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A Dual Fluorochrome Probe for Imaging Proteases Moritz F. Kircher, Ralph Weissleder, and Lee Josephson* Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129. Received August 27, 2003; Revised Manuscript Received December 12, 2003
Near-infrared fluorescence (NIRF) optical probes have been able to provide a noninvasive assessment of enzyme activity for a number of different enzymes and types of pathology. Here we describe a dual fluorochrome enzyme-activatable probe featuring one NIRF fluorochrome that is activated by protease activity and a second fluorochrome that is protease resistant and serves as an internal standard. The probe was prepared by attaching Cy7 directly to an amino-CLIO, an amine functional cross-linked iron oxide (CLIO) nanoparticle carrier, in a protease resistant manner. Cy5.5 was attached to a protease sensitive polyarginine peptide spacer, also attached to amino-CLIO. In vitro and in vivo the ratio of the Cy5.5 to Cy7 fluorescence was increased by protease, reflecting the increase in Cy5.5 fluorescence by protease in the vicinity of the probe. In vitro and in vivo the absolute values of the Cy5.5 and Cy7 fluorescence reflected lesion size and the distance of lesions from the surface, while the ratio of Cy5.5 to Cy7 fluorescence obtained was constant and independent of lesion size and depth. The dual fluorochrome probe, and related dual wavelength imaging method, represents a novel approach for imaging protease activity in vivo.
INTRODUCTION
In recent years a variety of so-called "smart” probes have been designed that respond to the presence of molecules in their environment with a change in signal. These include calcium indicators (1, 2), sensors of β-galactosidase activity (3), and fluorogenic substrates for a variety of hydrolytic enzymes (4). Recently, high molecular weight near-infrared fluorescent (NIRF) molecules have been synthesized which are quenched until activated by proteases, and these have been used to image protease activity in vivo (5, 6). These probes consist of a high molecular weight carrier, a cleavable peptide spacer or “stalk” attached through a C-terminal cysteine, and a fluorochrome attached to the N-terminus. These features of probe design are shown in Figure 1. Probes of this design have been used to visualize a variety of proteases including cathepsin D (7), MMP-2 (8), and thrombin (9). The high molecular carrier can be a graft copolymer or a magnetic nanoparticle, the latter serving as combined NIRF/optical and water relaxation enhancing/MR agent, a feature that is sometimes termed a multimodal imaging capability (10). Use of such probes to measure foci of enzyme activity in vivo was initially achieved with surface reflectancebased instrumentation (11), but more recent tomographic methods have been used to obtain tissue concentrations of the fluorochrome released as a product of protease activity (12). However, the absolute value of fluorescence obtained is a function of the intensity of incident light and the depth and size of the lesion. In addition, the amount of enzyme product produced by protease reflects not only the enzyme activity but also the delivery of substrate (substrate concentration). We previously reported on a dual probe-based imaging * Corresponding author: Lee Josephson, Ph.D., Center for Molecular Imaging Research, Massachusetts General Hospital, Bldg. 149 13th Street, #5406, Charlestown, MA 02129. Tel.: (617) 726 6478. Fax: (617) 726 5708. E-mail: ljosephson@partners. org.
Figure 1. Dual fluorochrome imaging strategies. Dual fluorochrome ratio imaging methods began with two probes with peptide stalks of different proteolytic susceptibility (13). Proteolytic cleavage of a peptide by an enzyme (scissors)-activated fluorescence. Here the dual fluorochrome probe consists of a single probe and two fluorochromes. One fluorochrome (Cy5.5) is activatable (cleaved by scissors), while a second (Cy7) serves as an internal standard. The probe is denoted Cy5.5-R4-Cy7CLIO where the peptide “stalk” is the L-arginyl peptide “R4” and the carrier is amino-CLIO.
