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Abstract

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)₄ presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin a_Vb₃ with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a “stealth” molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)₄-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)₄-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)₄-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)₄-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)₄-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.

MOLECULAR IMAGING OF RECEPTORS expressed on the surface of tumor cells is becoming a major field of investigation in clinical oncology, especially for the detection of cancer at its earliest stages. The development of specific tracers is thus of the utmost importance. Moreover, since they can target tumor cells specifically, these tracers could also be used as drug delivery vectors. Ultimately, it will be interesting to define a multifunctional molecule combining targeted drug delivery and imaging of its therapeutic activity. This requires the generation of multifunctional carrier molecules, onto which several functions can be loaded in a stereospecific and chemoselective manner. In this aim, our group developed a scaffold named RAFT (regioselectively addressable functionalized template), which is a cyclic decapeptide forming a ring with two faces.¹ On the upper face, four copies of the cyclo[-RGDfK-] peptide were grafted for recognition of the α_vβ₃-integrin receptor overexpressed on the tumor vasculature and on the surface of tumor cells. The bottom face of the RAFT can be used to link a drug and a “smart” contrast agent for in vivo molecular imaging.

In our recent publications, Cy5-labeled RAFT-c (-RGDfK-)₄ was shown to improve the tumor to normal tissue contrast compared with its monomeric analog, cyclic (Arg-Gly-Asp) (cRGD).^2,3 This was the result of its augmented specificity and longer time of retention in the tumor. The present study was aimed to improve RAFT-c(-RGDfK-)₄ performance by introducing an activatable linker and applying the concept of “smart-probes,” initially described by Weissleder and colleagues.⁴ “Smart” sensors reduce signal to noise ratios because they can be detected only once they have interacted with their substrate. These agents, primarily developed for optical and magnetic resonance imaging, are relatively undetectable in their native injected state. Examples of such agents include quenched near-infrared (NIR) fluorochromes that can be activated by tumor-associated enzymes such as cathepsin D and B,^5,6 matrix metalloproteinase 2,⁷ and caspases.⁸ The quenching can occur if the distance between two fluorescent molecules or between a fluorophore and a quencher molecule is very small. If a spatial separation of the two molecules occurs after cleavage of the covalent link, the fluorescence will not be quenched anymore. In Weissleder and colleagues' work, the design and synthesis of self-quenched multiple molecules of NIR cyanine dyes (Cy5.5) loaded on a long poly-l-lysine copolymer were described.⁴ Tumor accumulation was achieved by the long circulation time and passive leakage through tumor neovasculature, and internalization of polymer dye conjugate occured by fluid-phase endocytosis. After cleavage of the copolymer by the proteases, unquenched Cy5.5 molecules were released, leading to the site-specific apparition of photons.

In this study, we developed a self-quenched RAFT-c (-RGDfK-)₄-Cy5 by linking a fluorescence quencher (Q) to Cy5 via a disulfide bond (-SS-). This short linker was chosen because its cleavage is usually associated with the trafficking of endocytosed macromolecules.^9,10 We report the evaluation of RAFT-c(-RGDfK-)₄-Cy5-SS-Q as an activatable optical probe giving site-limited signals owing to α_vβ₃ integrin–mediated internalization and subsequent disulfide cleavage both in vitro and in vivo. RAFT-c (-RGDfK-)₄-Cy5-SS-Q may be helpful for in vivo monitoring of RGD/integrin intracellular trafficking and to achieve a higher tumor to noise ratio.

Materials and Methods

Synthesis of Quenched Cy5-RAFT-c(-RGDfK-)4 Conjugate

The synthesis of RAFT-c(-RGDfK-)₄ and its labeling with Cy5 have been reported.^1,2 The protocol for synthesis of cysteine(Cy5)-SS-cysteine(QSY21), which is conjugated to RAFT, is described as follows: Cy5 mono-NHS (N-hydroxysuccinimide) ester and quencher QSY21-NHS ester were purchased from Amersham Biosciences (Uppsala, Sweden) and Molecular Probes (Invitrogen SARL, Cergy Pontoise, France), respectively. Other reagents were from Aldrich (Sigma-Aldrich Chimie, Lyon, France) or Acros Organics (Geel, Belgium). Reverse phase high-performance liquid chromatography (RP-HPLC) was performed on Waters equipment fitted with a 600 controller and a Waters (Waters SAS, Guyancourt, France) 2487 Dual Absorbance Detector. The purity of peptide derivatives was analyzed on an analytic column (Macherey-Nagel Nucleosil 120–3 mm C₁₈ particles, 30 × 4.6 mm) using the following solvent system: solvent A, water containing 0.09% trifluoroacetic acid (TFA), and solvent B, acetonitrile containing 0.09% TFA and 9.91% H₂O; a flow rate of 1.3 mL min⁻¹ was employed with a linear gradient (5–100% B in 15 minutes). Ultraviolet absorbance was monitored at 214 and 250 nm simultaneously. Semipreparative column (Delta-Pak 100 Å 15 mm C₁₈ particles, 200 × 2.5 mm) was used to purify crude peptides (when necessary) by using an identical solvent system at a flow rate of 22 mL min⁻¹. Electrospray ionization (ESI) mass spectra were recorded on an Esquire 3000 (Bruker, Courtaboeuf, France) spectrometer. The analysis was performed in the positive mode for peptide derivatives using 50% aqueous acetonitrile as eluent.

