Radioactive Smart Probe for Potential Corrected ... - ACS Publications

Oct 2, 2012 - Radioactive Smart Probe for Potential Corrected Matrix Metalloproteinase Imaging ... Chemical Communications 2018 54 (26), 3227-3230 ...
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Radioactive Smart Probe for Potential Corrected Matrix Metalloproteinase Imaging Chiun-Wei Huang, Zibo Li,* and Peter S. Conti Molecular Imaging Center, Department of Radiology, University of Southern California, Los Angeles, California 90033, United States S Supporting Information *

ABSTRACT: Although various activatable optical probes have been developed to visualize metalloproteinase (MMP) activities in vivo, precise quantification of the enzyme activity is limited due to the inherent scattering and attenuation (limited depth penetration) properties of optical imaging. In this investigation, a novel activatable peptide probe 64Cu-BBQ650PLGVR-K(Cy5.5)-E-K(DOTA)-OH was constructed to detect tumor MMP activity in vivo. This agent is optically quenched in its native form, but releases strong fluorescence upon cleavage by selected enzymes. MMP specificity was confirmed both in vitro and in vivo by fluorescent imaging studies. The use of a single modality to image biomarkers/ processes may lead to erroneous interpretation of imaging data. The introduction of a quantitative imaging modality, such as PET, would make it feasible to correct the enzyme activity determined from optical imaging. In this proof of principle report, we demonstrated the feasibility of correcting the activatable optical imaging data through the PET signal. This approach provides an attractive new strategy for accurate imaging of MMP activity, which may also be applied for other protease imaging.



INTRODUCTION Advances in molecular imaging offer strategies for noninvasive, quantitative, and repetitive imaging of targeted macromolecules and biological processes in living organisms, which could provide an efficient early diagnosis, predict disease course, and finally lead to personalized medicine.1−4 Optical imaging with fluorescent probes has recently been proven to be a powerful tool with the advantages of lower cost, portability, and real-time capabilities.5−9 A unique feature of fluorescence is that optical probes can be designed so that they will emit light (“turning on”) only in the presence of a targeted enzyme or upon internalization. These activatable or “smart” agents could have low to no background signal and generate signal only after being exposed to the specific molecular target/process. The matrix metalloproteinase (MMP) family are important extracellular matrix remodeling proteases whose activity has been implicated in a number of key normal and pathologic processes.10−13 The latter include tumor growth, progression, metastasis, and dysregulated angiogenesis associated with these events.12,14−18 In fact, MMPs have come to represent important therapeutic and diagnostic targets for the treatment and detection of human cancers.19,20 For example, up-regulation of MMPs has been found to play an important role in tumor invasiveness, metastasis, and angiogenesis. Although the use of activatable probes seems to be a popular method to monitor enzyme activity, very limited platforms are available to clearly © 2012 American Chemical Society

image protease activity in vivo after systemic administration. The majority of the reported systems were tested as proof-ofconcept examples in in vitro or ex vivo conditions, such as after intratumoral injection in tumor models.21−23 For in vivo imaging, we believe one limitation of activatable probes is the lack of quantitative information. For example, if we assume two tumors, tumor-1 and tumor-2, with the same enzyme activity, when more probes are localized in tumor-1 due to better blood supply, we will observe higher optical signal in tumor-1 compared with tumor-2. This result if solely determined from absolute optical signal would be misleading, since tumor-2 has the same level of enzyme activity as tumor-1. Positron emission tomography (PET) is a noninvasive functional imaging technique with millimeter resolution and high sensitivity. An important advantage of PET is the quantitative information obtained with this imaging modality. In order to take advantage of the strengths of each imaging modality, we developed an activatable dual modality (PET/ fluorescent) imaging agent that could more accurately detect tumor MMP activity in vivo. This novel MMP activatable “smart” probe could be divided into three parts: the MMP cleavable peptide sequence, a pair of dye/quencher (activatable Received: April 9, 2012 Revised: September 30, 2012 Published: October 2, 2012 2159

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Figure 1. (a) The limitation of a fluorogenic probe is that the amount of probe cannot be accurately determined in tumor regions. Although two tumors may have the same enzyme activity, one may have higher optical signal if more probe is localized in the tumor region, which will result in misleading information. (b) A radioactive motif would allow us to more accurately determine how much probe is localized in a tumor region through quantitative PET imaging. This use of a multimodality imaging probe may provide corrected enzyme activity through the measurement of total probe concentration (from PET) and the cleavage rate (from optical).

