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Near-Infrared Fluorescent Nanoparticles as Combined MR/Optical Imaging Probes Lee Josephson,* Moritz F. Kircher, Umar Mahmood, Yi Tang, and Ralph Weissleder Center for Molecular Imaging Research, Massachusetts General Hospital, Charlestown, Massachusetts. Received September 14, 2001; Revised Manuscript Received December 21, 2001
A number of quantitative three-dimensional tomographic near-infrared fluorescence imaging techniques have recently been developed and combined with MR imaging to yield highly detailed anatomic and molecular information in living organisms (1, 2). Here we describe magnetic nanoparticle based MR contrast agents that have a near-infrared fluorescence (NIRF) that is activated by certain enzymes. The probes are prepared by conjugation of arginyl peptides to cross-linked iron oxide amine (aminoCLIO), either by a disulfide linkage or a thioether linker, followed by the attachment of the indocyanine dye Cy5.5. The NIRF of disulfide-linked conjugate was activated by DTT, while the NIRF of thioetherlinked conjugate was activated by trypsin. Fluorescent quenching of the attached fluorochrome occurs in part due to the interaction with iron oxide, as evident by the activation of fluorescence with DTT when nanoparticles that have less than one dye attached per particle. With a SC injection of the probe, axillary and brachial lymph nodes were darkened on MR images and easily delineated by NIRF imaging. The probes may provide the basis for a new class of so-called smart nanoparticles, capable of pinpointing their position through their magnetic properties, while providing information on their environment by optical imaging techniques.
INTRODUCTION
Magnetic nanoparticles are now well established as magnetic resonance (MR) reporters for detecting pathologies in the liver, spleen, and lymph nodes where, after phagocytosis, their presence is evident primarily by a darkening effect on T2 weighted images. After injection, magnetic nanoparticles accumulate in target tissues and are degraded over a period of several days, providing a convenient time interval to determine nanoparticle disposition. MR is now capable of providing high spatial resolution images (on the order of tens of microns per voxel resolution) of nanoparticle disposition in vitro, in small animals, and in humans. Nanoparticles can be identified in normal human lymph nodes, generally less than about 10 millimeters in diameter, and millimetersized metastases can be visualized within the node (35). Current magnetic nanoparticles, while providing superb information on their spatial disposition, are not designed to provide information on the molecular environment surrounding the nanoparticle. We have recently developed novel enzyme-activated near-infrared fluorescent (NIRF) probes that can image the activity of specific proteases in the local environment of the probe in vivo (6-8). Probes have been synthesized by the attachment of the NIRF dye Cy5.5 to a poly-lysine derivatized with PEG, a material termed poly(ethylene glycol) graft copolymer Cy5.5-PGC (PGC, PEG graft copolymer) (7, 9). In vivo the polylysine backbone is cleaved by cathepsin B, while in vitro it can be cleaved by trypsin. However, current optical imaging methods acquire the NIRF signal against a nonfluorogenic background and hence provide little spatial/anatomic inforCorresponding author: Lee Josephson, Ph.D., Center for Molecular Imaging Research, Massachusetts General Hospital, Bldg. 149 13th Street, #5406, Charlestown, MA 02129. Tel.: (617) 726 5788. Fax: (617) 726 5708. E-mail: josephso@ helix.mgh.harvard.edu.
mation. The development of optical tomographic imaging techniques offers the prospect of providing a lower detection limit for fluorochromes, and a higher spatial resolution, but a second imaging modality will still be required to obtain anatomical background. In this communication, we report on a new type of magnetic nanoparticle that acts as an MR contrast agent providing information on the location of the probe and, as a probe for optical imaging, providing information on the molecular environment of the probe. Contrast enhancement by MR results from a core of superparamagnetic iron oxide, while the NIRF signal results from cleavable Cy5.5-derivatized peptides attached to the surface of the nanoparticle. Amino-CLIO, a magnetic nanoparticle with a surface of aminated, cross-linked dextran, has been used to attach a variety of biological molecules, including the tat peptide of the HIV tat protein (10), transferrin (11), and oligonucleotides (12), and provides a convenient platform for the attachment of Cy5.5 peptides. Two chimeric nanoparticle probes are described. The first exhibits NIRF fluorescence when activated by a reducing environment, while the second exhibits NIRF fluorescence when activated by proteases. These novel probes may provide the basis for a new class of so-called smart nanoparticle probes, whose position can be ascertained by MR imaging and which provide information on their molecular environment through NIRF. EXPERIMENTAL PROCEDURES
Two dye-arginyl peptide nanoparticle conjugates were synthesized according to Figure 1 and are denoted Cy5.5R4-SS-CLIO and Cy5.5-R4-SC-CLIO. The designation “SS” denotes a disulfide linkage between the peptide and the iron oxide while “SC” denotes a thioether linkage, while the R4 stands for four arginyl peptides in the L configuration. The primary amine of a dextran-coated
10.1021/bc015555d CCC: $22.00 © 2002 American Chemical Society Published on Web 04/02/2002
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Figure 1. Scheme for synthesis of Cy5.5-arginyl-CLIO conjugates.