Starting with cyclic decapeptide c[-K(boc)-K(alloc)-K(boc)-P-G-K(boc)-A-K(boc)-P-G-] (520 mg, 0.34 mmol), Boc moieties were removed using a solution containing 50% TFA in CH₂Cl₂ (50 mL) for 15 minutes at room temperature (RT). The crude product was concentrated, triturated, and washed with ether to obtain a white powder that was subsequently treated for 30 minutes at RT with BocNHOCH₂CO-NHS (360 mg, 1.25 mmol, 4.3 equivalent) and diisopropylethylamine (DIPEA) (pH 8.0) in dimethy formamide (DMF) solution (30 mL). The alloc group was removed in dry CH₂Cl₂ (30 mL) under argon by adding successively phenylsilane (1.8 g, 17 mmol) followed after 3 minutes by Pd(PPh₃)₄ (79 mg, 68 mmol). The crude product was dissolved in CH₂Cl₂/CH₃OH (1:1), precipitated, and washed with ether (3×), affording a white powder (464 mg, 0.27 mmol, 80%, four steps). The crude peptide (162 mg, 100 μmol) was dissolved in 12 mL of dry DMF with DIPEA (46 μL, 0.26 mmol). After addition of BocCys(Npys) (25 mg, 0.066 mmol) and PyBOP (42 mg, 0.08 mmol), the pH was adjusted to 8.0 and the mixture was stirred at RT for 45 minutes. The solvent was then removed under reduced pressure. The residue was dissolved in dichoromethane (DCM), and the peptide was precipitated and washed with ether, affording a white solid (215 mg, 100 μmol). The mass (ES-MS, positive mode) was found (2,070.9) in perfect agreement with the calculated mass (2,070.4). The peptide was then dissolved in 12 mL of a solution containing TFA-DCM-triisopropylsilane (TIS)-H₂O (9:9:1:1), and the solution was stirred at RT for 40 minutes. The solvents were then removed under diminished pressure, and the residue was dissolved in H₂O/CH₃CN and purified by RP-HPLC, affording (78 mg, 43 mmol, 43%, two steps) the pure product as a white powder. The mass (ES-MS, positive mode) was found (1,569.4) in perfect agreement with the calculated mass (1,569.8). The purified cyclic decapeptide (78 mg, 43 mmol) dissolved in 2 mL of H₂O/CH₃CN (1:1) was then added dropwise to a solution of H₂O/CH₃CN (1:1) (9 mL) containing cyclic [-R-G-D-f-K(CO-CHO)-] (198 mg, 0.3 mmol). The solution mixture was stirred at RT for 4 days and then lyophilized to obtain the RGD-containing peptide as a white solid (200 mg, 43 mmol, 34% overall yield). The mass (ES-MS, positive mode) was found (2,068.8) in perfect agreement with the calculated mass (2,069.3).

The pure peptide (284 mg, 43 mmol) was dissolved in 16 mL of a degassed solution containing DMF and phosphate-buffered saline (PBS) (3:1, pH 4.8). Bocprotected cysteine (10.5 mg, 0.047 mmol) dissolved in 200 μL of DMF-PBS (3:1, pH 4.8) was then added, and the mixture was stirred under argon at RT for 1 hour. The product was purified by RP-HPLC to obtain the peptide as a white powder (131 mg, 27 μmol, 64%). The mass (ES-MS, positive mode) was found (2,101.1) in perfect agreement with the calculated mass (2,101.8).

