Targeted Molecular Imaging Agents for Cellular-Scale Bimodal Imaging

Oct 30, 2004 - Texas Tech University, Lubbock, Texas 79409, Section of Neurosurgical Oncology, Vanderbilt University,. Nashville, Tennessee 37232, and...
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Bioconjugate Chem. 2004, 15, 1488−1495

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Targeted Molecular Imaging Agents for Cellular-Scale Bimodal Imaging H. Charles Manning,† Timothy Goebel,‡ Reid C. Thompson,§ Ronald R. Price,| Haakil Lee,| and Darryl J. Bornhop*,† Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, Department of Chemistry, Texas Tech University, Lubbock, Texas 79409, Section of Neurosurgical Oncology, Vanderbilt University, Nashville, Tennessee 37232, and Department of Radiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232. Received April 16, 2004; Revised Manuscript Received June 28, 2004

Molecular imaging is a powerful tool that has the ability to elucidate biochemical mechanisms and signal the early onset of disease. Overexpression of the peripheral benzodiazepine receptor (PBR) has been observed in a variety disease states, including glioblastoma, breast cancer, and Alzheimer’s disease. Thus, the PBR could be an attractive target for molecular imaging. In this paper, the authors report cellular uptake and multimodal (MRI and fluorescence) imaging of PBR-overexpressing C6 glioblastoma (brain cancer) cells using a cocktail administration approach and a new PBR targeted lanthanide chelate molecular imaging agent.

INTRODUCTION

Recent discoveries in molecular imaging (MI) are certain to play a vital role in the early detection, diagnosis, and treatment of disease, as well as aid in the study of biological and biochemical mechanisms, immunology, and neuroscience (1-7). The ubiquitous clinical agent, Magnevist, is a perfusion agent that can give vascularity and fluid transport information, but has the limitation of lacking the ability to target specific tissues. Generally, targeted MI agents consist of a targeting functionality such as an antibody, peptide (8), sugar (9), or a ligand such as a peripheral benzodiazepine receptor (PBR) ligand (10) combined with a signaling moiety (a fluorophore, radioisotope, or Gd3+ ion, etc.). In some cases, the signal can be activated by a species found in the target environment such as an enzyme or high Ca2+ concentration (11-13). Targeted fluorescent MI agents show great promise for following gene therapy/delivery (14), for elucidating signaling pathways (1), and for performing disease diagnosis at the molecular level (6). Magnetic resonance MI agents have also been developed that provide unique insights on system physiology (11) and allow effective tumor imaging (5, 12). While existing MI agents have been functional, another limitation of most existing MI agents is that they provide a single signature. Multimodal agents have the potential to enhance visualization of diseased tissue by generating more than one signature from a single biological matrix. Two-signature integrated molecules for bimodal imaging have been used by Meade and coworkers to follow cell lineage in Xenopus laevis (15) and even stem cell migration (16). Figure 1 shows the result obtained when a X. laevis embryo was injected with * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Chemistry, Vanderbilt University. ‡ Department of Chemistry, Texas Tech University. § Section of Neurosurgical Oncology, Vanderbilt University. | Department of Radiology, Vanderbilt University Medical Center.

Gd-(DTPA)-tetramethylrhodamine-hydroxypropylpoly(D-lysine) (GRIPc, Figure 1A) into a marginal blastomere (15) and subsequently imaged with confocal microscopy (Figure 1B) and MRI (Figure 1C). Both MR and fluorescence signatures seem to colocalize in similar regions of the embryo. More recently, Josephson, Weissleder, and co-workers have reported an activatable polymeric agent that combines NIR fluorescence and MR properties (17). The agent, shown in Figure 2, utilizes a protease cleavable carrier that can be labeled with Cy5.5 and cross-linked iron oxide particles (CLIO). Two dye-arginyl peptide nanoparticle conjugates were reported, Cy5.5-R4-SSCLIO and Cy5.5-R4-SC-CLIO, where “SS” denotes a disulfide linkage between the peptide and the iron oxide while “SC” denotes a thioether linkage, while R4 stands for four L-arginyl peptides. As reported, the primary amine of a dextran-coated CLIO was reacted with one of two activating agents, either N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or succinimdyl iodoacetate (SIA), to produce two reactive forms of the CLIO as shown in Figure 2. The L-arginyl peptide is attached to the activated CLIO through a C-terminal cysteine residue while the N-hydroxysuccinimide ester of Cy5.5 is attached via the terminal amine, yielding the bifunctional species. Upon in vivo administration of Cy5.5-R4-SCCLIO, MR and fluorescence signals were observed to be colocalized in lymph nodes (Figure 2A-D). While both of these imaging strategies show promise, the aforementioned contrast agents are of a nontargeted (perfusion) nature. Bimodal targeted imaging using a PBR ligand would be particularly attractive due to the importance of this receptor in cell physiology (18). Thus, an alternate approach to bimodal imaging using “cocktails” of the PBR targeted fluorescent Eu-PK11195 and the MR active Gd-PK11195 complex is demonstrated for the first time. Multiple signature moieties could facilitate development of practical methodologies for correlating intraoperative in vivo fluorescence detection with MRI, thereby enhancing tumor resection and subsequently improving patient outcome.