Figure 2. Synthetic scheme of a MMP activatable probe.

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through the conjugation of BBQ650 NHS ester with the Nterminal amino group after the Fmoc protecting group removal. BBQ650 NHS ester (3 equiv dissolved in DMF) was added to the fully protected peptide (1 equiv still on the resin) in the presence of 10 equiv DIPEA. The reaction mixture was stirred overnight in the dark at room temperature and washed thoroughly with DMF (5×). The DOTA conjugates were achieved through the conjugation of DOTA-mono-NHS-tris (tBu) ester with BBQ650-PLGVR-K(ivDde)-E-K(NH2)-Wang resins after the removal of Mtt protecting group with 2% of TFA in DCM (3 min × 10). The DOTA-mono-NHS-tris (tBu) ester (3 equiv) dissolved in DMF was added to the BBQ650 conjugated peptide containing resins (1 equiv), in the presence of 10 equiv DIPEA. The reaction mixture was stirred in the dark for 3 h at room temperature. Unreacted DOTA chelator was removed by washing the resins with DMF thoroughly. Through a similar procedure as the BBQ650 conjugation, the fluorescence dye Cy5.5 was introduced to the partially deprotected peptide by coupling onto the exposed lysine amino group after the ivDde protecting group was regioselectively removed with 2% of hydrazine monohydrate in DMF. The conjugated peptides were cleaved from the resin and side chain protecting groups were simultaneously removed by treating with the cleavage solution (95% TFA, and 5% water) for 3 h. Peptide-containing supernatants were separated from the solid support by filtration and concentrated under a stream of nitrogen. Crude peptide was precipitated out and washed twice with ice-cold diethyl ether, which was then dissolved in 10% acetic acid in water before lyophilization. The desired products were purified and characterized by HPLC. The purity of conjugated peptides was greater than 95%, based on analytical HPLC. Fractions containing desired conjugates were collected, lyophilized, characterized by LTQ Orbitrap Hybrid mass spectrometer, and stored in the dark at −20 °C. Enzyme Cleavage Assay. At 37 °C, 1 μM peptide probe was incubated in triplicate with 5 nM human-recombinant MMP-13 using an assay buffer of 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, and 0.05% Brij-35 (w/v) at pH 7.5 with 500 rpm. Fluorescence intensity was monitored (excitation at 675, emission at 690 nm) at sequential time points (0 min, 10 min, 30 min, 60 min, and 120 min). Recombinant human MMP-13 was added to the peptide probe solution to test cleavage specificity and stability of the amino acid sequence. Formation of the hydrolyzed peptide domains was monitored by HPLC and compared with uncleaved peptide. Cell Lines. The human glioblastoma cell line, U87MG, was obtained from American Type Culture Collection (Manassas, VA) and maintained at 37 °C in a humidified atmosphere containing 5% CO2 in DMEM (Mediatech, Inc.) and 10% fetal bovine serum (Life Technologies, Inc., Grand island, NY). The U87MG cell line was chosen in this study for its high expression of a variety of MMPs, including MMP-2, MMP-9, and MMP-13.27,28 Tumor Xenografts. Animal procedures were performed according to an approved protocol by the University of Southern California Institutional Animal Care and Use Committee. Female athymic nude mice (BALB/c nu/nu) were obtained from Harlan (Indianapolis, IN) at 4 to 6 weeks of age and injected subcutaneously in the right shoulder with 5 × 106 of U87MG human glioblastoma cells suspended in 100 μL of PBS. When tumors reached 0.4 to 0.6 cm in diameter (28 days after implant), the tumor-bearing mice were subjected to in vivo fluorescence imaging studies.

