center (ICMIC) Grant P50 CA114747, and NCI Centers of Cancer Nanotechnology Excellence (CCNE) U54 Grant 1U54CA119367-01. We thank Pauline Chu for histology. This paper is dedicated to the memory of Professor Murray Goodman. Supporting Information Available: Cell-staining experiments and the autofluorescence and QD spectra used for spectral unmixing. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Choy, G.; Choyke, P.; Libutti, S. K. Mol. Imaging 2003, 2, 303312. (2) Massoud, T. F.; Gambhir, S. S. Genes DeV. 2003, 17, 545-580. (3) Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7, 626-634. (4) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (5) Chan, W. C.; Nie, S. Science 1998, 281, 2016-2018. (6) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544. (7) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435-446. (8) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 4146. (9) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47-51. (10) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630-638. 675

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