10.1021/bc049904q CCC: $27.50 © 2004 American Chemical Society Published on Web 10/30/2004

Technical Notes

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Figure 1. X. laevis embryo (stage 8) with GRIPc injected into the marginal blastomere at 16-cell stage. (A) Chemical structure of GRIPc. (B) Confocal fluorescence image. (C) MRI image show both signals propagate from the same population of cells. Image courtesy Tom Meade (42).

Figure 2. Synthesis and MR/NIRF images from animals injected with Cy5.5-R4-SC-CLIO. (A) Contrast-enhanced MR image of lymph nodes where ax ) axillary and br ) brachial. (B) Contrast-enhanced coronal image of axillary node. (C and D) White light and NIRF images, respectively, showing NIRF emission from the axillary and brachial nodes. Figure adapted from Josephson et al. (17).

Numerous targets exist for molecular imaging, including the sigma 2 (19), EGFr (20), and peripheral benzodiazepine receptor (PBR) (18, 21, 22). The 18 kDa protein, PBR, located in the mitochondria, has been demonstrated to be associated with numerous biological functions including regulation of cellular proliferation, immunomodulation, porphyrin transport, heme biosynthesis, anion transport, regulation of steroidogenesis, and even apoptosis (18, 23, 24). PBR density changes have important implications when acute and chronic human neurodegenerative pathologies are present. For example, temporal cortex obtained from Alzheimer’s patients showed an increase in PBR (25). Additionally, correlations with Huntington’s disease (26), multiple sclerosis

(27, 28), and gliosis (29, 30) have been observed. Combined with evidence that PBRs play a major role in proliferation of aggressive tumor cells (31), overexpression of PBR in these cells creates an excellent target for study of diseased human tissues and represents a potential tool for improving detection, diagnosis, prevention, and, very likely, treatment of certain cancers. Because PBR overexpression in diseased tissue has been suggested as an effective target for glioma and CNS disease (32), a number of ligands have been developed for targeting this receptor (10, 22, 33). Even though PBRs are expressed throughout the body (18), their density in the CNS is primarily limited to the ependymal cells and glia (25). Thus, compounds based on the well-character-

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Technical Notes

Scheme 1. Synthesis of Conjugable PBR Ligand (2) and PBR-Targeted Ligand (10)

ized, high affinity PBR ligand (33) PK11195 have been tested as PET agents for visualizing rat and human glia (22) and as a targeted therapeutic agent when conjugated to the drug gemcitabine (34). Furthermore, the 11C PET version is currently being tested in the clinic to study acute brain injury and neuroinflammation (35). Here we describe the first PBR-targeted MI agent that could facilitate MR imaging and, when delivered as a cocktail (Eu3+ and Gd3+) of a Ln-PK11195 complex, produces cellular-scale bimodal (MR and fluorescence) imaging. The MI agent described is a PK11195 (nanomolar binding PBR ligand) analogue conjugated to a trifunctional lanthanide chelate (Ln-QM-CTMC) (36-38). When prepared as the Eu3+complex, Eu-QM-CTMCPK11195 (Eu-PK11195), exhibited visibly bright fluorescence (38). By substitution with Gd3+, Gd-PK11195 then acts as a MRI agent with molar relaxivity comparable to Magnevist. This new MI agent could be useful for numerous applications in molecular imaging including disease mapping, surgical resection guidance through in vivo imaging, and the further elucidation of PBR function in cell biology. EXPERIMENTAL PROCEDURES