method where an optical probe originally designed for use as a single enzyme-activated probe (10) was used together with a second, nondegradable probe employing a second, optically distinct fluorochrome (see ref 13). The nondegradable probe served as a standard for internalization by macrophages; macrophages expressing cathepsin B phagocytosed both probes similarly but could only activate one probe, while macrophages without cathepsin B could not activate either probe. Hence, the ratio of fluorescence provided a measure of cathepsin B activity corrected for variable levels of macrophage internalization. These results indicated the feasibility of using dual fluorochrome ratio imaging methods for the improved
10.1021/bc034151d CCC: $27.50 © 2004 American Chemical Society Published on Web 02/28/2004
Dual Fluorochrome Probe for Measuring Enzyme Activity
quantification of enzyme activity, but the method required the synthesis of two probes and their use as a mixture. Here we report on an improvement of our previous dual fluorochrome, dual probe method consisting of a single probe that features two fluorochromes. A schematic representation of these different approaches to dual fluorochrome probes is shown in Figure 1. We attached one fluorochrome (Cy5.5) via a cleavable peptide linker while a second fluorochrome (Cy7) was attached directly to a macromolecular carrier and was resistant to proteolytic activation. The amino-CLIO nanoparticle (CLIO ) cross-linked iron oxide) was employed as a carrier. The ratio of Cy5.5 to Cy7 fluorescence reflected activation by protease and could be used to correct for differences in the size and depth of a target lesions. MATERIAL AND METHODS
The dual fluorochrome probe was synthesized by first attaching Cy7 directly to the amino-CLIO carrier and then by attaching Cy5.5 to the N-terminus of a proteolytically cleavable L-polyarginine peptide (“R4”). The probe is denoted Cy5.5-R4-Cy7-CLIO. Attachment of the peptide R4 (R-R-R-R-G-C) to the nanoparticle is through the thiol of the C-terminal cysteine and was accomplished by use of the bifunctional agent succinimidyl iodoacetate (SIA). Cy5.5 is attached to the Nterminal arginine. A more complete synthetic scheme of the probe Cy5.5-R4-CLIO, a probe identical to that used here but lacking the Cy7 attached to the CLIO, has been provided; see refs 10 and 13. Synthesis of Cy7-CLIO. Amino-CLIO, a magnetic nanoparticle with a coating of cross-linked dextran and primary amino groups was prepared as described (14, 15). To 1 mL of amino-CLIO (10 mg Fe/mL) was added 100 µL of 10x PBS and 2.5 µL 0.5 M EDTA. The mixture was added to one Cy7 dye tube (1 mg) (Amersham-Pharmacica, Piscataway, NJ) and incubated for 1 h at room temperature and then overnight at 4 °C. Unbound Cy7 was then removed by spin-separation over a PD-10 column (Amersham) equilibrated with PBS, 1.25 mM EDTA. Synthesis of the Peptide-Nanoparticle, R4-Cy7CLIO. The L-arginyl peptide R-R-R-R-G-C (“R4”) was synthesized as described previously (13, 14). To 1 mL Cy7-CLIO (10 mg Fe/mL) were added 330 µL of 0.1 M Na2HPO4 and 330 µL of 150 mM succinimidyl iodoacetate in DMSO. The mixture 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 run at 4 °C. To the void volume (3 mL) was added 10 mg of R4 peptide, and the reaction was incubated overnight at room temperature. Unreacted peptide was removed by dialysis against 3 L of 0.02 M citrate pH 8.2 using a 14 kDa cutoff membrane. Synthesis of the Dual Fluorochrome Probe, Cy5.5-R4-Cy7-CLIO. To attach Cy5.5, 140 µL of 1 M NaHCO3 (pH 8.3) was added to 3 mL of R4-Cy7-CLIO. The mixture was then distributed to 6 tubes of Cy5.5. After 2 h at room temperature, the tubes were pooled and 10% volume of 1.5 M hydroxylamine was added. The mixture was allowed to sit for an additional 2 h at room temperature and separated using PD-10 columns (equilibrated with 0.02 M citrate buffer, pH 8.2) run in a spin separation mode. One milliliter of sample was added to columns that were spun at 830g for 5 min. Physical Properties of Cy5.5-R4-Cy7-CLIO. Spectra of conjugates were taken using a Hitachi 3500
