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ß3-targeted Nanoparticle and 1.5 Tesla Magnetic Resonance Imaging1
Cardiovascular Magnetic Resonance Laboratories, Department of Medicine, Cardiovascular Division, Barnes-Jewish Hospital, Washington University School of Medicine [P. M. W., S. D. C., L. K. C., J. S. A., E. K. L., H. Z., S. A. W., G. M. L.] and Department of Biomedical Engineering [S. D. C., S. A. W., G. M. L.], Washington University, St. Louis, Missouri 63110; Philips Medical Systems, 5680 DA Best, the Netherlands [S. D. C., A. K.]; Bristol-Myers Squibb Medical Imaging, Inc., North Billerica, Massachusetts 01862 [T. D. H.]; and Analytical Chemistry Group, University of Missouri Research Reactor, Columbia, Missouri 65211 [J. D. R.]
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ABSTRACT |
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ß3-integrin remains an attractive biochemical epitope that is highly expressed on activated neovascular endothelial cells and essentially absent on mature quiescent cells. In this study, we report the first in vivo use of a magnetic resonance (MR) molecular imaging nanoparticle to sensitively detect and spatially characterize neovascularity induced by implantation of the rabbit Vx-2 tumor using a common clinical field strength (1.5T). New Zealand White rabbits (2 kg) 12 days after implantation of fresh Vx-2 tumors (2 x 2 x 2 mm3) were randomized into one of three treatment groups: (a) ß3-targeted, paramagnetic formulation; (b) nontargeted, paramagnetic formulation; and (c) ß3-targeted nonparamagnetic nanoparticles followed by (2 h) the ß3-targeted, paramagnetic formulation to competitively block magnetic resonance imaging (MRI) signal enhancement. After i.v. systemic injection (0.5 ml of nanoparticles/kg), dynamic T1-weighted MRI was used to spatially and temporally determine nanoparticle deposition in the tumor and adjacent tissues, including skeletal muscle. At 2-h postinjection, ß3-targeted paramagnetic nanoparticles increased MRI signal by 126% in asymmetrically distributed regions primarily in the periphery of the tumor. Similar increases in MR contrast were also observed within the walls of some vessels proximate to the tumor. Despite their relatively large size, nanoparticles penetrated into the leaky tumor neovasculature but did not appreciably migrate into the interstitium, leading to a 56% increase in MR signal at 2 h. Pretargeting of the ß3-integrin with nonparamagnetic nanoparticles competitively blocked the specific binding of ß3-targeted paramagnetic nanoparticles, decreasing the MR signal enhancement (50%) to a level attributable to local extravasation. The MR signal of adjacent hindlimb muscle or contralateral control tissues was unchanged by either the ß3-targeted or control paramagnetic agents. Immunohistochemistry of ß3-integrin corroborated the extent and asymmetric distribution of neovascularity observed by MRI. These studies demonstrate the potential of this targeted molecular imaging agent to detect and characterize (both biochemically and morphologically) early angiogenesis induced by minute solid tumors with a clinical 1.5 Tesla MRI scanner, facilitating the localization of nascent cancers or metastases, as well as providing tools to phenotypically categorize and segment patient populations for therapy and to longitudinally follow the effectiveness of antitumor treatment regimens. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Angiogenesis, the process in which preexisting capillaries proliferate and form new vascular networks in response to hypoxemic and nutritive stresses, progresses by the formation of neovascular sprouts or by the partitioning of the vessel lumen by tissue pillars and interstitial tissue structures (i.e., intussusceptive microvascular growth; Ref. 9
). These proliferating neovascular endothelial cells present unique, transient cell surface markers, such as integrins, which may be used to biochemically differentiate angiogenic vessels from maturing capillaries. In particular, ß3-integrin has been a well-recognized biomarker of angiogenesis (10)
that is relatively selective for activated endothelial cells while essentially unexpressed on mature, quiescent cells.
