M. Karen J. Gagnon

Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice

The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1+ cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1–coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[18F]fluorobenzoic acid ([18F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [18F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [18F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.

Targeted In vivo Imaging of Integrin αvβ6 with an improved Radiotracer and Its Relevance in a Pancreatic Tumor Model

The cell surface receptor alpha(v)beta(6) is epithelial specific, and its expression is tightly regulated; it is low or undetectable in adult tissues but has been shown to be increased in many different cancers, including pancreatic, cervical, lung, and colon cancers. Studies have described alpha(v)beta(6) as a prognostic biomarker linked to poor survival. We have recently shown the feasibility of imaging alpha(v)beta(6) in vivo by positron emission tomography (PET) using the peptide [(18)F]FBA-A20FMDV2. Here, we describe improved alpha(v)beta(6) imaging agents and test their efficacy in a mouse model with endogenous alpha(v)beta(6) expression. The modified compounds maintained high affinity for alpha(v)beta(6) and >1,000-fold selectivity over related integrins (by ELISA) and showed significantly improved alpha(v)beta(6)-dependent binding in cell-based assays (>60% binding versus <10% for [(18)F]FBA-A20FMDV2). In vivo studies using either a melanoma cell line (transduced alpha(v)beta(6) expression) or the BxPC-3 human pancreatic carcinoma cell line (endogenous alpha(v)beta(6) expression) revealed that the modified compounds showed significantly improved tumor retention. This, along with good clearance of nonspecifically bound activity, particularly for the new radiotracer [(18)F]FBA-PEG(28)-A20FMDV2, resulted in improved PET imaging. Tumor/pancreas and tumor/blood biodistribution ratios of >23:1 and >47:1, respectively, were achieved at 4 hours. Significantly, [(18)F]FBA-PEG(28)-A20FMDV2 was superior to 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) in imaging the BxPC-3 tumors. Pancreatic ductal adenocarcinoma is highly metastatic and current preoperative evaluation of resectability using noninvasive imaging has limited success, with most patients having metastases at time of surgery. The fact that these tumors express alpha(v)beta(6) suggests that this probe has significant potential for the in vivo detection of this malignancy, thus having important implications for patient care and therapy.

High-throughput in vivo screening of targeted molecular imaging agents

The rapid development and translation of targeted molecular imaging agents from bench to bedside is currently a slow process, with a clear bottleneck between the discovery of new compounds and the development of an appropriate molecular imaging agent. The ability to identify promising new molecular imaging agents, as well as failures, much earlier in the development process using high-throughput screening techniques could save significant time and money. This work combines the advantages of combinatorial chemistry, site-specific solid-phase radiolabeling, and in vivo imaging for the rapid screening of molecular imaging agents. A one-bead-one-compound library was prepared and evaluated in vitro, leading to the identification of 42 promising lead peptides. Over 11 consecutive days, these peptides, along with a control peptide, were successfully radiolabeled with 4-[18F]fluorobenzoic acid and evaluated in vivo using microPET. Four peptides were radiolabeled per day, followed by simultaneous injection of each individual peptide into 2 animals. As a result, 4 promising new molecular imaging agents were identified that otherwise would not have been selected based solely on in vitro data. This study is the first example of the practical application of a high-throughput screening approach using microPET imaging of [18F]-labeled peptides for the rapid in vivo identification of potential new molecular imaging agents.

Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE–/– Mice

The current study presents an effective and selective multifunctional nanoparticle used to deliver antiatherogenic therapeutics to inflamed pro-atherogenic regions without off-target changes in gene expression or particle-induced toxicities. MicroRNAs (miRNAs) regulate gene expression, playing a critical role in biology and disease including atherosclerosis. While anti-miRNA are emerging as therapeutics, numerous challenges remain due to their potential off-target effects, and therefore the development of carriers for selective delivery to diseased sites is important. Yet, co-optimization of multifunctional nanoparticles with high loading efficiency, a hidden cationic domain to facilitate lysosomal escape and a dense, stable incorporation of targeting moieties is challenging. Here, we create coated, cationic lipoparticles (CCLs), containing anti-miR-712 (∼1400 molecules, >95% loading efficiency) within the core and with a neutral coating, decorated with 5 mol % of peptide (VHPK) to target vascular cell adhesion molecule 1 (VCAM1). Optical imaging validated disease-specific accumulation as anti-miR-712 was efficiently delivered to inflamed mouse aortic endothelial cells in vitro and in vivo. As with the naked anti-miR-712, the delivery of VHPK-CCL-anti-miR-712 effectively downregulated the d-flow induced expression of miR-712 and also rescued the expression of its target genes tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) in the endothelium, resulting in inhibition of metalloproteinase activity. Moreover, an 80% lower dose of VHPK-CCL-anti-miR-712 (1 mg/kg dose given twice a week), as compared with naked anti-miR-712, prevented atheroma formation in a mouse model of atherosclerosis. While delivery of naked anti-miR-712 alters expression in multiple organs, miR-712 expression in nontargeted organs was unchanged following VHPK-CCL-anti-miR-712 delivery.

Accumulation, internalization and therapeutic efficacy of neuropilin-1-targeted liposomes

Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.

A Physiological Perspective on the Use of Imaging to Assess the In Vivo Delivery of Therapeutics

Our goal is to provide a physiological perspective on the use of imaging to optimize and monitor the accumulation of nanotherapeutics within target tissues, with an emphasis on evaluating the pharmacokinetics of organic particles. Positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound technologies, as well as methods to label nanotherapeutic constructs, have created tremendous opportunities for preclinical optimization of therapeutics and for personalized treatments in challenging disease states. Within the methodology summarized here, the accumulation of the construct is estimated directly from the image intensity. Particle extravasation is then estimated based on classical physiological measures. Specifically, the transport of nanotherapeutics is described using the concept of apparent permeability, which is defined as the net flux of solute across a blood vessel wall per unit surface area of the blood vessel and per unit solute concentration difference across the blood vessel wall. The apparent permeability to small molecule MRI constructs is accurately shown to be far larger than that estimated for proteins such as albumin or nanoconstructs such as liposomes. Further, the quantitative measurements of vascular permeability are shown to facilitate detection of the transition from a pre-malignant to a malignant cancer and to quantify the delivery enhancement resulting from interventions such as ultrasound. While PET-based estimates facilitate quantitative comparisons of many constructs, high field MRI proves useful in the visualization of model drugs within small lesions and in the evaluation of the release and intracellular trafficking of nanoparticles and cargo.

Abstract A227: Positron emission tomographic (PET) imaging of activated matriptase, a marker for cancer progression

The serine protease matriptase has been implicated in epithelial cancers, has been found in ∼45% of node‐negative breast cancers at high levels, and is indicated as a biomarker for survival independent of HER‐2/neu. While in vitro methods are invaluable, few breast cancer cell‐lines express matriptase, though 83% of breast cancer patients are positive for matriptase—this suggests that imaging the in vivo behavior of matriptase may aid in understanding its role in the context of the tumor system. Specifically, activated matriptase is associated with cancer progression. We have developed radiotracers against activated matriptase for in vivo imaging using PET. M69, an antibody against activated matriptase, was functionalized to capture [64Cu]copper or [89Zr]zirconium: 64Cu‐TETA‐M69 and 89Zr‐DF‐M69 were evaluated in a mouse model of human breast cancer. A dox‐regulable pair of PyVmT cell‐lines was bilaterally introduced into nude female mice to generate matriptase‐positive and control tumors. Mice were fed dox chow ad libitum, administered radiotracer, imaged using microPET at 1–4d p.i.; and through 336h for 89Zr; corresponding biodistribution was performed. PET images indicated specific tumor retention. Biodistribution at 4d revealed both 64Cu‐TETA‐M69 and 89Zr‐DF‐M69 retained two‐fold uptake ratios for the positive tumor over control; this was also observed at 336h for 89Zr‐DF‐M69. Immunohistochemistry on FFPE tissues confirmed human matriptase expression in the positive tumor. In summary, we have developed two novel radiotracers and performed the first in vivo imaging of activated matriptase. This approach has the potential for imaging metastasis, the primary cause of mortality in breast cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A227.