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spectrophotometer. The number of Cy5.5 and Cy7 dyes present was determined spectrophotometrically, using an extinction coefficient of 250 000 M-1 cm-1 at 675 nm for Cy5.5, and 200 000 M-1 cm-1 at 747 nm for Cy7. Iron concentration was determined spectrophotometrically (14). The ratio of fluorochromes to nanoparticles was obtained assuming 2064 iron atoms per crystal (16). R1 and R2 relaxivities were measured using a 0.47T MR relaxometer (14). Size was determined by laser light scattering using a Zetasizer 1000 (Malvern, Worcestershire, UK). Activation of Fluorescence by Trypsin. Cy5.5R4-Cy7-CLIO fluorescence was determined in a microtiter plate with 0.01 M Na2HPO4, pH 7.4, 50 µg Fe/mL of Cy5.5-R4-Cy7-CLIO, and 10 µg/mL trypsin. The plate was incubated at room temperature and Cy7 and Cy5.5 fluorescence determined (ex 747, em 777 and ex 645, em 695, respectively) using a fluorescent microtiter plate reader (SpectraMAXGeminiXS, Molecular Devices, Sunnyvale, CA). Optical Imaging. To examine the ability of the dual fluorochrome probe to correct for lesion size, a homogenate prepared from the liver from a mouse injected with Cy5.5-R4-Cy7-CLIO was used. Magnetic nanoparticle based probes are internalized by the liver with activation of their fluorescence (10, 13). C57BL/6 mice (National Cancer Institute, Bethesda, MD) were injected via tail vein with 10 mg Fe/mL of Cy5.5-R4-Cy7-CLIO, and 24 h later mice were sacrificed and the livers excised and homogenized. Volumes of homogenate containing activated probe were transferred to 384-well plates to serve as models of enzyme activated “lesions” in vivo. To mimic lesions of different sizes, volumes were chosen to yield wells containing between 2 mm and 10 mm high volumes of homogenate. To demonstrate the ability of the dual fluorochrome probe to correct for lesion depth, liver from a noninjected mouse was transferred to a 384-well plate to achieve heights of 0 to 7 mm in 1 mm increments. Subsequently, 15 µL of 1% agarose containing 10 µg of Cy5.5-R4-Cy7-CLIO was added to each well with liver samples. To demonstrate the ability of the dual fluorochrome probe to correct for lesion size and depth, the 9L rat gliosarcoma cell line stably transfected to express green fluorescence protein (GFP) (17) was cultured at 37 °C in a humidified 5% CO2 atmosphere in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin and 500 µg/mL geniticine G418 (all products from Cellgro, Herndon, VA). 9L gliosarcomas were induced by means of bilateral injection of 9L glioma cells deep into the mammary fat pad of nude mice (n ) 3, Charles River, Wilmington, MA). To obtain tumors of different sizes, 0.5 × 106 cells were implanted on the right, and 2 × 106 cells in the left mammary fat pad. After 12 days, animals were anesthetized (ketamine/xylazine, 65/10 mg/kg) and 10 mg Fe/kg body weight Cy5.5-R4-Cy7-CLIO injected by tail vein. Twenty-four hours later, mice were imaged, euthanized by halothane inhalation, and underwent progressive dissection and further NIRF imaging. Optical imaging of the plates and mice was performed using a whole mouse imaging system as described (11). Cy5.5 fluorescence was obtained with an excitation bandpass filter at 630 ( 15 nm (630RDF30; Omega Optical, Brattleboro, VT) and emission band-pass filter at 700 ( 20 nm (700DF40; Omega Optical). Cy7 fluorescence was obtained with an excitation band-pass filter at 736 ( 20 (736AF40; Omega Optical) and emission band-pass filter at 800 ( 20 (800AF40; Omega Optical). Images were
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Table 1. Physical Properties of Cy5.5-R4-Cy7-CLIO size (nm)
R1 (s-1 mM-1)
R2 (s-1 mM-1)
Cy5.5 fluorochromes per particle
Cy7 fluorochromes per particle
28
16
45
2.3
2.5
acquired using a cooled CCD camera with 800 × 600pixel resolution (SenSys; Photometrics, Tucson, AZ). Kodak Digital Science 1D Software (Eastman Kodak; Rochester, NY) was used for acquisition and data analysis. RESULTS