Numerous clinical trials using antiangiogenic therapies are in progress worldwide, but there is a paucity of robust mechanisms to assess the net angiogenic activity of a tumor to aid investigators in the rational design of treatment schemes. To date, MVD has been used as a prognostic indicator for many cancers and assumed by some as a surrogate marker of angiogenesis (11) . In a recent review, Folkman et al. (12) considered the clinical application of MVD and suggested that MVD was an inadequate measure of the functional or angiogenic status of tumor neovasculature. Moreover, they proposed that MVD decreases may reflect the antivascular activity of a particular agent, but as a single end point, fails to adequately quantify the vascular response of antiangiogenic agents in general.
Molecular imaging of angiogenic vasculature presents a significant opportunity to detect nascent tumors or metastases, characterize and segment patients a priori into appropriate antiangiogenic treatments, and evaluate the effectiveness of these antineovascular therapeutic regimens. We have reported previously in vivo MRI detection at 4.7 Tesla of ß3-integrin expression in angiogenic vessels stimulated within a rabbit corneal micropocket model by exogenous basic fibroblast growth factor using paramagnetic nanoparticles targeted by an anti-ß3-antibody (DM101; Ref. 13
). In the present study, we extend this research to demonstrate the specific molecular imaging of ß3-integrin expression by angiogenic vasculature induced by nascent Vx-2 rabbit tumors using a commercially available MRI scanner (1.5 Tesla) and current clinical imaging techniques.
The objectives of the present study were: (a) to develop a novel ß3-targeted paramagnetic nanoparticle agent capable of detecting ß3-integrins expressed on neovasculature induced by early Vx-2 tumor growth using a clinical MRI (1.5 Tesla) scanner; (b) to quantify the magnitude and specificity of signal enhancement achieved with the angiogenesis-targeted agent; and (c) to demonstrate the use of ß3-targeted paramagnetic nanoparticles to delineate the anatomical distribution of the rabbit angiogenic response to Vx-2 tumor hind-limb implantation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 kg) were anesthetized with i.m. ketamine and xylazine (65 and 13 mg/kg, respectively). The left hind leg of each animal was shaved, sterile prepped, and infiltrated locally with Marcaine before placement of a small incision above the popliteal fossa. A 2 x 2 x 2 mm3 Vx-2 carcinoma tumor fragment, freshly obtained from a donor animal, was implanted at a depth of 0.5 cm. Anatomical planes were reapproximated and secured with a single absorbable suture. Finally, the skin incision was sealed with Dermabond skin glue. After the tumor implantation procedure, the effects of xylazine were reversed with yohimbine, and animals were allowed to recover. Twelve days after Vx-2 implantation, rabbits were anesthetized with 1–2% Isoflurane, intubated, ventilated, and positioned within the bore of the MRI scanner for study. i.v. and intraarterial catheters, placed in opposite ears of each rabbit, were used for systemic injection of nanoparticles and arterial blood sampling. Animals were monitored physiologically throughout the study in accordance with a protocol and procedures approved by the Animal Studies Committee at Washington University Medical School.
Experimental Design.
Twelve New Zealand rabbits implanted with Vx-2 tumors, as described above, were randomized into three treatment regimens and received either: (a) ß3-integrin-targeted paramagnetic nanoparticles (ß3-targeted, n = 4); (b) nontargeted paramagnetic nanoparticles (i.e., control group, n = 4); or (c) ß3-integrin-targeted nonparamagnetic nanoparticles followed by ß3-integrin targeted paramagnetic nanoparticles (i.e., competition group, n = 4). In treatment groups 1 and 2, rabbits received 0.5 ml/kg ß3-integrin-targeted or control paramagnetic nanoparticles after the acquisition of baseline MR images. In group 3, all rabbits were pretreated 2 h before MR imaging with 0.5 ml/kg ß3-integrin-targeted nonparamagnetic nanoparticles to competitively inhibit specific binding of the ß3-integrin-targeted paramagnetic nanoparticles (0.5 ml/kg). Dynamic MR images were obtained at injection and every 30 min for each animal over 2 h to monitor initial changes in signal enhancement in the tumor and muscle regions. All tumors were resected and frozen for histology to corroborate MR molecular imaging results.