cross-linked iron oxide (amino-CLIO) was reacted with one of two activating agents, either N-succinimidyl 3-(2pyridyldithio)propionate (SPDP) or succinimidyl iodoacetate (SIA), to produce two reactive forms of CLIO as shown in Figure 1. The L-arginyl peptide R-R-R-RG-C (“R4”) is attached to the activated CLIO’s through a C-terminal cysteine residue to the SPDP or succinimidyl iodoacetate CLIO, followed by the N terminal residue with N-hydroxylsuccinimidyl (NHS) ester of Cy5.5 to yield Cy5.5-R4-SS-CLIO and Cy5.5-R4-SC-CLIO. Synthesis of Aminated-CLIO. Aminated CLIO was prepared as described (10, 11). It consists of a core of superparamagnetic iron oxide, a cross-linked coating of dextran with amino groups. Peptide Synthesis. Peptides were synthesized on an automatic synthesizer (PS3, Rainin, Woburn, MA) at a 0.1 mmol scale using Fmoc chemistry with HBTU and HOBT. They were cleaved from Rink amide HBHA resin (Novabiochem, San Diego, CA) with 5 mL of TFA/ thioanisole/ethanedithiol/anisole (90/5/3/2) and purified by C18 reversed phase chromatography. Peptides were the L-arginyl peptide R-R-R-R-G-C-NH2 (“R4”) and the d-arginyl peptide r-r-r-r-G-C-NH2 (“r4”). By MALDI-MS R4 was 802.93 calculated versus 803.3 found (M + 1). For the peptide r4 M + 1 was 803.63. Synthesis of Cy5.5 Nanoparticle Conjugates. To synthesize the thioether-bonded conjugate R4-SC-CLIO, amino-CLIO (22 mg Fe, 1.5 mL) was added to 0.5 mL of
0.1M Na2HPO4 and 0.5 mL of 150 mM SIA in DMSO. The reaction was allowed to sit for 1 h at room temperature and the addition of succinimidyl-iodoacetate repeated. Iodoacetyl-CLIO was separated from iodoacetic acid using a Sephadex G-25 column equilibrated with 0.025 M Citrate pH 6.5 at 4 °C. To the void volume, 5 mL, was added 6-7 mg of R4 in 0.6 mL of citrate buffer and the reaction incubated overnight at room temperature. Unreacted peptide was removed by dialysis against 3 L of 0.025 M citrate pH 8.2 using a 14 kDa cutoff membrane. To synthesize the disulfide linked conjugate R4-SSCLIO, amino-CLIO was activated with SPDP as described (10) and reacted with R4 as above except that the pH of the citrate buffer was 8.2 for chromatography and storage (Figure 1). To attach Cy5.5 to arginyl-CLIO conjugates, 200 µL of 1 M NaHCO3 (pH 8.3) was added to 2 mL of R4-SCCLIO (7.2 mg Fe). Four hundred forty microliters was then added to each of the five tubes of lyophilized Cy5.5 (Amersham Pharmacia, Piscataway, NJ). After 2 h at room temperature, unreacted dye was removed using PD10 columns run in a spin separation mode. Columns were washed with 0.025 M citrate pH 8.2 and spun dry. A one milliliter sample was added to columns that were spun at 2500g for 7 min. To synthesize Cy5.5-R4-SS-CLIO conjugates with varying ratios of dye attached, various amounts of dye
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Table 1. Physical Properties of Cy5.5-arginyl-CLIO Conjugates conjugate
size (nm)
R1 (s-1 mM-1)
R2 (s-1 mM-1)
peptides per particle
fluorochromes per particle
Cy5.5-R4-SS-CLIO Cy5.5-R4-SC-CLIO
68 ( 13 62 ( 7
27.8 29.9
91.2 92.5
11.9 15.5
1.19 1.79