To a solution containing the peptide (19.9 mg, 4.45 μmol) in 400 μL of dry DMF was added a solution of Cy5-N-hydroxysuccinimide (NHS) (3.1 mg, 3.91 μmol) in 250 μL of dry DMF with DIPEA (2.6 μL, 15 mmol). The solution mixture was stirred at RT for 48 hours. The solvent was then removed under reduced pressure to obtain the peptide as a deep blue solid. The crude peptide was then dissolved in 6 mL of solution containing TFA-H₂O-TIS (95:2.5:2.5), and the solution was stirred at RT for 45 minutes. The solvents were removed under reduced pressure. The residue was dissolved in DMF-H₂O-CH₃CN and purified by RP-HPLC to afford the product as a deep blue powder (11.5 mg, 2.4 mmol). The mass (ES-MS, positive mode) was found (2,370.2) in perfect agreement with the calculated mass (2,370.7). To a solution containing the Cy5-containing peptide (5.4 mg, 1.06 μmol) in 300 μL of dry DMF was added a solution of QSY21-NHS (1.13 mg, 1.39 μmol) in 200 μL of dry DMF with DIPEA (0.9 μL, 5.3 μmol). The solution mixture was stirred at 45°C for 16 hours. The product was then purified by RP-HPLC to produce RAFT-c(-RGDfK-)₄-Cy5-SS-Q (Figure 1A) as a deep blue powder (1.2 mg, 0.22 μmol, 13% overall yield) of the pure product (Figure 1B). Mass spectrum: calculated 2,703.1 (M+2/2), found 2,702.8 (Figure 1C).

Throughout the following experiments, RAFT-c (-RGDfK-)₄-Cy5-SS-Q and RAFT-c(-RGDfK-)₄-Cy5 were dissolved in a PBS solution containing 10% dimethyl sulfoxide and 10% ethyl alcohol. The working concentration for each probe was checked by RP-HPLC by comparing its peak area with that of a control solution with a known concentration. In addition, the absorption and fluorescence emission properties of the two probes were very similar to those of free Cy5.

Cell Lines and Culture Conditions

HEK293(β₃) cells, stable transfectants of the human integrin β₃ subunit from the human embryonic kidney cell line (kindly provided by Dr. J.-F. Gourvest, Aventis, France), were cultured in Dulbecco's Modified Eagle's Medium (DMEM) enriched with 4.5 g L⁻¹ glucose and supplemented with 1% glutamine, 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, and 700 μg/mL Geneticin (G418 sulfate, Gibco, Paisley, UK). Human ovarian adenocarcinoma IGROV1 cells were kindly provided by Dr. Laurent Poulain (Centre de lutte contre le cancer François Baclesse, Caen, France) and maintained in RPMI 1640 supplemented with 10% FBS and the antibiotics mentioned above. All cell lines were cultured at 37°C in a humidified 95% air–5% CO₂ atmosphere.

In Vitro Optical Imaging of -SS- Cleavage

Fourteen microliters of a 15 μM solution of RAFT-c (-RGDfK-)₄-Cy5-SS-Q or RAFT-c(-RGDfK-)₄-Cy5 was loaded in a 0.5 mL Eppendorf tube and imaged using our optical imaging system. Two microliters of 2-mercaptoethanol (β-ME), a reducing reagent for -SS-, was then added into the tube at a final concentration of 178 mM and mixed immediately. Dynamic imaging was then continued for 10 minutes.

Real-Time Imaging of Live Cells in Culture

HEK293(β₃) or IGROV1 cells were seeded on Lab-Tek (Bioblok Scientific, Illkirch, France) chambered coverglass and incubated overnight at 37°C. The culture medium was then replaced with fresh phenol red–free DMEM/F-12 medium containing RAFT-c(-RGDfK-)₄-Cy5-SS-Q or the control probe at final concentrations of 0.1 and 0.5 μM for HEK293(β₃) and IGROV1 cells, respectively. Immediately after probe loading, the cells were observed using confocal laser scanning microscopy (CLSM) (LSM510, Zeiss, France) using a Plan-Neofluar 40×/1.30 oil objective in a culture chamber at 37°C in a humidified 95% air–5% CO₂ atmosphere. Time series of images were recorded to monitor the kinetics of Cy5 fluorescence, and simultaneous z-stacks of images were also performed to adjust the signal shifting owing to the slight movement of medium. The combination of a 635 nm He/Ne laser and 685 nm emission filters was used to visualize the Cy5 signal. Simultaneous phase contrast images were also acquired to distinguish the signal location. Image processing was handled by LSM5 Image Browser software (Zeiss, Le Pecq, France). For colocalization studies, RAFT-c(-RGDfK-)₄-Cy5-SS-Q and 50 nM LysoTracker Green DND-26 (Invitrogen Molecular Probes, Carlsbad, CA) were coincubated for 90 minutes. The LysoTracker probe is known to accumulate in acidic organelles, including lysosomes.

Figure 1.

Schematic molecule structure (A), reverse phase high-performance liquid chromatogram (B), and electrosprayionisation mass spectrum (C) of RAFT-c(-RGDfK-)₄-Cy5-SS-Q. UV = ultraviolet.