Nanoparticle Preparation.
Paramagnetic nanoparticles were produced as described previously (14)
. The nanoparticulate emulsions were comprised of 40% (volume for volume) perfluorooctylbromide, 2% (w/v) of a surfactant commixture, 1.7% (w/v) glycerin and water representing the balance. The surfactant of control, i.e., nontargeted, paramagnetic emulsions, included 60 mol% lecithin (Avanti Polar Lipids, Inc., Alabaster, AL), 8 mol% cholesterol (Sigma Chemical Co., St. Louis, MO), 2 mol% dipalmitoyl-phosphatidylethanolamine (Avanti Polar Lipids, Inc., Alabaster, AL), and 30 mol% Gd-DTPA-BOA (Gateway Chemical Technologies, St. Louis, MO; Ref. 15
). ß3-targeted paramagnetic nanoparticles were prepared as above with a surfactant commixture that included: 60 mol% lecithin, 0.05 mol% N-[{w-[4-(p-maleimidophenyl)butanoyl]amino} poly(ethylene glycol)2000]1,2-distearoyl-sn-glycero-3-phosphoethanolamine covalently coupled to the ß3-integrin peptidomimetic antagonist (Bristol-Myers Squibb Medical Imaging, Inc., North Billerica, MA; Ref. 16
), 8 mol% cholesterol, 30 mol% Gd-DTPA-BOA, and 1.95 mol% DPPE. ß3-targeted nonparamagnetic nanoparticles were prepared in an identical fashion to the targeted formulation excluding the addition of a lipophilic Gd3+ chelate, which was substituted in the surfactant commixture with increased lecithin (70 mol%) and cholesterol (28 mol%). The components for each nanoparticle formulation were emulsified in a M110S Microfluidics emulsifier (Microfluidics, Newton, MA) at 20,000 pounds per square inch for 4 min. The completed emulsions were placed in crimp-sealed vials and blanketed with nitrogen.
Particle sizes were determined at 37°C with a laser light scattering submicron particle size analyzer (Malvern Instruments, Malvern, Worcestershire, United Kingdom), and the concentration of nanoparticles was calculated from the nominal particle size (i.e., particle volume of a sphere). Retained bioactivity of the ß3-integrin peptidomimetic antagonist on the nanoparticle surface was confirmed with a vitronectin cell adhesion assay. Briefly, ß3-targeted nanoparticles significantly inhibited adhesion of human melanoma (C32) cells to vitronectin-coated culture wells (<5% attached compared with controls).
Perfluorocarbon concentration was determined with gas chromatography using flame ionization detection (Model 6890; Agilent Technologies, Inc., Wilmington, DE). One ml of perfluorocarbon emulsion combined with 10% potassium hydroxide in ethanol and 2 ml of internal standard (0.1% octane in freon) was vigorously vortexed and then continuously agitated on a shaker for 30 min. The lower extracted layer was filtered through a silica gel column and stored at 4°C-6°C until analysis. Initial column temperature was 30°C and ramped upward at 10°C/min to 145°C.
The gadolinium content of the emulsions was determined by neutron activation analysis in a 10 MW nuclear reactor (17)
at the University of Missouri Research Reactor. The mass of Gd3+ was determined by measuring the 361 keV 
rays from the ß decay of 161Gd (t1/2 = 3.66 min) produced through neutron capture on 160Gd. Individual samples and standards were irradiated in a thermal neutron flux of ca. 5 x 1013 n/(cm2 x s) for 7 s, allowed to decay for 30 s, and counted on a high-resolution -ray spectrometer for 300 s. The number of Gd3+ complexes per nanoparticle was calculated from the ratio of the concentrations of Gd3+ and the estimated number of nanoparticles in the emulsion. In addition, the relaxivities of each paramagnetic nanoparticle formulation were measured at 0.47 Tesla and 40°C with a Minispec Analyzer (Bruker, Inc., Milton, Ontario, Canada).
