An unmet clinical need: The history of thrombus imaging.

Robust thrombus imaging is an unresolved clinical unmet need dating back to the mid 1970s. While early molecular imaging approaches began with nuclear SPECT imaging, contrast agents for virtually all biomedical imaging modalities have been demonstrated in vivo with unique strengths and common weaknesses. Two primary molecular imaging targets have been pursued for thrombus imaging: platelets and fibrin. Some common issues noted over 40 years ago persist today. Acute thrombus is readily imaged with all probes and modalities, but aged thrombus remains a challenge. Similarly, anti-coagulation continues to interfere with and often negate thrombus imaging efficacy, but heparin is clinically required in patients suspected of pulmonary embolism, deep venous thrombosis or coronary ruptured plaque prior to confirmatory diagnostic studies have been executed and interpreted. These fundamental issues can be overcome, but an innovative departure from the prior approaches will be needed.

Dual-therapy with αvß3-targeted Sn2 lipase-labile fumagillin-prodrug nanoparticles and zoledronic acid in the Vx2 rabbit tumor model.

Fumagillin, an unstable anti-angiogenesis mycotoxin, was synthesized into a stable lipase-labile prodrug and incorporated into integrin-targeted lipid-encapsulated nanoparticles (αvß3-Fum-PD NP). Dual anti-angiogenic therapy combining αvß3-Fum-PD NP with zoledronic acid (ZA), a long-acting osteoclast inhibitor with proposed anti-angiogenic effects, was evaluated. In vitro, αvß3-Fum-PD NP reduced (P<0.05) endothelial cell viability without impacting macrophage viability. ZA suppressed (P<0.05) macrophage viability at high dosages but not endothelial cell proliferation. 3D MR neovascular imaging of rabbit Vx2 tumors showed no effect with ZA, whereas αvß3-Fum-PD NP alone and with ZA decreased angiogenesis (P<0.05). Immunohistochemistry revealed decreased (P<0.05) microvascularity with αvß3-Fum-PD NP and ZA and further microvascular reduction (P<0.05) with dual-therapy. In vivo, ZA did not decrease tumor macrophage numbers nor cancer cell proliferation, whereas αvß3-Fum-PD-NPs reduced both measures. Dual-therapy with ZA and αvß3-Fum-PD-NP may provide enhanced neo-adjuvant utility if macrophage ZA uptake is increased. From the Clinical Editor: Although anti-angiogenesis is one of the treatment modalities in the fight against cancer, many cancers become resistant to VEGF pathway inhibitors. In this article, the authors investigated the use of dual therapy using fumagillin, integrin-targeted lipid-encapsulated nanoparticles (αvß3- Fum-PD NP) and zoledronic acid (ZA), in both in-vitro and in-vivo experiments. This combination approach may provide an insight to the design of future drugs against cancers.

Atherosclerotic neovasculature MR imaging with mixed manganese-gadolinium nanocolloids in hyperlipidemic rabbits.

A high r1 relaxivity manganese-gadolinium nanocolloid (αvß3-MnOL-Gd NC) was developed and effectively detected atherosclerotic angiogenesis in rabbits fed cholesterol-rich diets for 12 months using a clinical MRI scanner (3T). 3D mapping of neovasculature signal intensity revealed the spatial coherence and intensity of plaque angiogenic expansion, which may, with other high risk MR bioindicators, help identify high-risk patients with moderate (40% to 60%) vascular stenosis. Microscopy confirmed the predominant media and plaque distribution of fluorescent αvß3-MnOL-Gd NC, mirroring the MR data. An expected close spatial association of αvß3-integrin neovasculature and macrophages was noted, particularly within plaque shoulder regions. Manganese oleate bioelimination occurred via the biliary system into feces. Gd-DOTA was eliminated through the bile-fecal and renal excretion routes. αvß3-MnOL-Gd NC offers an effective vehicle for T1w neovascular imaging in atherosclerosis. From the clinical editor: Cerebrovascular accidents are a leading cause of mortality and morbidity worldwide. The acute formation of thrombus following atherosclerotic plaque rupture has been well recognized as the etiology of stroke. The authors studied microanatomical features of vulnerable atherosclerotic plaque in this article, in an attempt to identify those with high risk of rupture. Gadolinium-manganese hybrid nanocolloid (MnOL-Gd NC) was developed as a novel contrast agent for MRI. They show that this agent is effective in providing neovascular imaging.

Synergy between surface and core entrapped metals in a mixed manganese-gadolinium nanocolloid affords safer MR imaging of sparse biomarkers.