Properties of the Dual Fluorochrome Probe Cy5.5-R4-Cy7-CLIO. The physical properties of Cy5.5-R4-Cy7-CLIO are summarized in Table 1. The probe uses a thioether linkage and an enzyme-cleavable L-arginine peptide (10, 13). Figure 2A shows the absorption spectrum of Cy5.5-R4-Cy7-CLIO, with a characteristic absorption of the iron oxide in the ultraviolet region of the spectrum and with the absorption maxima of Cy5.5 and Cy7 at 674 and 748 nm, respectively. Figure 2B shows the absorption spectra of Cy5.5-R4-Cy7CLIO in relation to the excitation and emission filters used. Also shown are the emission maxima for Cy5.5 (694 nm) and Cy7 (737 nm). As shown in Figure 2C an excellent separation of fluorescent signals from Cy5.5 and Cy7 was obtained with the optical imaging system used in this study. Enzyme Activation of Cy5.5-R4-Cy7-CLIO. Figure 3 shows the effect of treating Cy5.5-R4-Cy7-CLIO with trypsin. Trypsin cleaved the R4 peptide, producing a 700% increase in the Cy5.5 fluorescence after 60 min and 850% increase after 24 h (data not shown). In contrast, Cy7 fluorescence was nearly constant, as expected, because of the direct attachment of Cy7 to the cross-linked dextran coating of CLIO and inability to undergo enzymatic cleavage. The small increase of about 20% in Cy7 fluorescence may reflect a small quenching effect resulting from slight interaction between the two fluorochromes when in close physical proximity to each other (on the same probe). We next performed a series of experiments to determine whether the light associated with Cy5.5 fluorescence and Cy7 fluorescence would be attenuated (or transmitted) similarly in biological tissues. If Cy5.5 fluorescence and Cy7 fluorescence are attenuated similarly, the ratio of their fluorescence should reflect the activation of the probe and be independent of size and depth of the fluorescent lesion. Imaging with Cy5.5-R4-Cy7-CLIO in Vitro. To assess whether the ratio of Cy5.5 to Cy7 fluorescence depended on the size or magnitude of the fluorescence, we imaged phantoms consisting of a 384-well plate containing variable amounts of liver homogenate (Figure 4). A mouse was injected with 10 mg Fe/kg Cy5.5-R4Cy7-CLIO and liver homogenate containing probe plus a mixture of naturally occurring proteins and light absorbing materials, was placed in the wells of the plate at different heights (Figure 4A). The fluorescence from Cy5.5 and Cy7 and the ratio of Cy5.5 to Cy7 fluorescence was obtained as shown in Figures 4B and 4C. Cy5.5 and Cy7 fluorescence intensities increased with increasing amounts of homogenate, however, for both fluorochromes fluorescence tended to plateau at higher amounts of homogenate. This reflects the fact that the NIRF signal obtained is strongly surface weighted with this type of instrumentation. However, the ratio of Cy5.5 to Cy7 fluorescence was nearly constant over a 7-fold change in
Figure 2. (A) Absorption spectra of Cy5.5-R4-Cy7-CLIO. (B) Magnification of (A) with indication of emission and excitation filters used in the imaging experiments. (C) Spectral separation of imaging channels. Images of a 96-well plate containing free Cy5.5, free Cy7, or buffer only, respectively. Note the clear channel separation despite high fluorochrome concentration.
homogenate height (Cy5.5/Cy7 ) 6.43 at 1 mm versus 6.13 at 7 mm). With the liver homogenate the dual fluorochrome probe and ratio imaging method yielded a value of Cy5.5/Cy7 fluorescence of 6 to 7 which was characteristic of the activated probe in vitro, see Figure 3. We next assessed whether the ratio of Cy5.5 to Cy7 fluorescence could correct for a constant lesion at variable depths from the surface. For this purpose, a constant amount of agar containing unactivated Cy5.5-R4-Cy7CLIO was placed on top of variable amounts of liver tissue from a noninjected animal (Figure 5A). Figures 5B and 5C show that Cy5.5 and Cy7 fluorescence decreased with increasing depths of liver. However, the ratio of Cy5.5 to Cy7 fluorescence was nearly constant when light passed through 1 to 10 mm of liver (Cy5.5/Cy7 ) 1.70 at 1 mm versus 1.64 at 10 mm). The Cy5.5/Cy7 ratio of
Dual Fluorochrome Probe for Measuring Enzyme Activity
Figure 3. Activation of the dual fluorochrome probe Cy5.5R4-Cy7-CLIO by trypsin. (A) Time-course measured with fluorescent plate reader. Cy5.5 fluorescence (∆), Cy7 fluorescence (9). (B) Images representing ratio Cy5.5/Cy7 at 0 and 60 min of activation by trypsin. Orange color corresponds to probe activation or to a Cy5.5/Cy7 fluorescence ratio of about 7. Cy5.5 fluorescence is increased by proteolytic cleavage while Cy7 fluorescence is nearly constant.