Uptake Experiments. C6 glioblastoma cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)F12 medium (Gibco/Invitrogen) supplemented with 15% horse serum (ATCC), 2.5% FBS (Gibco/Invitrogen), and 0.1% 50 mg/mL gentamicin sulfate (Biowhittaker). Standard medium pH was between 7.2 and 7.4. Cells were maintained in a Forma Scientific incubator at 37 °C in a sterile air environment containing 5% CO2 and were

plated for imaging on glass coverslips just prior to confluency. Next, cells were dosed with 1 mL of the contrast agent in 0.9% saline (25 µM Eu-PK11195) and allowed to incubate at either 37 °C (or 4 °C, cold study) for various times, including 35 min, 45 min, 60 min, and beyond. At the end of the incubation time, the cells were thoroughly rinsed with saline and imaged in white light and fluorescence directly using a standard epi-illumination fluorescence microscope (Carl Zeiss, Axioskop). The microscope was configured to facilitate the imaging of our complex. The optical train consisted of a UG11 filter inserted into the excitation path. The subsequent light was reflected onto a dosed sample using a 400 nm cutoff dichroic mirror. Finally, fluorescence was collected through a 40× objective (NA ) 0.5), passed through a 612 × 25 nm emission filter, and directed onto a Sinsys CCD camera. Images were collected and stored on a pc. Displacement Experiments. C6 glioblastoma cells were plated out on glass coverslips and incubated at 37 °C until just prior to confluency. Next, cells were dosed with 1 mL of contrast agent in 0.9% saline (25 µM Eu-PK11195) and allowed to incubate at 37 °C for 60 min. At the end of the incubation time, the cells were thoroughly rinsed with saline and imaged in white light and fluorescence directly using a standard epi-illumination fluorescence microscope (Carl Zeiss, Axioskop). Next, a 6-fold molar excess of PK11195 was allowed to incubate with the fluorescing cells at 37 °C for 1 h. At the appropriate time, the cells were again rinsed with saline and imaged directly under the fluorescence microscope. To calculate the change in fluorescence intensity between the dosed and displaced experiments, the total gray scale

Technical Notes

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Figure 3. Comparison of MR contrast between a commercially available agent and Gd-PK11195. Note, unlike the former agent (Magnevist), the latter compound affords the ability to utilize both fluorescence and MR signatures as diagnostic tools. Scheme 2. Complexation of Ln-PK11195 Facilitating the Preparation of a PBR-Targeted MR Agent

intensity was integrated over both pictures (same integration times) using Adobe Photoshop. Values were recorded, and the displaced intensity was compared to the dosed intensity to give the percent fluorescence change [100 - (displaced/dosed) × 100]. MR Imaging. Sample solutions (Magnevist and Gd-PK11195) were prepared in water and imaged via a T1-weighted Spin-Echo pulse sequence in a standard 4.7 T instrument where TE was kept constant and TR was varied 100-3000 ms. Cocktail Imaging. Approximately 106 C6 glial cells were inoculated with a 40-60 cocktail (1 mM total Ln-PK11195 concentration) of the Eu-PK11195 and the Gd-PK11195 complexes. Directly after 60-min incubation time, fluorescence microscopy was performed on a small number of cell cultures that were flash frozen for subsequent MR imaging. Images of thawed cells (in µL centrifuge tubes) were acquired using a 4.7 T instrument (Figure 8, TE/TR ) 11/1000 ms, FOV ) 4 cm, slice thickness ) 1 mm). The data matrix was 256 × 128, extrapolated to 256 × 256 for the final images. RESULTS AND DISCUSSION

Initially, we prepared the PBR targeted ligand by coupling our conjugable form of PK11195 to a trifunctional lanthanide chelate (38) (Scheme 1). The conjugable PBR ligand was realized by first synthesizing 1-(2chlorophenyl)isoquinoline-3-carboxylic acid using established procedures (39). We completed the conjugable molecule by appending the carboxylic acid residue with a diamine linker (38). Previously, we have reported the synthetic methodology leading to the trifunctional lanthanide chelator {Griffin, 2001 #48; Manning, 2004 #46}. Briefly, cyclen is first selectively di-protected with benzyloxycarbonyl groups at low pH (40). Next, the 1, 7-diprotected macrocycle can then be mono-alkylated with an antenna to later be used for lanthanide sensitization. The macrocycle is then fitted with a pendant carboxylic ester residue for conjugation (36). The protecting groups are removed under mild hydrogenation condi-