MRI.
Twelve days after tumor implantation, the animals underwent MRI scanning on a 1.5 Tesla clinical scanner (NT Intera with Master Gradients, Philips Medical Systems, Best, the Netherlands). Each animal was placed inside a quadrature head/neck birdcage coil with an 11-cm diameter circular surface coil positioned against the hindlimb near the tumor. The quadrature body coil was used for all radiofrequency transmission, the birdcage coil was used for detection during scout imaging, and the surface coil was used for detection during high-resolution imaging. A 10-ml syringe filled with gadolinium diethylenetriaminepentaacetic acid doped water was placed within the high-resolution FOV and served as a signal intensity standard.
Tumors were initially localized at the site of implantation with a T2-weighted turbo spin-echo scan (TR: 2000 ms, TE: 100 ms, FOV: 150 mm, slice thickness: 3 mm, matrix: 128 by 256, signal averages: 2, turbo factor: 3, scan time: 3 min). A high-resolution, T1-weighted, fat suppressed, three-dimensional, gradient echo scan (TR: 40 ms, TE: 5.6 ms, FOV: 64 mm, slice thickness: 0.5 mm, contiguous slices: 30, in-plane resolution: 250 µm, signal averages: 2, flip angle: 65°, scan time: 15 min) of the tumor was collected at baseline and repeated immediately and 30, 60, 90, and 120 min after paramagnetic nanoparticle injection.
MR Image Analysis.
Tumor volumes were calculated on an offline image processing workstation (EasyVision v5.1; Philips Medical Systems, Best, the Netherlands). ROIs were applied manually around the tumor in each slice of the T1-weighted baseline scan, were combined into a three-dimensional object, and the volume was calculated.
To quantify image enhancement over time, we developed an unbiased image analysis program. T1-weighted images (three contiguous slices through the center of each tumor) collected before, immediately after, and 30, 60, 90, and 120 min after i.v. nanoparticle injection were analyzed with MATLAB (The MathWorks, Inc., Natick, MA). The image intensity at each time point was normalized to the baseline image via the reference gadolinium standard. Serial images were spatially coregistered, and contrast enhancement was determined for each pixel at each postinjection time point. An ROI was manually drawn around a portion of the hindlimb muscle in the baseline images, and the average pixel-by-pixel signal enhancement inside the ROI was calculated at each time point. A second ROI was manually drawn around the tumor, and the SD of the tumor signal was calculated in the baseline image for each animal. Pixels were considered enhanced when signal intensity was increased by greater than three times the SD of the tumor signal at baseline (i.e., enhancement > 99% of the variation seen at baseline). Solitary enhancing pixels, those in which all surrounding in-plane pixels did not enhance, were removed from the calculations as noise. The remaining enhancing pixel clusters were mapped back to the immediate, 30-, 60-, and 90-min images, and the average signal increase at each interval was determined. Statistical comparisons were performed for tumor and muscle for each time point using ANOVA (SAS, SAS Institute, Cary, NC). Treatment means were separated using the least significant difference (LSD) procedure (P < 0.05).
Histology and Immunohistochemistry.