about 1.7 was slightly higher than the ratio of native probe (1.0-1.2, see Figure 2). The slight probe activation presumably results from mixing at the interface between native probe (clear solution) with enzymes in liver tissue (brown tissue). Imaging with Cy5.5-R4-Cy7-CLIO in Vivo. We next tested the ability of the dual fluorochrome probe to correct for differences in tumor size and depth in vivo. Nude mice bearing 9L GFP expressing gliosarcomas of different sizes (Figure 6) were injected with Cy5.5-R4Cy7-CLIO. Cy5.5 and Cy7 fluorescence from tumors were then determined, where tumor borders (regions of interest) were defined by tumor GFP fluorescence using our previously described technique (18). Figure 6 shows white light, fluorescent, and Cy5.5/Cy7 ratio images for an intact animal with tumors of different size as dissection progressed. With the intact animal, the Cy5.5 and Cy7 fluorescence intensities of the two tumors were markedly different, a difference which was not seen on the ratio of Cy5.5/Cy7 image (top row, right image). The ratio of Cy5.5 to Cy7 fluorescence for the two tumors (12 mg and 195 mg) was further independent of dissection level. Results with variable tumor size were similar to in vitro results obtained with variable amounts of homogenate (Figure 4). DISCUSSION
Here we report on a novel type of multimodal probe that features two fluorochromes covalently linked to an MRI detectable carrier (amino-CLIO), in which each fluorochrome responds differently to proteases. While we have utilized only the optical properties of the Cy5.5R4-Cy7-CLIO probe, the superparamagnetic iron oxide
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core of the CLIO nanoparticle has potent relaxation enhancing properties and can be used in conjunction with MRI to determine probe location (14, 15). The feasibility of optical- or magnetic resonance-based imaging in vivo using a probe similar to Cy5.5-R4-Cy7-CLIO but lacking Cy7 has been shown (10). We have shown that fluorescent activation occurs, not by direct interaction between proteases or reducing agents and the fluorochrome, but with the transfer of the fluorochrome from the local environment of the nanoparticle to the bulk solvent (10). Cleavage of the bond between a fluorochrome and a nanoparticle has been achieved with proteases such as trypsin or with DTT (dithiothreitol). Use of the latter reagent requires disulfide-linked fluorochromes, synthesized by use of the bifunctional cross-linking agent N-succinimidyl 3-(2pyridyldithio)propionate (SPDP), in contrast to the bifunctional agent succinimidyl iodoacetate (SIA) used in the current study. DTT could not activate fluorescence when a thioether linkage was used to attach the fluorochrome to the nanoparticle (see Figure 3 of ref 10), and proteases could not activate fluorescence when protease resistant D-arginyl peptides were used to couple the fluorochrome to the nanoparticle (13). The activation of Cy5.5 fluorescence by trypsin of (700%, Figure 3A) therefore results from the proteolytic cleavage of the R4 peptide, which transfers Cy5.5 from the local environment of the nanoparticle to the bulk solvent. The much lower activation of Cy7 fluorescence (about 20% Figure 3A) reflects Cy7’s direct attachment to the nanoparticle, its resistance to proteolysis, and the inability of the protease to affect a transfer to the bulk solvent. A second feature of fluorescence quenching by nanoparticles is that when fluorochromes with widely different optical properties (fluorescein, Cy5.5, or Cy3) are attached to CLIO nanoparticles, significant quenching results (see ref 10 and Josephson, unpublished observations). Fluorescence quenching also occurs when fluorochromes are attached to colloidal gold (19). In the case of CLIO nanoparticles, quenching reflects two types of effects: interactions between fluorochromes on the nanoparticle, and interactions between fluorochromes and the nanoparticle itself. The effects of quenching due to fluorochrome/nanoparticle interaction, rather than fluorochrome/fluorochrome interaction, were evident from the synthesis of nanoparticles with as little as 0.14 fluorochromes (average per nanoparticle). Such probes still showed greater than 200% activation of fluorescence when treated by DTT, and the fluorochrome was transferred to the bulk solvent (10). For colloidal gold and the colloidal CLIO nanoparticle, a major mechanism of quenching is the interaction between the fluorochrome and the nanoparticles, which may reflect collisions of the dye against the nanoparticle surface (collisional or dynamic quenching) (19). The value of the dual fluorochrome probe and related imaging methods is not related to the specific protease(s) responsible for fluorescence activation. As noted above, probes of this general design (high molecular weight carrier, linker peptide, fluorochrome) have been designed to image a wide variety of proteases by varying the structure of the linker peptide. Several factors suggest that the activation of the Cy5.5-R4-Cy7-CLIO probe described here results from cathepsin B activity. First, using specific protease inhibitors and macrophages in vitro, we have shown that the protease activating Cy5.5R4-CLIO, the single fluorochrome version of the dual fluorochrome probe of the current study, was cathepsin B (13). Second, as noted, a nanoparticle probe made with
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Figure 4. Dual fluorochrome probe imaging with different amounts of liver homogenate. (A) White light image of wells containing different amounts of liver homogenate. Liver is from mouse injected with Cy5.5-R4-Cy7-CLIO. (B) NIRF images of wells in A in the Cy5.5 and Cy7 channels and resulting ratio Cy5.5/Cy7 image (color-coded). Orange color corresponds to a Cy5.5/Cy7 ratio of about 7, which is characteristic of the activated probe. (C) Values of fluorescence intensity and the ratio of Cy5.5/Cy7 fluorescence obtained by region of interest analysis from images in B.