tions using cyclohexene and Pd/C, and complexation arms are then added. Initially, we chose to alkylate the remaining cyclen nitrogen atoms with phosphonate acidester pendant arms, which, upon hydrolysis have very good chelation ability (41). After hydrolysis, the completed trifunctional lanthanide chelator was coupled to the conjugable form of PK11195 in mixed aqueous organic solvent (38). Complexation of the ligand with europium resulted in a fluorescent derivative that gave the expected lanthanide luminescence (38). The Gd3+ derivative was prepared as shown in Scheme 2. While the substitution may seem trivial, simply exchanging the metal ion in the complex results in another agent with an entirely different signature. In this way, one scaffold offers multiple facets of imaging opportunity. For example, aside from fluorescence (M ) Tb or Eu) and MRI (Gd), additional metal substitution (M ) Ga, Cu, Ho, etc.) can facilitate applications for PET imaging and ultimately, targeted therapeutic dose delivery. Next, we evaluated the magnetic resonance signature of this new PBR-targeted compound (Gd-PK11195) by correlation to the clinical MRI agent Magnevist. A series of dilutions of each contrast agent at similar concentrations was used to measure the molecular relaxivity at 4.7 T. From the linear calibration plot (r2 ) 0.999, data not shown), r1Gd-PK11195 ) 5.94 s-1 mM-1 and r1Magnevist ) 6.45 s-1 mM-1. These data show that the new PBR targeted MI has a T1 relaxivity value comparable to a commonly used perfusion agent. To evaluate functional contrast, the same solutions were imaged as phantoms in a standard MR imaging system, again at 4.7 T. Figure 3 shows the result of this experiment, the T1 weighted phantom images of Gd-PK11195 and Magnevist solutions over a range of concentrations. As the images demonstrate, Gd-PK11195 gives similar MR contrast to Magnevist. Therefore, Gd-PK11195 delivered to the specific target, PBR, results in contrast comparable with an accepted agent. Such specificity for PBR-overexpressing cells or tissues could

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Technical Notes

Figure 4. (A) C6 glioblastoma cells incubated with Eu-PK11195 for 60 min. (B) Blank, undosed C6 cells.

Figure 5. (A) C6 glioma cells incubated with Eu-PK11195 at 37 °C, demonstrating appreciable uptake and fluorescence. (B) C6 cells incubated with Eu-PK11195 at 4 °C, demonstrating minimal fluorescence.

Figure 6. (A) C6 glioblastoma cells after 60-min incubation with Eu-PK11195 and viewed in fluorescence mode (False Colorized). Inset: undosed (blank) C6 glioblastoma cells. (B) C6 culture first incubated 60 min with Eu-PK11195, then counter-incubated with PK11195 for 60 min.

prove useful in elucidating the critical functions of PBR, dealing with reactive oxygen species (31), and even improved detection of mitochondrial misfunction diseases (21). To evaluate Ln-PK11195-targeted delivery, we performed uptake and displacement studies. PBR-overexpressing C6 glioma cells (10, 22) were dosed with Eu-PK11195 at concentrations of 10 µM to 1.0 mM for times ranging from 20 to 60 min. Quinoline-sensitized fluorescence from the Eu3+ complex (38) was readily detectable in the cells at all incubation times and concentrations using a modified epi-illumination fluorescence

microscope affixed with a CCD camera. For 10-500 µM concentrations of the agent, the optimum dosage time was found to be between 45 min and 1 h. As illustrated in Figure 4A, there is substantial uptake of Eu-PK11195 by C6 glioma cells, with fluorescence being associated with distinct internal structures, suggesting specific organelle location of the protein. PBRs are in fact known to be expressed in the outer mitochondrial membrane (18, 42). Preliminary coregistration studies indicate that Ln-PK11195 is localized in the same region of the cell as the mitochondrial dye JC-1 (43). Figure 4B presents the control, an undosed population of C6 cells of comparable

Technical Notes

Figure 7. Average raw fluorescence of agent-dosed C6 glioblastoma cells. Bar A is the raw fluorescence after dosing with Eu-PK11195. Bar B is the raw fluorescence after PK11195 displacement of contrast agent from PBR. Error bars represent the standard deviation of triplicate experiments.