In Vivo Optical Imaging

Animal procedures were in agreement with the European community guidelines. Female athymic Swiss nude mice, purchased from IFFA-Credo (Marcy l'Etoile, France) at 6 to 8 weeks of age, were used and maintained under specific pathogen-free conditions. Mice were injected subcutaneously around the right flank with 5 × 10⁶ IGROV1 cells resuspended in 200 mL of PBS. When the tumors reached 6 to 8 mm in diameter (14–21 days after implantation), the mice were used for optical imaging after intravenous injection of RAFT-c(-RGDfK-)₄-Cy5-SS-Q (n = 4 mice) or the control probe (n = 3 mice) at 10 nmol per mouse.

Fluorescence reflectance imaging was performed using a Hamamatsu optical imaging system described previously.^2,3 In brief, imaging was carried out in a dark box, and the anesthetized animal or probe-loaded tubes, as described previously, were illuminated with a monochromatic 633 nm light (50 μW cm⁻²). The reemitted fluorescence was filtered using a colored glass filter RG 665 (optical density > 5 at the excitation wavelength 633 nm) and collected with a cooled (–70°C) digital charge-coupled device camera (Hamamatsu digital camera C4742-98-26LWGS, Hamamatsu, Massy, France). Image acquisition parameters were kept constant throughout the experiment, and exposure time was set at 100 milliseconds for the animal and 20 milliseconds for the probe solution–loaded tubes. Images were acquired as 16-bit TIFF files, which can provide a dynamic of up to 65,535 gray levels. Image processing used in this study, including setting LUT (look-up-table) range and measurement of the fluorescence intensity for each region of interest, was performed using Wasabi software (Hamamatsu). It is also important to note that all of the images are presented without background subtraction. For quantifying tumor contrast, the mean fluorescence intensities of the tumor areas (T) and those of the distant skin areas (S) were calculated; dividing T by S yielded the ratio between tumor tissues and background level.

Histologic Distribution of RAFT-c(-RGDfK-)4-Cy5-SS-Q in Tumors

Three hours after intravenous injection of 10 nmol RAFT-c(-RGDfK-)₄-Cy5-SS-Q or of the control probe in mice bearing IGROV1 subcutaneous tumors, tumors were sampled, frozen in liquid nitrogen, and stored at −80°C. Thick sections (20–30 μm) were fixed with 2% paraformaldehyde at RT for 10 minutes. The nuclei were stained with 5 μM Hoechst 33342, and the coverslips were mounted using MOWIOL (Calbiochem, San Diego, CA) and kept at 4°C in the dark until observation. Endothelial cells present in the blood vessels of IGROV1 subcutaneous tumors were visualized on methanol-fixed cryosections (5–7 μm thick) by immunostaining with a rat antimouse CD31 monoclonal antibody (clone MEC13.3 diluted 3,000 times; BD Biosciences PharMingen, San Diego, CA), followed by incubation with Strept-ABComplex/HRP (DakoCytomation, Glostrup, Denmark) and counterstaining of the nuclei with hematoxylin.

Statistical Analysis

All data are given as mean ± standard deviation of n independent measurements. Statistical analysis was performed using the two-tailed nonparametric Mann-Whitney t-test. Statistical significance was assigned for values of p < .05.

Results

Quenching and Dequenching Efficiency of RAFT-c(-RGDfK-)4-Cy5-SS-Q

When 14 μL of a 15 μM solution of RAFT-c(-RGDfK-)₄-Cy5 was exposed to the camera, a very strong Cy5 signal could be measured (Figure 2). This signal was weakly reduced after the addition of 2 μL of β-ME.

In contrast, when RAFT-c(-RGDfK-)₄-Cy5-SS-Q was used, the number of emitted photons was reduced 13.5 times, demonstrating the quenching efficiency. Moreover, addition of 2 μL of β-ME restored almost completely the fluorescence coming from the 15 μM of Cy5 after reduction of the -SS- bridge (see Figure 2), indicating that a complete dequenching effect can be achieved from RAFT-c(-RGDfK-)₄-Cy5-SS-Q.

Figure 2.

In vitro fluorescence dequenching in the presence of a reducing agent. RAFT-c(-RGDfK-)₄-Cy5-SS-Q and RAFT-c(-RGDfK-)₄-Cy5 solutions loaded in an Eppendorf tube were imaged before and after the addition of 2-mercaptoethanol (β-ME) at a final concentration of 178 mM for up to 10 minutes. A, Fluorescence images (no background subtraction) before and 10 minutes after the addition of β-ME. All images were normalized to the same scale of gray levels. B, Quantification of fluorescence intensity. The fluorescence intensity, recorded as total photons in a specified region of interest (ROI), was calculated after subtraction of the background fluorescence in a similar ROI. The arrow on the x-axis indicates when β-ME was added.