After imaging, tumors were resected for histology and immunohistochemistry to verify tumor pathology and assess associated vascularity and angiogenesis. Tumors were frozen (-78°C) in ornithine carbamyl transferase medium with known orientation relative to original anatomical position and the MRI image planes. Four micron frozen sections (Leica Microsystems, Inc., Bannockburn, IL), fixed in acetone at -20°C for 15 min and air dried overnight (4°C), were stained with H&E, murine antihuman/rabbit endothelium antibody (QBEND/40, 1:10 dilution; Research Diagnostics, Inc., Flanders, NJ), or a murine antihuman ß3-integrin (LM-609, 1:200 dilution; Chemicon International, Temecula, CA). Immunohistochemistry was performed using the Vectastain Elite avidin-biotin complex method kit (Vector Laboratories, Burlingame, CA), developed with the Vector VIP kit, and counterstained with Vector methylgreen nuclear counterstain. Slides were reviewed with a Nikon Eclipse E800 research microscope (Nikon USA, Melville, NY) equipped with a Nikon digital camera (Model DXM 1200) and captured with Nikon ACT-1 software.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß3-targeted or nontargeted paramagnetic nanoparticles and dynamically imaged in vivo by MRI at 1.5 Tesla for 2 h to detect and characterize developing angiogenic vasculature. The physical and chemical properties of the two paramagnetic formulations were similar with respect to particle size distribution, gadolinium content, 19fluorine concentration, and relaxivity (Table 1
; Fig. 1
). At 12-days postimplantation, Vx-2 tumor volumes of animals receiving the ß3-targeted (130 ± 39 mm3) or nontargeted nanoparticles (148 ± 36 mm3) were not different (P > 0.05).
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ß3-targeted paramagnetic nanoparticles revealed a marked increase in MR contrast primarily, although not exclusively, located asymmetrically along the tumor periphery. ß3-integrin enhancement was typically seen in a patchy distribution adjacent to blood vessels and along tissue fascial interfaces (Fig. 2)
. Histology and immunocytochemical assessments of the Vx-2 tumors corroborated that angiogenesis was predominately distributed within a few independent regions along the tumor periphery and was found less often within intratumoral connective tissue tracts interspersed between tumor cell lobules (Fig. 3)
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ß3-targeted paramagnetic nanoparticles was detected in regions of angiogenesis soon after injection at relatively low levels, which was presumably attributed to a local extravasation of nanoparticles through a fenestrated neovasculature at 30 min (Fig. 4)
. No intravascular blood pool contrast effect was detectable after 30 min. After 2 h, the magnitude of signal enhancement among rabbits treated with the ß3-targeted nanoparticles increased (56%) relative to the nontargeted nanoparticle effect (P < 0.05). Blockage of ß3-integrin sites with pretargeted nonparamagnetic ß3-nanoparticles 2 h before injection of the ß3-targeted paramagnetic particles reduced the targeted contrast signal enhancement in half (P < 0.05), to a signal effect slightly below that attributed to localized neovascular leakage, confirming the specificity of the targeted nanoparticles.
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ß3-targeted or nontargeted paramagnetic nanoparticles.
All Vx-2 rabbits routinely underwent baseline T2-weighted MRI imaging at the known site of surgery to localize tumor at 12-days postimplantation. In some rabbits, no tumor was detected, and so these were excluded from the study. In a few other animals, a mass which appeared appropriate based on size and T2-image characteristics was observed, but later, histology revealed it to be a tumor remnant with heavy infiltrates of inflammatory cells (Fig. 5)
; these animals were excluded from the study as well. The hyperintense appearance on T2-weighted MRI is attributable to edema associated with inflammation. Interestingly, among this subset of animals, some randomly received ß3-targeted paramagnetic nanoparticles, and no MR contrast enhancement was appreciated within the periphery of the mass nor within nearby vasculature (Fig. 6)
. This lack of signal enhancement associated with a popliteal mass or adjacent vasculature was clearly distinct from the molecular imaging features routinely obtained in animals with histologically verified tumor. Histology and immunohistochemical analysis of the remnant tissues confirmed a paucity of vascularity in the tumor periphery and adjacent tissues with negligible staining for the ß3-integrin. These findings illustrate the specificity of molecular imaging to help differentiate viable Vx-2 masses from tumor remnants.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sensitive molecular imaging and detection of tumors or their supporting neovascularity requires high avidity, target-specific probes, which produce robust signal amplification compatible with a sensitive high-resolution imaging modality. Until recently, the signal amplification required to detect and visualize important molecular or cellular moieties present in nano and picomolar concentrations in vivo was obtainable only with nuclear imaging modalities, which were plagued by high background noise levels. More recently, molecular imaging with magnetic resonance has shown great promise (3 , 4 , 13 , 14 , 19 , 20) .