component), and the bifunctional chelator for radiometal labeling (Figure 2). The MMP-cleavable sequence PLGVR is preferentially cleaved by MMP-7, -9, -12, and-13. The quencher, BBQ650, is nonfluorescent, thus contributing no background fluorescence upon release. The imaging agent is optically quenched in its native form, but provides strong nearinfrared fluorescence signal (700−900 nm) upon cleavage by selected MMP enzymes such as MMP-13. The latter has been shown to be highly expressed in various aggressive phenotypes of tumors,24−26 making MMP-13 a potential prognostic marker between benign and malignant tumors. Although most peptidebased activatable probes provide their fluorescence signal through the release of their near-infrared dyes, in this study, all of the activated fluorescence remains closely conjugated with the radiometal labeled segments, which would allow us to track both dissociated and quenched probe with PET imaging. We expect that the enzyme activity determined from optical imaging could be corrected through PET imaging. The basic principle is illustrated in Figure 1.



EXPERIMENTAL PROCEDURES General. All commercially available chemical reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. 9-Fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids and Rink amide resin were purchased from either Novabiochem (San Diego, CA) or Bachem (Torrance, CA). DOTA(OBu-t)3-NHS(1,4,7,10-tetraazacyclododecane-1-(N-hydroxysuccinimi-deacetate)-4,7,10tris(tert-butyl acetate)) was purchased from Macrocyclics Inc. (Dallas, TX). Human recombinant MMP enzymes were purchased from R&D Systems (Minneapolis, MN). The enzymes were activated according to the vendor’s protocols, if necessary. The broad-spectrum MMP inhibitor III was obtain from EMD Chemicals (San Diego, CA). 64CuCl2 solution was purchased from University of Wisconsin (Madison, WI). Water was purified using a Milli-Q ultrapure water system from Millipore (Milford, MA, USA), followed by passage through a Chelex 100 resin before bioconjugation and radiolabeling. The purification of the crude product was carried out on an analytical reversed-phase high-performance liquid chromatography (HPLC) system equipped with a Waters 2487 dual UV absorbance detector (Waters, Milford, MA) using a Phenomenex C18 RP (250 × 4.6 mm 5 μm) column. The flow rate was 1 mL/min using a gradient mobile phase from 98% solvent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in acetonitrile) (0−2 min) to a mobile phase of 40% solvent A and 65% solvent B at 32 min. The radioactivity was detected with a Ludlum 2200 single-channel radiation detector. Synthesis of BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH Probe. BBQ650-PLGVR-K(Cy5.5)-EK(DOTA)-OH was synthesized based on the procedure illustrated in Figure 2. In brief, peptides were synthesized though a standard Fmoc solid-phase peptide synthesis method with 4-fold excess amounts of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBt), and 8fold molar excess of diisopropylethylamine (DIPEA). Acylation was carried out for 60 min and completion of the conjugation was confirmed by the trinitrobenezene sulfonic acid test (TNBS). Removal of the Fmoc protective group on the αamino group was achieved with 20% piperidine in dimethylformamide (DMF) (v/v). DMF was used to wash the resin between each acylation and deprotection step. The synthesis of BBQ650-PLGVR-K(ivDde)-E-K(Mtt)-resin was achieved 2161