Active Dequenching of RAFT-c(-RGDfK-)4-Cy5-SS-Q during Intracellular Trafficking

RAFT-c(-RGDfK-)₄ probes were then incubated with live IGROV1 or HEK293(β₃) cells and observed using a confocal microscope. It is important to note that the incubation media were not changed during the completion of these experiments (Figure 3A). In addition, it was necessary to use a low concentration of peptides to reduce as much as possible the shrinking of the cells normally observed when RGD peptides are binding to integrins. To visualize the presence of 0.5 μM Cy5-labeled peptide on IGROV1 cells expressing low levels of α_vβ₃,³ the laser power had to be elevated (30% of its full power). This was associated with a very intense signal coming from the RAFT-c(-RGDfK-)₄-Cy5 in solution, greatly hampering the visualization of the specific signal coming from labeled cells (see Figure 3A, I). In contrast, this background signal was absent when using the quenched molecule (see Figure 3A, II), and the intracellular vesicles containing the “activated smart probe” were then easily detected.

A similar experiment performed on HEK293(β₃) cells produced identical results (see Figure 3A, III and IV). But since HEK293(β₃) cells expressed very high levels of α_vβ₃,^2,3 the peptide concentrations were reduced to 0.1 μM and the laser power to 1%. In these conditions, the background signal coming from the free RAFT-c(-RGDfK-)₄-Cy5 was low, but a strong fluorescence coming from a large excess of peptides accumulated on the cell surface still affected the visualization of intracellular vesicles (see Figure 3A, III). Again, when using RAFT-c(-RGDfK-)₄-Cy5-SS-Q, the unbound molecules in suspension remained undetectable (see Figure 3A, IV), and only a faint signal was coming from the membrane-associated molecules. Nonetheless, most of the Cy5 signal was coming from intracellular vesicles. Thus, Cy5-SS-Q allows the visualization of an intracellular signal despite the presence of a large excess of extra- and pericellular probe concentration.

The kinetic of the internalization process was then observed on live HEK293(b₃) cells by real-time imaging (Figure 3B). The intracellular signal became visible 10 minutes after addition of RAFT-c(-RGDfK-)₄-Cy5-SS-Q and augmented regularly during the first 2 hours.

Finally, live IGROV1 or HEK293(β₃) cells were incubated simultaneously with the quenched probe and the green-fluorescent LysoTracker probe during 90 minutes and examined by CLSM. As shown in Figure 3C, a large proportion of the red Cy5 signal colocalized with the green fluorescence coming from LysoTracker in both cell lines (see Figure 3C, d and h). This suggested that the dequenching effect occurs mainly in acidic vesicles.

Figure 3.

Confocal microscopy of intracellular Cy5 dequenching in live cells. A, Representative images of IGROV1 and HEK293(β₃) cells taken after 2 hours of incubation with RAFT-c(-RGDfK-)₄-Cy5 or RAFT-c(-RGDfK-)₄-Cy5-SS-Q at a final concentration of 0.5 μM for IGROV1 or 0.1 μM for HEK293(β₃). IGROV1: + RAFT-c(-RGDfK-)₄-Cy5 (I) or RAFT-c(RGDfK)₄-Cy5-SS-Q (II). II‘: phase contrast image of II; HEK293(β₃): + RAFT-c(-RGDfK-)₄-Cy5 (III) or RAFT-c(RGDfK)₄-Cy5-SS-Q (IV). IV‘: phase contrast image of IV. Cy5 fluorescence was excited with 1% of the full power of the 633 nm laser for HEK293(β₃) cells or 30% for IGROV1 cells. The detected signal was then pseudocolored red. Original objective: Plan-Neofluar 40×/1.30 oil ph3; zoom: ×3. Note that the medium was not changed after adding each probe and during this experiment. B, Realtime imaging of Cy5 fluorescence in HEK293(β₃) cells (phase contrast image) cultured with 0.1 μM RAFT-c(-RGDfK-)₄-Cy5-SS-Q. Cy5 fluorescence was excited by 633 nm laser with 1% of full power and pseudocolored red. Original objective: Plan-Neofluar 40×/1.30 oil ph3; zoom: ×1.

Figure 3.

(Continued) C, Colocalization of Cy5 and LysoTracker Green. Live IGROV1 (a–d) or HEK293(β₃) (e–h) cells were incubated simultaneously with RAFT-c(-RGDfK-)₄-Cy5-SS-Q (at concentrations mentioned above) and LysoTracker Green (50 nM) for 90 minutes. Cy5 fluorescence was pseudocolored red. Emission of LysoTracker Green, pseudocolored green, was collected by excitation at 488 nm. Colocalization of Cy5 and LysoTracker Green was observed as a yellow color in the merged images of Cy5 and LysoTracker Green. Original objective: Plan-Neofluar 40×/1.30 oil ph3; zoom: ×2.