density, which is relatively devoid of fluorescence. To demonstrate that the Eu-PK11195 molecule was being transported into the cells as opposed to simply adhering to the surface, we performed a standard biological colduptake mechanism study where C6 glioma cells were incubated with the same concentration of Eu-PK11195 at both 37 °C and 4 °C. As seen in Figure 5A, the cells incubated with the contrast agent at 37 °C show appreciable fluorescence, while those incubated at 4 °C (Figure 5B) show very little. These data suggests that the molecule is labeling the cell internally, consistent with endocytosis, although the exact transport mechanism remains unknown (44). Next, a preliminary displacement study to obtain an estimate for the binding affinity of Ln-PK11195 for PBR relative to the high affinity ligand PK11195 was undertaken. Utilizing procedures similar to Pramanik (45), we labeled cells with our agent and quantified the binding via fluorescence intensity. We then treated the same cells with a 6-fold excess of the native, nonlabeled PBR ligand, PK11195. The reduction in fluorescence intensity was used to give an estimate of ligand displacement (45). One result of these experiments is displayed in Figure 6. The false colorized image in Figure 6A is of cells dosed with Eu-PK11195, while Figure 6B shows the cells after 60min competition with excess PK11195. Cell repositioning, due to rinsing procedures, accounts for the differences in appearance of the images. The average reduction in fluorescence intensity (triplicate determinations) after displacement of Eu-PK11195 with PK11195 was 37% (Figure 7), indicating that Eu-PK11195 functions as a PBR ligand. Yet, there is still appreciable fluorescence remaining in the cells after the displacement dosage of a 6-fold excess of the native PK11195, suggesting that our complex is binding to the PBR with substantial affinity as desired (10). Additional studies are currently underway to quantify binding affinity, with nanomolar Kd values expected. Next, cocktail mixtures were prepared in varying ratios (20:80, 30:70, 40:60, and 50:50, Eu3+:Gd3+) and used for

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examination of C6 glioma cell colonies in order to demonstrate the “cocktail” approach. All ratios gave both signatures, with Figure 8 illustrating that the 40% Eu3+: 60% Gd3+ cocktail has readily quantifiable fluorescence and MR contrast from the same population of C6 cells. A cursory evaluation of these preliminary data indicates contrast enhancement (signal-to-background ratio) by the MI agent to be 9:1 for fluorescence and 1.7:1 for MR. Of particular intrigue is the observation of significant MR contrast using Gd-PK11195 given the controversy surrounding quantitative receptor imaging using Gd3+ complexes. To be sure, MR imaging of receptors is only possible if the receptor of interest is expressed at a sufficiently high rate, and if the agent of interest can be delivered and retained in sufficient concentration. Tweedle and co-workers have estimated that at least 106 receptor-bound Gd3+ agents are required per cell to achieve approximately 2:1 S/N in a conventional 2T MR imager (46). Thus, assuming a 1:1 receptor-contrast agent pair, there would have to be on the order of 106 PBR in a cell to observe MR contrast. In fact, Papadopoulos has indeed reported PBR concentrations in cancer cells on the order of 106 receptors/cell, assuming 1010 cells/g (42, 47). Therefore, given the expression profile reported for PBR, in conjunction with our experiments being performed at high field (4.7 T), it would appear that PBR can be imaged in cells with Gd-PK11195. In conclusion, we have demonstrated targeted delivery of Ln-PK11195 to PBR-overexpressing C-6 glioma cells. Bright, localized fluorescence of the Eu-PK11195 was observed with standard microscopy and displacement studies indicated PBR ligand functionality. Furthermore, it was shown that the Gd3+ complex of Ln-PK11195 has a relaxivity and an MR signature comparable in brightness to the clinical agent Magnevist but with the benefit of specificity. Through internalization and association with the PBR within the mitochondria, we can now use MRI to study expression, identify up-regulating cells, and further elucidate PBR functions, including its association with apoptosis (48). Finally, bimodal cellular-scale contrast-enhancement was afforded using a cocktail of Eu-PK11195 and Gd-PK11195. These investigations demonstrate that both fluorescence and MRI signatures are possible and were observed in C6 glioma cells using a PBR ligand-based MI agent. To expand on these findings, experiments are underway to determine if PBR expression can be imaged in realtime using Ln-PK11195. Other investigations with this new MI agent include both relaxivity determination (contrast enhancement) in PBR-overexpressing tissues and quantification of Kd for PBR. Other variations of the metal-PK11195 complex could potentially facilitate targeted imaging with PET (M ) Ga, Cu, etc.) and eventu-

Figure 8. (A) White-light image of C6 glioma cells (40×) dosed with a cocktail of Ln-PK11195 (40% Eu:60% Gd). (B) Cocktail-dosed C6 glioma cells from A imaged in fluorescence mode. (C) Undosed (blank) C6 glioma cells. (D) MRI image of cocktail-dosed C6 glioma cells. (E) MRI image of undosed (blank) cells.

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ally site-directed therapy delivery of a radioactive isotope (M ) 166Ho, 177Lu). We envision that Eu-PK11195 can be useful as a histopathological stain, and that this unique strategy of integrating optical imaging with the specificity of a targeted delivery could ultimately facilitate intraoperative multimodal imaging, impacting diagnosis and treatment of malignant brain cancer and other neurological dysfunctions. ACKNOWLEDGMENT

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