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In Vivo NIR Imaging Evaluation of MMP Activation. In vivo fluorescence imaging was performed with an IVIS Lumina (Xenogen, Alameda, CA) equipped with 21 emission filter sets that can be used to image optical signals from green to nearinfrared. In vivo imaging of activatable peptide probes were performed to evaluate activation specificity. Blocking studies were performed with the broad-spectrum MMP inhibitor III (EMD Bioscience) (0.4 mM; 50 μL) through intratumor injection 30 min before the injection of imaging probes. Scans were performed with U87MG xenograft mice (n = 3) after intratumor injection of activatable peptide probes (2 nmol) with and without MMP inhibitor at serial time points (5 min,10 min, 15 min, 30 min, 60 min, and 90 min). Identical illumination settings (lamp voltage, filters, f/stop, field of views, binning) were used for acquisition of all images, and fluorescence emission scans were normalized to photons per second per centimeter squared per steradian (p/s/cm2/sr). Images were analyzed using the Living Image software (Xenogen, Alameda, CA) with spectral unmixing to reduce tissue autofluorescence. Mean activated fluorescent intensities and corresponding standard deviation were calculated for each group of animals. 64 Cu Labeling. BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)OH was radiolabeled with 64Cu2+ according to our previously reported method.29,30 Briefly, 64CuCl2 was added to DOTA conjugated peptides in 0.1 M sodium acetate buffer (pH 5.5) and incubated at 40 °C for 1 h. The radiochemical purity of the final product was >95% as confirmed by radio-HPLC. The radioactive peak containing the desired product was collected and rotary-evaporated to remove solvent. The HPLC-purified product was then reconstituted in phosphate-buffered saline to 1 mCi/mL and passed through a 0.22 mm Millipore filter into a sterile multidose vial for in vitro and in vivo experiments. In Vitro Stability Assay. The in vitro stability of 64CuBBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH was studied at different time points. Briefly, 3.7 MBq of labeled peptide was pipetted into 1 mL of PBS and mouse serum (1:1). After incubation at 37 °C for 4, 12, and 24 h, an aliquot of the mixture was removed from the PBS solution and the radiochemical purity was determined with radio-HPLC. For the stability test in serum, aliquots were added to 100 μL of PBS with 50% TFA. After centrifugation, the supernatant solution was removed and filtered for radio-HPLC analysis. Proof of Concept in Vivo Multimodality Imaging Studies. Mice (n = 3) were subcutaneously injected with 64CuBBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH (15 μCi (0.13 nmol) at sites 1 and 3, and 2.5 μCi (0.022 nmol) at sites 2 and 4, respectively) and the same amount of MMP-13 enzyme (sites 1−4) with or without the MMP inhibitor III (Table 1). Mice were anesthetized with 2% isoflurane and imaged with static scans at sequential time points (10 min, 60 min, 120 min, and 180 min). For each optical imaging scan, regions of interest (ROIs) were drawn over each injection site to measure optical

signal. Small-animal PET imaging was performed on a microPET R4 rodent model system (Concorde Microsystems, Knoxville, TN). Images were acquired after each optical imaging scan and reconstructed by a two-dimensional ordered subsets expectation maximum (2D-OSEM) algorithm. Regions of interest (ROI) were drawn over injected sites on decaycorrected whole-body coronal images. The average radioactivity concentration was obtained from mean pixel values within three-dimensional ROI volumes. The accuracy of noninvasive microPET imaging was used to assist the quantification of optical signals. Data Processing and Statistics. All data were measured in mean ± SD using three independent measurements. Statistical analysis was performed using the Student’s t test. Statistical significance was assigned for P values less than 0.05.



Table 1. In Vivo Proof-of-Concept Quantification Correction Study Layout of PET/Optical Probe, MMP-13 Enzyme and Inhibitor

PET/optical probe MMP-13 enzyme MMP inhibitor

site 1

site 2

site 3

site 4

15 μCi 10 ng -

2.5 μCi 10 ng -

15 μCi 10 ng 20 nM

2.5 μCi 10 ng 20 nM

RESULTS

Chemistry and Radiolabeling. The MMP sensitive peptide probe BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH was synthesized in high purity (>98%) by solid-phase peptide synthesis. In this design, resonance energy transfer occurs between a donor group (NIR Cy5.5 dye) and a fluorescence quencher group (BQQ-650). The chemical structure of BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH and activation mechanism are shown in Figure 3. The identity of peptide probe was confirmed by HPLC and electrospray ionization (ESI-MS) mass spectra. The HPLC retention time was (Rt) = 25.3 min and mass was m/z: [M+3H]+ = 937.06 (calculated: 937.14) (Figure S1). The 64Cu-labeled BBQ650-PLGVRK(Cy5.5)-E-K(64Cu-DOTA)-OH was achieved in more than 95% decay-corrected yield with >98% radiochemical purity. The specific activity of 64Cu-BBQ650-PLGVR-K(Cy5.5)-EK(DOTA)-OH was around 4.2 × 1015 Bq/mol. In Vitro Stability. The in vitro stability of 64Cu labeled probe was studied in PBS (pH 7.4) and mouse serum at physiological temperature (37 °C). The purpose of these studies was to demonstrate that the 64Cu radiotracer remains intact before being injected into the mice. The stability was presented as the percentage of intact peptide probe on the basis of the radio-HPLC analysis. As shown in Figure S2, after 24 h of incubation, more than 97% of imaging agent remained intact in PBS, and more than 80% of peptide remained intact in mouse serum. It is clear that the radiotracer has reasonable stability for in vivo imaging. In Vitro Studies. In order to verify that the DOTA motif has no major effect on the “smart probe” property, the newly synthesized BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH was tested for its cleavage specificity by MMP-13 enzyme in vitro. In our design, the MMP enzyme would cleave the peptide substrate that bridged a fluorescent dye and quencher, and release of the quencher will provide the fluorescence from the probe. Specific hydrolysis of peptides by MMPs was evaluated and characterized. The cleavage specificity of fluorescent probes was assessed in triplicate by optical imaging with the IVIS Lumina scanner. As shown in Figure 3c, fluorescent intensity was time dependent; the signal has an 8.2-fold increase from 0 to120 min, which can be blocked in the presence of MMP inhibitors. In addition, the HPLC profile also demonstrated two peptide fragments after the enzymatic cleavage (Figure 3b). These results showed that modification of the cleavage site with additional amino acids, fluorescence compounds, and DOTA chelator did not affect the MMP enzyme cleavage reaction. 2162