In Vivo Imaging

Ten nanomoles of each molecule was then injected intravenously into nude mice bearing subcutaneous IGROV1 tumors. As shown in Figure 4, both probes accumulated specifically in the tumor masses and were also visible in the kidneys. Tumors were stained as early as 10 minutes after injection of RAFT-c(-RGDfK-)₄-Cy5, but not with the quenched probe. Visually, good tumor contrasts were observed from 3 to 24 hours for RAFT-c(-RGDfK-)₄-Cy5 and from 3 to 72 hours for the quenched probe. Note that the range of gray levels was adjusted to its best values and is indicated for each image. A quantitative analysis was then performed (see Figure 4, right panel). RAFT-c(-RGDfK-)₄-Cy5 reached maximum values in the tumors and normal tissues as soon as 5 to 10 minutes after injection and then decreased over time. When divided by the amount of fluorescence in normal tissues, the tumor to skin ratio augments progressively and reaches its best value after 6 hours. The Cy5 signal coming from the quenched probe is building up more slowly than with the original probe. Whereas the RAFT-c(-RGDfK-)₄-Cy5 signal in the tumor is decreasing as soon as 10 minutes after injection, the fluorescence intensity is increasing regularly during the first 3 hours into the tumors treated by RAFT-c(-RGDfK-)₄-Cy5-SS-Q (see Figure 4, inset). Although the tumor to skin ratio is not significantly different during the first 6 hours for both molecules, it augments and peaks at 10 hours with the quenched probe, although it is already significantly lower with the conventional one. As an example, the tumor to skin ratio is 3.18 ± 0.17 for RAFT-c(-RGDfK-)₄-Cy5-SS-Q and 1.78 ± 0.23 for RAFT-c(-RGDfK-)₄-Cy5 24 hours after injection.

Tumors sampled at 3 hours after intravenous injection of each molecule were then analyzed by confocal microscopy using constant acquisition parameters optimized for imaging RAFT-c(-RGDfK-)₄-Cy5-SS-Q. The results showed that the quenched probe produced a much stronger fluorescence signal in tumor cells or in the stroma than RAFT-c(-RGDfK-)₄-Cy5 (Figure 5, A vs B), supporting the macroscopic measures shown in Figure 4. As shown in the inset at high magnification, RAFT-c(-RGDfK-)₄-Cy5-SS-Q was also actively internalized by tumor cells in vivo. Beside the staining of the tumor cells, the stroma was also fluorescent, with a pattern resembling that of CD31 immunostaining (Figure 5C). This suggested that RAFT-c(-RGDfK-)₄-Cy5-SS-Q was also binding to endothelial cells.

Figure 4.

Representative fluorescence images of Swiss nude mice bearing IGROV1 subcutaneous tumors after intravenous injection of 10 nmol RAFT-c(-RGDfK-)₄-Cy5 or RAFT-c(-RGDfK-)₄-Cy5-SS-Q. All images were displayed at their best look-up-table values, providing optimal tumor to background contrast. The values shown in each image represent the range of minimal to maximal signal intensities. K = kidney; T = tumor. Right panel: time courses of fluorescence intensities in tumors and skin and the tumor to skin ratios in these nude mice. The fluorescence intensities were recorded as photons per pixel in a specified region of interest (as shown in the insert). Data are expressed as means ± SD (n = 3 or 4 mice per group).

Discussion

RGD peptides have been intensively investigated as targeting vectors for delivery of therapeutic agents, including small molecular drugs, therapeutic peptides and proteins, therapeutic nucleic acids, deoxyribonucleic acid (DNA), and even viruses (for a review, see Temming and colleagues¹¹). Most of these agents are biologically active after internalization only. In vitro studies have shown that a multivalent presentation of the cRGD peptide greatly augments its integrin-dependent internalization,^1,12 but this internalization process is difficult to address in vivo. In this article, we present a novel α_vβ₃ integrin–targeted activatable optical probe that can be turned on once internalized by target cells.