were reacted with a fixed amount of conjugate. R4-SSCLIO (0.1 mg) was added to between 10 and 160 µL of a tube of NHS-Cy5.5 reconstituted with 500 µL of water immediately before use. Additional water and buffer was added so that the reaction with dye occurred in a volume of 275 µL of 0.05 M NaHCO3. After 2 h at roomtemperature, unreacted dye was removed using PD-10 columns as above. Physical Properties of Nanoparticles. The number of peptides per CLIO was determined as the number of reactive primary amino groups using fluorescamine (13, 14). Five to fifty microliters of arginyl-CLIO nanoparticles or various amounts of the standard peptide (R4) were diluted into 500 µL of 0.1 M borate, pH 8.5. The weight of R4 was determined by amino acid analysis. Two hundred fifty microliters of fluorescamine (4 mg in 20 mL of acetyl nitrile) was added, and the mixture was allowed to stand overnight at room temperature. Fifty microliters of trypsin (1 mg/mL in 0.1 M phosphate) was added, and samples were allowed to stand for 2 h at room temperature. Fluorescamine fluorescence, ex 390, em 475, was measured after separating the iron oxide from low molecular weight materials by ultrafiltration (10). Iron concentration was determined by absorbance (11). The peptide-to-crystal ratio was obtained from concentrations of peptide and iron, assuming 2064 iron atoms per crystal (15). Dye/Particle Ratios. Spectra of conjugates were taken using a Hitachi 3500 spectrophotometer. The number of dyes per crystal attached was taken from the absorption at 675 nm and an extinction coefficient of 250000 M-1cm-1 and assuming 2064 iron atoms per crystal. Activation of NIRF. For Cy5.5-R4-SS-CLIO or Cy5.5R4-SC-CLIO, activation by trypsin or DTT was determined in a microtiter plate with volume of 350 µL of 0.01 M phosphate pH 7.4 with a concentration conjugate of approximately 80 µg Fe/mL. Either trypsin (10 µg/mL, Sigma Chemical St. Louis, MO) or DTT (5 mM) were present. The plate was incubated at room temperature for various times up to 200 min and Cy5.5 fluorescence determined (ex 660, em 695) using a fluorescent microtiter plate reader (Spectra Max, Molecular Devices, Sunnyvale, CA). Activation was defined as a dimensionless number obtained by dividing fluorescence obtained with trypsin (or DTT) by fluorescence obtained without trypsin (or DTT), after subtraction of background fluorescence (16). Magnetic and Size Measurements. The R1 and R2 relaxivities were measured using a 0.47 T tabletop relaxometer as described (10, 11). Size was determined by laser light scattering using a Coulter N4 particle size analyzer. MR and Optical Imaging. For the determination of tissue signal intensities after intravenous (iv) injection (Figure 4), C57 BL/6 mice (National Cancer Institute) were injected through the tail vein with Cy5.5-R4-SCCLIO (5.1 mg Fe/kg, 2 nmoles dye, 150µL). After 18 h, animals were euthanized with pentobarbital sodium (200 mg/kg, ip), and tissues (approximately 200 mg) were placed in a 96-well microtiter plate. MR imaging was performed on a clinical GE Signa 1.5T imager using a standard 3 in. surface coil to acquire signal. A T2
Figure 2. Absorption spectra of Cy5.5, amino-CLIO and Cy5.5R4-SC-CLIO.