Figure 5. Dual fluorochrome probe imaging through different depths of liver tissue. (A) White light image of wells containing liver from a noninjected animal overlayed with a constant amount of Cy5.5-R4-Cy7-CLIO in 1% agar. (B) NIRF images of wells in A in the Cy5.5 and Cy7 channels and resulting ratio Cy5.5/Cy7 image (color-coded). (C) Values of fluorescence intensity and ratio of Cy5.5/Cy7 fluorescence obtained from images in B.
noncleavable D-arginine peptide spacer could not be activated by macrophages (13). Third, macrophages including Kupffer cells of the liver are high in cathepsin B (20-22). After injection, CLIO nanoparticles are removed by phagocytosis and are concentrated in high
cathepsin B activity containing macrophages (17, 23, 24). Finally, cathepsin B hydrolyzes peptides with arginine in the S2 or S1 subsites of the protease, which are provided by L-polyarginine peptides used to couple the Cy5.5 due to the nanoparticle (25, 26).
Dual Fluorochrome Probe for Measuring Enzyme Activity
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Figure 6. Dual fluorochrome ratio imaging of implanted 9L GFP expressing gliosarcoma tumors. A nude mouse bearing tumors of different sizes (12 mg (left) and 195 mg (right), respectively) implanted in the mammary fat pads was injected with Cy5.5-R4Cy7-CLIO. From left to right are shown the white light image, the Cy5.5 fluorescent image, the Cy7 fluorescence image and the ratio of Cy5.5 to Cy7 fluorescence (color-coded). Note that despite the variation in signal intensity caused by the differences in tumor size and differences in dissection level (depth), the resulting ratio is similar reflecting the ability of the intenal standard Cy7 to correct for size and depth.
The optical properties of the Cy5.5 (ex 675, em 694) and Cy7 (ex 747, em 777) were found to be well suited to their use in dual fluorochrome probes. First, the fluorescence from each fluorochrome was detected without significant interference from each other (Figure 2). Second, the interaction between the two fluorochromes when attached to the probe was minimal. Proteolytic cleavage and transfer of Cy5.5 to the bulk solvent was associated with a 700% increase in fluorescence, while the increase in Cy7 fluorescence was only about 20% (Figure 3A), which presumably reflects interactions between Cy5.5 and Cy7. If Cy5.5 and Cy7 interacted strongly to quench each other, then both would be dequenched by proteolytic cleavage of either. Third, the ratio of Cy5.5 to Cy7 fluorescence reflected probe activation both in vitro and in vivo. Thus a value of 1 was characteristic of the probe before exposure to proteases but increased to 7 in the presence of protease (Figure 1). A similar value was obtained in liver homogenates after iv injection of the probe (Figure 4). Fourth, the ratio of Cy5.5 to Cy7 fluorescence was independent of sample size (Figures 4 and 6) and tissue depth (Figure 5). The dual fluorochrome multimodal probe using the Cy5.5/Cy7 fluorochrome pair, and related dual fluorochrome imaging, may permit use of this type of probe to gather a new type of information with in vivo imaging, in addition to the corrections for lesion size and depth demonstrated here. Currently the interaction of a probe with a molecular target, such as a protease, can reflect both the activity of the molecular target enzyme in a specific tissue, and the transport and concentration of the probe in the vicinity of the molecular target. Factors affecting probe transport include blood flow, capillary permeability, capillary blood volume, and other physiological factors. In these studies the dual fluorochrome probe and imaging method provided information on probe activation that is independent of the absolute value of Cy5.5 fluorescence, which depended on parameters such as lesion size and depth. Hence with the dual fluoro-
chrome probe design used here, the Cy7 fluorescence might provide an internal standard for determining probe concentration, allowing Cy5.5 fluorescence to be corrected for variable levels of probe transport. However, proof of this hypothesis that Cy7 fluorescence can yield probe concentration in vivo will require determination of probe concentrations by a suitable reference “gold standard” bioanalytical method. Such studies now appear warranted based on the properties of the Cy5.5-R4-Cy7CLIO probe described here. ACKNOWLEDGMENT
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