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Figure 3. Cleavage of BBQ650TM-PLGVR-K(Cy5.5)-E-K(DOTA)-OH by MMP-13: (a) Reaction scheme. (b) HPLC analysis of the crude reaction mixture. (c) Reaction was followed by optical imaging at different time points. (0 min, 10 min, 30 min, 60 min, and 120 min). (d) Quantification plot of the fluorescence intensity of MMP-13 with and without a broad spectrum MMP inhibitor III.

In Vivo Evaluation of MMP Activation. In vivo MMP activation of BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH was evaluated in nude mice bearing U87MG human glioma xenograft tumors (n = 3) at sequential time points with the IVIS Lumina small animal optical imaging system. Representative images at different time points were shown in Figure 4. The probes demonstrated an instant positive fluorescent signal upon injection, which could be attributed to the MMP enzymes secreted from the tumor. The U87MG tumor was clearly visualized with high tumor-to-background contrast and steady increase in fluorescent intensity during the time course of scans. On the contrary, the control group (preinjected with MMP inhibitor) showed dramatically lower intensity at the tumor area at all time points and confirmed the cleavage specificity of the peptide probe. Proof of Concept In Vivo Multimodality Imaging Studies. As the enhanced optical signal may depend on both MMP enzyme activity and the amount of “smart” imaging probes localized in tumor region, we expect that the introduction of PET could assist in the correction of enzyme activity determined from optical imaging. In order to support our assumption, we injected the same amount of MMP-13 enzyme with different amounts of substrate (64Cu-BBQ650PLGVR-K(Cy5.5)-E-K(DOTA)-OH) under the mouse skin. As shown in Figure 5, the optical signal reached a plateau at sites 1, 2, 3, and 4, which was 1.08 × 104, 6.47 × 103, 6.66 × 103, and 5.78 × 103 (a.u.), respectively. Comparison of sites 1 and 2 simulates one scenario where results may vary due to

substrate delivery. Although sites 1 and 2 have the same MMP activity, site 1 gave much higher optical signal than site 2 due to the larger amount of substrate. Based on optical imaging alone, one may conclude that the enzyme activity at site 1 is higher than site 2 since increased optical signal was observed (1.08 × 104 v 6.47 × 103 (a.u.)). In contrast, the substrate difference was clearly reflected in PET imaging, which would warrant the correction of the observed optical signal with PET imaging. Additional confusion came from the blocking study with the MMP inhibitors. The same amount of substrate was localized at the injection sites 1 and 3 (or 2 and 4, also reflected in PET imaging). As expected, we did not observe prominent optical signal at the sites 3 and 4 due to the inhibition of the MMP enzyme activity. However, as shown in Figure 5, the blocking effect of MMP inhibitor of each pair calculated from the optical signal values was 38.33% (site 1 v site 3) and 10.66% (site 2 v site 4), respectively. It seems that the pair of sites 1 and 3 demonstrated greater than 3-fold higher inhibition compared with the pair of sites 2 and 4. Since we injected the same amount of MMP enzyme and inhibitor at both sites, the discrepancy of the blocking efficiency obtained from the optical data is quite confusing and misleading, which may limit the measurement of MMP activity by activatable optical probes. Introduction of PET imaging provided an opportunity to coregister the optical signal with the PET signal. In order to perform these corrections, we made the following assumptions: (1) For site 1 and site 2, activatable peptide probes had been completely hydrolyzed by MMP-13 at 180 min postinjection. 2163