Our novel approach entails the use of a ligand-directed paramagnetic molecular imaging platform technology designed to improve detection and quantification of occult tumors and metastases. The contrast agent is a lipid-encapsulated liquid perfluorocarbon nanoparticle (250 nm nominal diameter) that has high target avidity, a prolonged systemic half-life, and can carry enormous paramagnetic ion payloads (>90,000 Gd3+ atoms per particle) for high detection sensitivity.
Molecular Imaging of Angiogenesis.
In the present study, New Zealand White rabbits implanted with nascent Vx-2 tumors were treated systemically with an ß3-integrin-targeted paramagnetic nanoparticle agent that bound to and significantly increased the MR detection of the developing neovasculature using a commercially available, clinical magnetic resonance scanner (1.5 Tesla). Targeted contrast signal enhancement was specific to sites of angiogenesis associated directly with the tumor and adjoining vasculature and fascia stimulated by the developing tumor. Signal intensity of the targeted angiogenic endothelium increased steadily and rapidly to >125% after 2 h. In parallel unpublished studies, nanoparticle systemic half-life was estimated in excess of 2 h, which suggests that MRI of the tumors at later time points may yield greater signal enhancement from angiogenic sites and provide an opportunity for unbound nanoparticles to wash out from the local interstitium.
These results are consistent with our previous experiments in which paramagnetic perfluorocarbon nanoparticles coupled to monoclonal antibodies specific for ß3-integrin and targeted to angiogenic vessels artificially induced by basic fibroblast growth factor in a rabbit corneal micro-pocket model increased the signal steadily for 3 h with a significant increase in contrast detected after 1 h (13)
. In contradistinction to this earlier study in which the antibody was coupled to the nanoparticles through avidin-biotin interactions and injected as a "single-step" system, in the current experiments, a small arginine-glycine-aspartic acid (RGD)-peptidomimetic covalently attached to the nanoparticle was used, which increased the number of ligands per particle 10-fold. In addition, in the current paramagnetic formulation, each ß3-integrin-bound nanoparticle carries a 50% greater payload of gadolinium to better enhance the signal from angiogenic vessels. These two factors have been predominantly responsible for a 400% improvement in contrast signal enhancement compared with the earlier corneal micro-pocket model results.
In seminal work reported by Sipkins et al. (19)
, ß3-targeted paramagnetic polymerized liposomes were used to detect angiogenesis in the Vx-2 tumor model. In that study, adequate contrast enhancement was appreciated only after 24 h, and the pattern of neovascular contrast enhancement reported was different from that observed in the present study. Sipkins et al. noted contrast enhancement throughout the entire tumor mass, whereas, in the present study, angiogenesis was predominately detected along the tumor periphery by MR imaging within 2 h and by immunohistochemistry. In part, this discrepancy may be attributable to variations in tumor implantation technique between the two studies. However, progressive leakage of the smaller liposomes used by Sipkins through the angiogenic vasculature over the longer time course of circulation and later imaging time point (24 h) is probably much greater than sterically possible with the larger perfluorocarbon nanoparticles used in the present study. Although the larger size of our nanoparticles minimizes extravasation into the tumor interstitium, a subpopulation was nonspecifically retained locally, which provided a passively targeted, lower level of signal enhancement in the neovascular regions.
Neovascular Permeability.