weighted pulse sequence TR/TE 3000/100 was used (1.0 mm slice thickness, 256 × 256 matrix, 8 cm FOV). For imaging lymph nodes after subcutaneous (SC) injection (Figure 5), a nu/nu nude mouse (Taconic Farms, Tarrytown, NY) was injected with Cy5.5-R4-SC-CLIO (2.2 mg Fe/kg, 0.85 nmol dye, 30 µL) under the skin of the left front extremity. After 24 h, animals were anesthetized with ketamine/xylazine (80/10 mg/kg, ip) and imaged in the magnet with the coil above. A T1 weighted pulse sequence (TR/TE 500/17) was used (1.5 mm slice thickness, 256 × 256 matrix, 6 cm FOV). Optical imaging was performed using a whole mouse imaging system, a modification of a commercially available chemiluminescence system, as described (17). Cy5.5 fluorescence was obtained with a band-pass filter at 630 ( 20 nm (model # 630RDF30 Omega Optical, Brattleboro, VT) and a 700 nm long pass filter (700 EFLP; Omega Optical). Animals were handled according to institutional guidelines. RESULTS
The physical properties of Cy5.5-R4-SC-CLIO and Cy5.5-R4-SS-CLIO are summarized in Table 1. The nanoparticles are similar in size and have the high relaxivity characteristics of superparamagnetic iron oxides (15). The absorption spectrum of Cy5.5-R4-SS-CLIO is shown in Figure 2. Cy5.5-R4-SS-CLIO had an absorption maximum of 680 nm, which is slightly shifted from the maximum of 675 nm for Cy5.5. The spectrum of amino-CLIO is also shown, which is dominated by the absorption of iron oxide in the ultraviolet region of the spectrum and which shows little absorption in the nearinfrared portion of the spectrum. The distinct spectral properties of amino-CLIO and Cy5.5 suggest that the iron oxide would have little effect on the NIRF of Cy5.5 though evidence for such an interaction is presented below. Figure 3A shows the effect of treating the nanoparticle conjugates Cy5.5-R4-SS-CLIO and Cy5.5-R4-SCCLIO with DTT. DTT cleaves disulfide bonds and provides gentle conditions for separating intact molecules from the surface of magnetic nanoparticles (11, 12).
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Figure 4. Activation of the disulfide linked nanoparticle conjugate Cy5.5-R4-SS-CLIO with different numbers of fluorochromes (Cy5.5) attached. Activation was with DTT.
Figure 3. Activation of optical magnetic nanoparticle conjugates by DTT (A) or trypsin (B).
Incubation of Cy5.5-R4-SS-CLIO with DTT resulted in a 12.8-fold increase in NIRF. On the other hand, the NIRF of the thioether-linked nanoparticle Cy5.5-R4SC-CLIO was not activated by DTT. Trypsin incubation activated the near-infrared fluorescence of Cy5.5-R4SS-CLIO and Cy5.5-R4-SS-CLIO 36.2 and 27.4-fold (Figure 3B), respectively, by cleaving peptide bonds and releasing a number of degradation products (Cy5.5-RR, Cy5.5-R-R-R-). Activation by trypsin was notably slower than that with DTT presumably because the concentration of DTT employed (5 mM) was high enough to produce a nearly instantaneous cleavage of disulfide bonds. Activation was notably higher with trypsin than with DTT (36.2 versus 12.8), which may reflect intramolecular interactions between the negatively charged Cy5.5 dye and positively charged peptide obtained when Cy5.5-R4-SS-CLIO is treated with DTT (Cy5.5-R-RR-R-G-C-NH2). To further prove the specificity of conjugate activation, a conjugate denoted Cy5.5-r4-SSCLIO was synthesized, where r4 indicates a peptide identical to “R4” but with all amino acids in the proteaseresistant D configuration. Cy5.5-r4-SS-CLIO was activated by DTT but was resistant to activation by trypsin (Josephson, unpublished observations). Enzyme-activatable Cy5.5 probes generate signals when exposed to proteases primarily because multiple fluorochromes attached to a single carrier molecule quench due to interactions with each other (3, 8, 17). Proteolysis cleaves bonds between fluorochromes, increasing the distance between them and activating the