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Figure 4. (a) Representative NIR fluorescence images of MMP-positive U87MG tumor-bearing mice after intratumoral injection of the probe without or with MMP inhibitor. (b) Quantification analysis of NIR fluorescence intensity of U87MG tumor in vivo.

value as a quantification baseline, the MMP activity can be further validated more precisely. This conversion factor allowed recalculation of the inhibition effect at site 3 (or 4) by converting the radioactivity value to equivalent optical signal. For example, the real fluorescent signal at inhibition site can be calculated as the following: [(the fluorescent signal at site 3 (or 4) − autofluorescent signal)/ PET activity × 346.4]. The inhibition percentage was calculated at site 3 and site 4 to be 79.6% and 79.7%, respectively. The blocking efficacy was more precisely quantified after the coregistration of PET/fluorescence signals. These results demonstrated the advantage of our smart-optical/PET design.

(2) The optical signal of each site composed of autofluorescence and MMP probe fluorescence. (3) Autofluorescent background was similar at all sites. With these assumptions in mind, we determined the increased fluorescent signal (4.33 × 103 (a.u.)) at site 1 compared to site 2 was caused by probes corresponding to 12.5 μCi activity, as determined with PET. The normalized fluorescence signal (a.u.) per unit of radioactivity (μCi) can be calculated to be 346.4 (a.u./μCi) (Figure 6). Skin autofluorescence can be estimated by subtracting the converted fluorescent signal with acquired fluorescent signal (1.08 × 104 to 5.19 × 103) as 5.6 × 103 (a.u.). Therefore, the “real” fluorescent signal coming from the hydrolysis of substrate would only be 5.19 × 103 (a.u.) as determined from site 1 with 15 μCi. Using this autofluorescent 2164

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Figure 5. In vivo multimodality imaging studies. (a) Representative PET/NIR images of mice injected subcutaneously with 64Cu-BBQ650-PLG VRK(Cy5.5)-E-K(DOTA)-OH probes (15 μCi and 2.5 μCi at sites 1 and 3 and 2 and 4, respectively) and the same amount of MMP-13 without (sites 1 and 2) or with (sites 3 and 4) the MMP inhibitor. (b) Inhibition analysis of MMP inhibitor based on NIR fluorescence results in vivo.



DISCUSSION Matrix metalloproteinases (MMPs) have been identified as a key modulators in extracellular matrix remodeling and shown to play a pivotal role in promoting various stages of tumor invasion and metastasis. The recognition of MMPs as disease biomarkers stimulated the development of imaging strategies targeting these proteases for improved diagnosis and staging of tumors.31−33 Although various activatable optical probes have been developed to visualize the MMP activities in vivo, precise quantification of the enzyme activity is limited due to the inherent scattering and attenuation (limited depth penetration) properties of optical imaging. We hoped this challenge could be solved at least in part by introducing a secondary imaging modality: PET. The positron signal from the radioisotope can help determine the total amount of probes located in a tumor region (both quenched and unquenched), which could be used to correct the enzyme activity determined from optical imaging. Thus, measurement of PET signal will yield information about the precise distribution of the agent, while measurement of the optical signal will yield information about the MMP activity. This coincident measurement technique using both PET and activatable optical imaging will help us draw a correlation involving the distribution pattern of agents and activity level of MMP enzymes simultaneously. In addition, this method can be performed in the same animal at similar time points, thus eliminating problems that may occur when trying to compare across animals or time points. In this study, the designed probe BBQ650-PLGVRK(Cy5.5)-E-K(DOTA)-OH was obtained though solid-phase peptide synthesis in high purity (>98%). In our proof-ofconcept study, we first tested the enzyme sensitivity of this probe through in vitro cleavage assays and in vivo intratumoral injection imaging. It was demonstrated that the MMP enzyme