Abnormal capillary permeability of tumors is typically studied by two general MRI approaches: dynamic imaging using extracellular fluid agents in conjunction with kinetic analysis (21)
and time-resolved T1-weighted MRI of the uptake of macromolecular contrast agents, typically Mr >20,000 (22)
. These results correlate with changes in tumor angiogenesis, but issues of potential toxicity and prolonged imaging times have limited clinical acceptance of these methods. Medium molecular weight agents (e.g., NMS60, Mr 2,000; Ref. 23
) have much lower leakage rates (50%) than Gd-DTPA, yet provide 20% higher peak contrast signal intensity because of their greater molecular T1-relaxivity [9.1 versus 3.5 (s x mM)-1 at 1.5 Tesla and 37°C].
Our nanoparticle contrast agent exhibits an "ultraparamagnetic" MR character with a molecular relaxivity of >1,800,000 (s x mM)-1, which is essential for molecular imaging of biochemical epitopes expressed on cell surfaces only in nanomolar concentration. A permeability pilot study was conducted in which the leakage of Gd-DTPA was compared with paramagnetic nanoparticles. The preliminary results confirm that paramagnetic nanoparticle leakage along the rim is extremely low, very delayed, and highly restricted in comparison with Gd-DTPA.4 As expected, nanoparticle leakage in the tumor is much greater than muscle, similar to the results obtained by Crespigny et al. (23) , reflecting the increased permeability of angiogenic vessels and the "ultraparamagnetic" effects of the extravasated nanoparticles.
Clinical Implications.
Molecular imaging of angiogenesis with targeted paramagnetic nanoparticles could allow the early detectability of solid tumors or metastases in myriad clinical circumstances. Ligand-directed paramagnetic nanoparticles may be adapted to noninvasively detect, quantify, and biochemically characterize a variety of biomarkers on tumor neovasculature, which may help to segment patient populations for therapeutic regimens or to noninvasively monitor tumor progression during treatment. In particular, these features could address some of the limitations of MVD measurements in the design, evaluation, and clinical implementation of novel antiangiogenic therapies (12)
.
We have recently reported the potential of ligand-directed paramagnetic nanoparticles to carry and deliver antiproliferative agents, e.g., doxorubicin and paclitaxel, specifically to targeted cells (20) . Additionally, we demonstrated the concept that targeted drug delivery with nanoparticles can be confirmed with 1H MRI, as illustrated in the current study, and provide quantification of delivered drug dosage using noninvasive MR 19fluorine spectroscopy of the perfluorocarbon core. This unique combination of molecular imaging and rational targeted therapy offers additional broad clinical possibilities for the future.
Study Limitations.
In the present study, dynamic imaging of angiogenic signal enhancement continued for 2 h postinjection by experimental design. However, given the marked increase in contrast detected after the 2 h, evaluation of longer time courses is warranted with the expectation that even greater signal enhancement may be achieved.
The dosage of nanoparticles in the present study was 0.5 ml/kg, well within the expected safety range for perfluorocarbon emulsions. However, preliminary results suggest that much lower dosages of targeted nanoparticles may be equally effective.
Finally, the molecular relaxivity of paramagnetic nanoparticles used in the present study, already well beyond the relaxivity of any other known paramagnetic agent, has recently been improved by 150% (24)
, which should further increase the sensitivity of this MR molecular imaging agent to detect endothelial cell surface biomarkers and delineate tumor physiology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported, in part, by Grant CO-07121 from the National Cancer Institute (to G. M. L.), Grant 0235125N from the American Heart Association (to P. M. W.), and a grant from Philips Medical Systems (to S. A. W.). 
2 To whom requests for reprints should be addressed, at Washington University School of Medicine, Cardiovascular Division, 660 South Euclid Avenue, Campus Box #8086, St. Louis, MO 63110. Fax: (314) 454-7490; E-mail: patrick{at}cvu.wustl.edu
3 The abbreviations used are: MR, magnetic resonance; MRI, magnetic resonance imaging; MVD, microvessel density; Gd-DTPA-BOA, gadolinium diethylenetriaminepentaacetic acid-bisoleate; Gd-DTPA, gadolinium diethylenetriaminepentaacetic acid; FOV, field of view; ROI, region-of-interest.