cRGD peptides have been labeled with various radioisotopes or NIR dye Cy5.5 or Cy5 for in vivo and noninvasive nuclear or optical imaging of integrin α_Vβ₃ expressions in solid tumors.^2,3,13–17 Furthermore, imaging quality can be improved by using multimeric cRGD peptides with favorable pharmacokinetics.^2,3,17 However, the signals obtained always correspond to the sum of the signals coming from the RGD molecules specifically binding to their receptor, plus the one coming from the nonspecific binding and of the freely circulating molecules. As initially established by Weissleder and colleagues,⁴ optical probes are interesting because their signal emission can be controlled by chemical modification of the fluorescent dye.^5,18–22 In addition, it was established that introducing an -SS- link could augment Cy5.5 signal in the presence of dithiothreitol (DTT), another reducing reagent.²² Taking advantage of this property, we introduced an -SS- between Cy5 and a fluorescence quencher molecule Q forming an activatable unit. The fluorescence emission of Cy5 on Cy5-SS-Q is intensively quenched because of the spatial proximity between Cy5 and Q, a light-absorbing, nonemitting dye.²³ Cleavage of the disulfide bridge by reduction restores the Cy5 fluorescence. Although most of the previously published activatable fluorescent probes used a proteolytic cleavage of specific peptide sequences for imaging intra- or extracellular proteases but were not targeted to a specific cell type, our Cy5-SS-Q unit was grafted onto the RAFT-c(-RGDfK-)₄ targeting vector. Thus, fluorescence can be activated during RGD-mediated cellular internalization in α_Vβ₃-positive cells only. In addition, since a multivalent presentation of cRGD by RAFT-c(-RGDfK-)₄ is efficiently triggering an active endocytosis of this vector,² the RAFT-c(-RGDfK-)₄-Cy5-SS-Q is expected to combine specific targeting and active induction of fluorescence during endocytosis.

Figure 5.

Confocal laser scanning microscopic images of IGROV1 subcutaneous tumors dissected 3 hours after intravenous injection of 10 nmol (A) RAFT-c(-RGDfK-)₄-Cy5 or (B) RAFT-c(-RGDfK-)₄-Cy5-SS-Q. Paraformaldehyde-fixed cryosections were incubated with Hoechst 33342 for nuclear staining (blue). Signals from Cy5 were pseudocolored red. Original objective: Plan-Neofluar 40×/1.30 oil ph3; zoom: ×1 (inset: zoom: ×4). C, Antimouse CD31 staining of an untreated IGROV1 subcutaneous tumor. Original magnification: ×40 objective lenses. S = stroma; T = tumor cells. Tumor nodules are delimited by dashed lines or circles.

Endocytosis is known to lead to inactivation of the drug; thus, many chemical approaches aimed to facilitate endosome rupture and liberation of the active drug. This was often performed by incorporating a disulfide linker between the vector and the drug and inspired by indirect evidence suggesting that the redox potential within the endosomal system is reducing, but direct demonstration of such a mechanism is lacking.^24–26 A mechanism for the maintenance of lysosome reductive potential was proposed after the discovery of a rapid lysosomal importer specific for cysteine and possibly cysteamine, both of which have a free thiol group able to mediate reduction of disulfide bonds,²⁷ but another report suggested that these intracellular compartments are not always reducing.²⁸ Our results demonstrate that the quenching/dequenching efficiency of Cy5-SS-Q is very good. This was quantified in vitro after the addition of b-ME in cell culture using live cells and in vivo in tumor cells.

Using real-time CLSM on live cells in the presence of RAFT-c(-RGDfK-)₄-Cy5-SS-Q, we show that this quenched probe is a very efficient tool for the study of integrin-mediated endocytosis on two different cell lines expressing variable amounts of the integrin. In HEK293(b₃) cells that strongly express α_Vβ₃, low energy of the exciting laser beam was sufficient for optimal signal detection without activating the background fluorescence coming from the RAFT-c(-RGDfK-)₄-Cy5-containing medium. However, for IGROV1 cells with a low level of expression of α_Vβ₃, higher power of excitation was required and the culture medium containing RAFT-c (-RGDfK-)₄-Cy5 became highly fluorescent. This prevented the detection of the specific signal. This problem was not occurring anymore with the RAFT-c(-RGDfK-)₄-Cy5-SS-Q probe, and it was thus possible to investigate the internalization process. Schraa and colleagues reported that the cultured primary human umbilical vein endothelial cells internalized and degraded a multivalent cRGD peptide–protein conjugate, and this process could be down-regulated by cations through inhibition of internalization or by the lysosomal degradation inhibitors chloroquine and ammonium chloride.¹² We also observed colocalized signals by CLSM of the living cells in the simultaneous presence of RAFT-c(-RGDfK-)₄-Cy5-SS-Q and a lysosomotropic compound, LysoTracker Green. These studies suggested that the lysosomal compartment could be an important site for cellular handling of the internalized RGD peptides or peptide-drug conjugates. Further work should be done to investigate if the agents reported to affect internalization or degradation could influence the intracellular signal produced by RAFT-c (-RGDfK-)₄-Cy5-SS-Q in vitro and in vivo. The combined use of a competitive dose of nonlabeled cRGD peptide may also be helpful to confirm the receptor-mediated internalization.