NIRF signal. The quenching/activation attained with less than two dye molecules per nanoparticles suggests that quenching may result from an interaction of Cy5.5 with CLIO rather than between Cy5.5 molecules, an interaction which if present occurs despite the lack of spectral overlap between iron oxide and Cy5.5. To further analyze the activation of fluorescence obtained, a series of Cy5.5-R4-SS-CLIO conjugates were made at different ratios of Cy5.5 to iron oxide. Figure 4 shows the activation of Cy5.5-R4-SS-CLIO conjugates ranging from 0.14 Cy5.5 molecules per particle to 1.19 Cy5.5 molecules per particle. Remarkably, fluorescence increased 2.28-fold with a conjugate that had an average of 0.14 mol of Cy 5.5 per particle, suggesting that quenching is occurring, at least in part, from an interaction between the dye and the iron oxide. Activation increased to 12.8-fold as the number of Cy5.5 molecules was increased to 1.19 Cy5.5’s per particle. To demonstrate the ability of Cy5.5-R4-SC-CLIO to act as a combined MR/optical imaging probe, we injected the agent into mice by the IV and SC routes of administration and obtained MR images and NIRF images. With the IV injection (Figure 5), the animal was sacrificed and MR and NIRF images were obtained from tissues after dissection. Uptake of Cy5.5-R4-SC-CLIO, through the presence of magnetic iron oxide in the spleen and lung, is evident from the lower signal intensity (darkening) of those tissues on MR images, compared to tissues from an uninjected animal. With NIRF fluorescence, no signal was obtained with tissues from an uninjected animal, reflecting the absence of fluorochromes in the range of 610 to 650 nm used to excite Cy5.5. With the injected animal on the other hand, a high intensity NIRF signal was obtained from the liver and spleen and a weak NIRF signal was obtained from the kidney. The low intensity signal from the kidney probably results from proteolytic degradation of the conjugate and excretion of low molecular degradation products. With the SC injection (Figure 6), MR imaging and NIRF images were obtained on a single live mouse. Figure 6A shows transverse MR images of the axillary and brachial nodes after injection into the front extremity. Axillary and brachial nodes are evident as points of very low signal intensity in two different transverse planes. The darkening of the axillary node due to Cy5.5R4-SC-CLIO is further shown in the coronal view in Figure 6B. Similar points of low signal intensity are not evident for the contralateral, uninjected side of the animal. Figure 6C shows a white light, reflectance image determined by the surface topography of the animal. However, with the corresponding NIRF image (Figure 6D) background light is negligible, and fluorescent signal
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Figure 5. MR and NIRF images from tissues of animals injected iv with Cy5.5-R4-SC-CLIO.
from the brachial and axillary nodes passes through the skin, permitting the nodes to be seen as bright centers or points of light. DISCUSSION
We have designed magnetic nanoparticles that serve both as magnetic resonance contrast agents and NIRF optical probes. With SC administration the conjugate denoted Cy5.5-R4-SC-CLIO produced contrast-enhanced MR images of lymph nodes that were free of the susceptibility artifact that can accompany the use of iron oxide based contrast agents (18, 19). The procedure also provided a NIRF signal sufficiently strong to be seen with our simple imaging equipment consisting of a relatively low power halogen lamp and CCD camera described in detail elsewhere (17). However, SC administration used here is a technique that allows nanoparticles to concentrate at high doses in lymph nodes, such as the brachial and axillary nodes, that drain the injection sites of the front extremity (20). More sensitive means of visualizing fluorochrome distribution, such as diffuse optical tomography (1, 21), may enable the detection of far lower tissue concentrations of Cy5.5 than possible with our equipment. It has recently been recognized that colloidal materials, rather than organic molecules, can act as efficient quenchers of fluorescence. For example molecular beacons based on the hybridization of target oligonucleotides to single stranded hairpins use a pair of organic molecules (EDANS/DABCYL) as a donor/quencher pair (22). However, 1.4 nm colloidal gold can replace DABCYL for hairpin-based molecular beacons (23). In this example oligonucleotides are attached to gold through a 5′ terminal phosphate through a (CH2)6-SH group, while a dye couple is coupled to a 3′ hydroxyl group through a (CH2)6-NH2 linker. Fluorescence is quenched until the hairpin opens due to hybridization, which increases the distance between gold and the fluorophore. With magnetic nanoparticles, treatment with DTT or trypsin cleaves covalent bonds between the fluorophore and the
Figure 6. MR and NIRF images from tissues animals injected with Cy5.5-R4-SC-CLIO after SC injection. A. Contrast enhanced MR images of lymph nodes where ax ) axillary and br ) brachial. B. Contrast enhanced coronal image of the axillary node. C and D. White light and NIRF images, respectively, showing intense regions of NIRF in axillary and brachial nodes.