4 Patrick M. Winter, Shelton D. Caruthers, Andrea Kassner, Samuel A. Wickline, Gregory M. Lanza, unpublished data.
Received 3/26/03. Revised 6/ 6/03. Accepted 6/13/03.
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W. Cai, Y. Wu, K. Chen, Q. Cao, D. A. Tice, and X. Chen In vitro and In vivo Characterization of 64Cu-Labeled AbegrinTM, a Humanized Monoclonal Antibody against Integrin {alpha}v{beta}3 Cancer Res., October 1, 2006; 66(19): 9673 - 9681. [Abstract] [Full Text] [PDF]
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S. Geninatti Crich, B. Bussolati, L. Tei, C. Grange, G. Esposito, S. Lanzardo, G. Camussi, and S. Aime Magnetic resonance visualization of tumor angiogenesis by targeting neural cell adhesion molecules with the highly sensitive gadolinium-loaded apoferritin probe. Cancer Res., September 15, 2006; 66(18): 9196 - 9201. [Abstract] [Full Text] [PDF]
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 P. M. Winter, A. M. Neubauer, S. D. Caruthers, T. D. Harris, J. D. Robertson, T. A. Williams, A. H. Schmieder, G. Hu, J. S. Allen, E. K. Lacy, et al. Endothelial {alpha}{nu}{beta}3 Integrin-Targeted Fumagillin Nanoparticles Inhibit Angiogenesis in Atherosclerosis Arterioscler Thromb Vasc Biol, September 1, 2006; 26(9): 2103 - 2109. [Abstract] [Full Text] [PDF]
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 M. Atri New Technologies and Directed Agents for Applications of Cancer Imaging J. Clin. Oncol., July 10, 2006; 24(20): 3299 - 3308. [Abstract] [Full Text] [PDF]
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S. A. Wickline, A. M. Neubauer, P. Winter, S. Caruthers, and G. Lanza Applications of Nanotechnology to Atherosclerosis, Thrombosis, and Vascular Biology Arterioscler Thromb Vasc Biol, March 1, 2006; 26(3): 435 - 441. [Abstract] [Full Text] [PDF]
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R. Rossin, D. Pan, K. Qi, J. L. Turner, X. Sun, K. L. Wooley, and M. J. Welch 64Cu-Labeled Folate-Conjugated Shell Cross-Linked Nanoparticles for Tumor Imaging and Radiotherapy: Synthesis, Radiolabeling, and Biologic Evaluation J. Nucl. Med., July 1, 2005; 46(7): 1210 - 1218. [Abstract] [Full Text] [PDF]
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 P. J Cassidy and G. K Radda Molecular imaging perspectives J R Soc Interface, June 22, 2005; 2(3): 133 - 144. [Abstract] [Full Text] [PDF]
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S. M. Moghimi, A. C. Hunter, and J. C. Murray Nanomedicine: current status and future prospects FASEB J, March 1, 2005; 19(3): 311 - 330. [Abstract] [Full Text] [PDF]
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 J. C. Miller, H. H. Pien, D. Sahani, A. G. Sorensen, and J. H. Thrall Imaging Angiogenesis: Applications and Potential for Drug Development J Natl Cancer Inst, February 2, 2005; 97(3): 172 - 187. [Abstract] [Full Text] [PDF]
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K. Kelly, H. Alencar, M. Funovics, U. Mahmood, and R. Weissleder Detection of Invasive Colon Cancer Using a Novel, Targeted, Library-Derived Fluorescent Peptide Cancer Res., September 1, 2004; 64(17): 6247 - 6251. [Abstract] [Full Text] [PDF]
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) paramagnetic nanoparticles, nontargeted (
) paramagnetic nanoparticles, or competition group (
). Significantly higher signal enhancement was observed in tumor 2 h postinjection of 