In addition, since the Cy5 function is permanently coupled by a stable covalent peptidic link to the RAFT vector, its fluorescence is expected to follow the intracellular trafficking of the targeting vector. But it is also possible to attach the quencher onto the RAFT instead of Cy5 and to form a molecule RAFT-c(-RGDfK-)₄-Q-SS-Cy5-X, where X could be a drug linked covalently to Cy5. In this case, after reduction of the -SS- link, the fluorescence signal will come from the “free” Cy5-X. This may allow a direct identification of the targeted tissues, cells, and even the nature of the subcellular compartment into which the labeled drug would accumulate. Such molecules are currently under investigation and could participate to the better definition of targeting vectors for drug delivery, as well as efficient tools for the study of RGD-mediated internalization.

The in vivo stability of the Cy5-SS-Q unit in the absence of the RAFT-c(-RGDfK-)₄ was first tested by incubation of Cy5-SS-Q in freshly collected murine blood and after systemic administration into normal mice and whole-body imaging.²³ Whole-body optical imaging of IGROV1 tumor–bearing mice injected with RAFT-c (-RGDfK-)₄-Cy5-SS-Q confirmed that the quenching effect was very efficient in vivo since very low levels of fluorescence were measured in the skin during the first hours using RAFT-c(-RGDfK-)₄-Cy5-SS-Q compared with the RAFT-c(-RGDfK-)₄-Cy5 analog. Later, this signal, coming from circulating and nonspecifically adsorbed molecules, stabilized at values close to the natural background levels related to autofluorescence. Immediately after intravenous injection, the quantitative analysis of tumor-associated signal showed higher photon counts in the RAFT-c(-RGDfK-)₄-Cy5 treated animals compared with the RAFT-c(-RGDfK-)₄-Cy5-SS-Q ones. Indeed, RAFT-c(-RGDfK-)₄-Cy5-SS-Q signal was building up more slowly than its unquenched analog and increased steadily within the first 3 hours. Although it is not possible to hypothesize that the two probes share similar pharmacokinetic parameters, it is reasonable to assume that RAFT-c(-RGDfK-)₄-Cy5-SS-Q will accumulate, at best, as efficiently as RAFT-c(-RGDfK-)₄-Cy5 into the tumor. Thus, this delay in signal apparition should be coming from the internalization and reduction steps necessary for establishment of fluorescence. Microscopic fluorescence imaging corroborated this result and revealed intracellular Cy5 dequenching, in both tumor and stromal cells. However, we cannot rule out that RAFT-c(-RGDfK-)₄-Cy5-SS-Q could have a longer accumulation time. When it comes to the tumor to skin ratio, the RAFT-c(-RGDfK-)₄-Cy5-SS-Q showed an improved contrast and a significant amelioration of its duration. This indicated that the quenched probe presented better tumor imaging properties, with enhanced tumor contrasts at later time points from 6 to 72 hours postinjection. Indeed, a longer duration of high tumor contrast may be important for future clinical purposes because this removes the necessity to choose a precise time point providing the higher ratio. In addition, this will allow multiple and repeated imaging after a single injection, an important issue, for example, during imaging-guided surgery of tumor masses.

Altogether, this study clearly shows the importance of introducing a disulfide function for imaging RGD-mediated tumor-specific targeting plus its internalization. It also provides a strong molecular rationale for improving the design of targeted drug delivery vectors and optical imaging of complex events, such as drug internalization and release.

Footnotes

Acknowledgments

We gratefully acknowledge “La région Rhône-Alpes” (France) for financial support to Zhao-Hui Jin. We are grateful to Dr. Laurent Poulain (Centre de lutte contre le cancer Francois Baclesse, Caen, France) for giving us the IGROV1 cells and Dr. J.-F. Gourvest (Aventis, France) for the HEK293(β₃) cells. We are also very grateful to Philippe Rizo (CEA Leti, France) for offering the optical imaging system. We thank Corine Tenaud and Dominique Desplanques in our laboratory for technical assistance.

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In Vivo Noninvasive Optical Imaging of Receptor-Mediated RGD Internalization Using Self-Quenched Cy5-Labeled RAFT-c(-RGDfK-) 4

Abstract

Materials and Methods

Synthesis of Quenched Cy5-RAFT-c(-RGDfK-)4 Conjugate

Cell Lines and Culture Conditions

In Vitro Optical Imaging of -SS- Cleavage

Real-Time Imaging of Live Cells in Culture

In Vivo Optical Imaging

Histologic Distribution of RAFT-c(-RGDfK-)4-Cy5-SS-Q in Tumors

Statistical Analysis

Results

Quenching and Dequenching Efficiency of RAFT-c(-RGDfK-)4-Cy5-SS-Q

Active Dequenching of RAFT-c(-RGDfK-)4-Cy5-SS-Q during Intracellular Trafficking

In Vivo Imaging

Discussion

Footnotes

Acknowledgments

References