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nanoparticle, permitting the fluorophore to leave the local environment of the nanoparticle and distribute through the bulk media. Interestingly, fluorophores ranging from fluorescein (ex 494, em 520) to Cy 5 (ex 649, em 670) were highly quenched by colloidal gold (23). We have noted that the attachment of fluorescein to magnetic nanoparticles is associated with a profound quenching effect (Josephson, unpublished observations). The fluorescence quenching of magnetic nanoparticles may be due to nonradiative energy transfer between the dye and iron oxide or to collisions between the Cy5.5 and the nanoparticle. In the case of colloidal gold nonradiative energy transfer is believed to be the dominant mechanism of fluorescence quenching (23). The chemistry we have employed (Figure 1) offers many options for the design of optically activatable nanoparticles. It can be used to synthesize nanoparticles designed for activation by any protease by selecting peptides that are selective substrates for any protease. The method requires only that the peptide have a C-terminal cysteine, a reactive N-terminal amino that reacts with the N-hydroxysuccinimide esters of fluorochromes and a lack of primary amino groups elsewhere on the peptide. The R4 peptide meets these requirements, since the guanidyl groups of the arginine are unreactive NHS esters (24). The optical nanoparticle probes we have designed utilize polyarginyl peptides to link the fluorophore Cy5.5 to the nanoparticle. Sequences of basic amino acids such as polyarginine can be cleaved by trypsin, a convenient protease for in vitro studies with specificity for basic amino acids. In vivo Cy5.5-R4-SC-CLIO can be activated by proteases with a specificity for basic amino acids such as cathepsin B, which cleaves optical probes such as Cy5.5-PGC which has a polylysine backbone (7, 17). Cathepsin B is a widely distributed enzyme found in high concentrations in the organs of the reticuloendothelial system (RES) (25, 26). It has been proposed to play a role in tumor invasiveness (27, 28) and been examined as a prognostic marker in breast cancer (29, 30) and small cell lung carcinomas (31, 32). The attachment of polyarginyl nanoparticles to nanoparticles may result in particles with membrane translocating properties similar to tat-CLIO (10, 33). Positively charged peptides derived from the tat protein of HIV (GRKKRRQRRRPPQ) or from homeoprotein transcription factors (RQIKIWFQNRRMKWKK) act as internalization signals for a wide variety of cells (33-35). We have recently demonstrated that after iv injection a TatCLIO nanoparticle is translocated into cells and concentrated in the nucleus, in a manner similar to that obtained when the reagent is incubated with cells in culture (Wunderbaldinger, Josephson and Weissleder, in preparation). Since the guanidine headgroup of arginine seems to be all that is required for a substantial degree of membrane translocating activity (36, 37) nanoparticles with simple polyarginyl peptides may be able to efficiently enter cells other than macrophages. The importance of macrophage activation for a variety of medically important conditions, the association of macrophage activation with elevated levels of proteases, and the ability of macrophages to concentrate nanoparticles after intravenous injection suggest that the optical magnetic nanoparticle probes described here might initially be employed to measure macrophage activation in vivo. The activation state of macrophages is a key element in a wide range of diseases, ranging from the host defense against cancer cells (38) to the response to infectious microorganisms (39). Immunological activation
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of macrophages in response to stimuli such as mineral oil, thioglycolate and gamma-interferon is associated with increased levels of proteases such as cathepsins B and D (40, 41). The development of macrophage-targeted optically active nanoparticles is aided by the fact that similar particles are in clinical use and accumulate in macrophages of the liver, spleen, and lymph nodes (4, 5, 42). Following the chemistry developed here, nanoparticle probes might be designed which reveal the location of macrophages that clear nanoparticles from circulation, while providing a fundamentally new type of information on the immunological activation state of the macrophages that have sequestered them. The ability to obtain satisfactory MR and optical images with the nanoparticle/ optical probe described here, together with use of more sensitive and sophisticated equipment for fluorochrome detection in vivo, suggests that future materials of this type might be developed for eventual clinical use. ACKNOWLEDGMENT
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