sensitivity was maintained after the introduction of additional amino acids and DOTA chelator to the PLGVR sequence. In addition, cleavage was effectively blocked with MMP inhibitor both in vitro and in vivo. To demonstrate the important role of PET on correcting the enzyme activity determined from optical imaging, we injected the same amount of MMP-13 enzyme with different amounts of substrate (64Cu-BBQ650 -PLGVR-K(Cy5.5)-E-K(DOTA)OH) under the mouse skin. Although injection sites 1 and 2 have the same level of MMP enzyme, site 1 gave much higher optical signal simply due to the increased localization of imaging probe. However, the substrate difference was clearly reflected in PET imaging. By using PET signal as a converting baseline, we can correct the optical signal from enzyme activities and eliminate the autofluorescence signal from mouse skin. On the basis of the optical/PET signal conversion, the inhibition ratio between site 1 and site 3 (or site 2 and site 4) can also be appropriately corrected. These experiments demonstrated the advantage of our smart-optical/PET design. We would also like to point out that there are many other issues that need to be addressed in order to obtain a translatable probe for in vivo imaging of MMP activity through systemic administration: short half-life, poor pharmacokinetic profiles, instability, and high background induced by a nonspecific degradation of fluorogenic substrates. A targeting ligand may efficiently increase the retention time of the probe in the target region, minimize nonspecific accumulation caused by global MMP activity, and enhance tumor-to-background signal. In our followup studies, a potent targeting structure will be introduced to the MMP probe design to avoid the fast clearance of activated imaging fragments from tumor lesions. The targeting ligand not only can provide an anchoring capability of probes in tumor lesions, but also will produce the 2165

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Figure 6. Diagram for the normalization of fluorescence signal (a.u.) based on radioactivity (μCi). Assuming that the autofluorescent signals are similar at all sites, thus, the conversion factor can be gained by dividing the difference of the acquired optical signals with the radioactivity difference, which is 346.4 (a.u./μCi).



quantification of selected receptors. Previously, Ogawa et al (2009) had synthesized a dual labeled anti-HER1 (or -HER2) antibody for dual-modality tumor targeting imaging.34 In that study, an activatable fluorescent property of ICG dye was used to introduce additional information in addition to the “on” signal from the 111indium radionuclide. The nuclear imaging successfully provided the quantification data in detail to compensate the attenuation of fluorescent signal. However, antibody based probes generally accumulate in tumor regions slowly and circulate in the blood pool for a relatively long time. For an MMP targeted probe, the effect of global MMP activity needs to be carefully considered. Therefore, a peptide-based targeting unit may be preferred due to its quick accumulation in tumors and fast clearance from the blood. Currently, we are improving our probe design through development of targeted radioactive MMP probes.

ASSOCIATED CONTENT

S Supporting Information *

ESI-MS of BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH, in vitro stability curve of 64Cu-BBQ650-PLGVR-K(Cy5.5)-EK(DOTA)-OH in PBS (pH = 7.4) or mouse serum. This material is available free of charge via the Internet at http:// pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Telephone: 323-442-3252; Fax: 323-442-3253; e-mail: [email protected]. Notes

The authors declare no competing financial interest.





ACKNOWLEDGMENTS This study was supported by the USC Molecular Imaging Center, the Department of Energy (DE-SC0002353), and the American Cancer Society (121991-MRSG-12-034-01-CCE). We would also like to thank Mr. Ryan Park for proofreading the manuscript.

CONCLUSIONS The use of a single modality, such as optical imaging, to image biomarkers/processes may lack detail and even be misleading in many cases. A coincident measurement technique using an additional imaging modality could be advantageous. In this proof of principle report, we demonstrated the feasibility of correcting the optical imaging data through PET imaging. This approach stands as an attractive new strategy for accurate measurement of MMP activity, which can also be applied for other protease imaging.



REFERENCES

(1) Richmond, T. D. (2008) The current status and future potential of personalized diagnostics: Streamlining a customized process. Biotechnol. Annu. Rev. 14, 411−422.

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Bioconjugate Chemistry

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dx.doi.org/10.1021/bc3001968 | Bioconjugate Chem. 2012, 23, 2159−2167