High-relaxivity T1-weighted (T1w) MR molecular imaging nanoparticles typically present high surface gadolinium payloads that can elicit significant acute complement activation (CA). The objective of this research was to develop a high T1w contrast nanoparticle with improved safety. We report the development, optimization, and characterization of a gadolinium-manganese hybrid nanocolloid (MnOL-Gd NC; 138±10 (Dav)/nm; PDI: 0.06; zeta: -27±2 mV). High r1 particulate relaxivity with minute additions of Gd-DOTA-lipid conjugate to the MnOL nanocolloid surface achieved an unexpected paramagnetic synergism. This hybrid MnOL-Gd NC provided optimal MR TSE signal intensity at 5 nM/voxel and lower levels consistent with the level expression anticipated for sparse biomarkers, such as neovascular integrins. MnOL NC produced optimal MR TSE signal intensity at 10 nM/voxel concentrations and above. Importantly, MnOL-Gd NC avoided acute CA in vitro and in vivo while retaining minimal transmetallation risk. From the clinical editor: The authors developed a gadolinium-manganese hybrid nanocolloid (MnOL-Gd NC) in this study. These were used as a high-relaxivity paramagnetic MR molecular imaging agent in experimental models. It was shown that MnOL-Gd NC could provide high T1w MR contrast for targeted imaging. As the level of gadolinium used was reduced, there was also reduced risk of systemic side effects from complement activation.

Characterization of early neovascular response to acute lung ischemia using simultaneous (19)F/ (1)H MR molecular imaging.

Angiogenesis is an important constituent of many inflammatory pulmonary diseases, which has been unappreciated until recently. Early neovascular expansion in the lungs in preclinical models and patients is very difficult to assess noninvasively, particularly quantitatively. The present study demonstrated that (19)F/(1)H MR molecular imaging with αvß3-targeted perfluorocarbon nanoparticles can be used to directly measure neovascularity in a rat left pulmonary artery ligation (LPAL) model, which was employed to create pulmonary ischemia and induce angiogenesis. In rats 3 days after LPAL, simultaneous (19)F/(1)H MR imaging at 3T revealed a marked (19)F signal in animals 2 h following αvß3-targeted perfluorocarbon nanoparticles [(19)F signal (normalized to background) = 0.80 ± 0.2] that was greater (p = 0.007) than the non-targeted (0.30 ± 0.04) and the sham-operated (0.07 ± 0.09) control groups. Almost no (19)F signal was found in control right lung with any treatment. Competitive blockade of the integrin-targeted particles greatly decreased the (19)F signal (p = 0.002) and was equivalent to the non-targeted control group. Fluorescent and light microscopy illustrated heavy decorating of vessel walls in and around large bronchi and large pulmonary vessels. Focal segmental regions of neovessel expansion were also noted in the lung periphery. Our results demonstrate that (19)F/(1)H MR molecular imaging with αvß3-targeted perfluorocarbon nanoparticles provides a means to assess the extent of systemic neovascularization in the lung.

MR cholangiography demonstrates unsuspected rapid biliary clearance of nanoparticles in rodents: implications for clinical translation.

Due to their small size, lower cost, short reproduction cycle, and genetic manipulation, rodents have been widely used to test the safety and efficacy for pharmaceutical development in human disease. In this report, MR cholangiography demonstrated an unexpected rapid (<5 min) biliary elimination of gadolinium-perfluorocarbon nanoparticles (approximately 250 nm diameter) into the common bile duct and small intestine of rats, which is notably different from nanoparticle clearance patterns in larger animals and humans. Unawareness of this dissimilarity in nanoparticle clearance mechanisms between small animals and humans may lead to fundamental errors in predicting nanoparticle efficacy, pharmacokinetics, biodistribution, bioelimination, and toxicity. From the clinical editor: Comprehensive understanding of nanoparticle clearance is a clear prerequisite for human applications of nanomedicine-based therapeutic approaches. Through a novel use of MR cholangiography, this study demonstrates unusually rapid hepatic clearance of gadolinium-perfluorocarbon nanoparticles in rodents, in a pattern that is different than what is observed in larger animals and humans, raising awareness of important differences between common rodent-based models and larger mammals.

Molecular MR imaging of neovascular progression in the Vx2 tumor with αvß3-targeted paramagnetic nanoparticles.

PURPOSE: To assess the dependence of neovascular molecular magnetic resonance (MR) imaging on relaxivity (r1) of αvß3-targeted paramagnetic perfluorocarbon (PFC) nanoparticles and to delineate the temporal-spatial consistency of angiogenesis assessments for individual animals. MATERIALS AND METHODS: Animal protocols were approved by the Washington University Animal Studies Committee. Proton longitudinal and transverse relaxation rates of αvß3-targeted and nontargeted PFC nanoparticles incorporating gadolinium diethylenetrianime pentaacedic acid (Gd-DTPA) bisoleate (BOA) or gadolinium tetraazacyclododecane tetraacetic acid (Gd-DOTA) phosphatidylethanolamine (PE) into the surfactant were measured at 3.0 T. These paramagnetic nanoparticles were compared in 30 New Zealand White rabbits (four to six rabbits per group) 14 days after implantation of a Vx2 tumor. Subsequently, serial MR (3.0 T) neovascular maps were developed 8, 14, and 16 days after tumor implantation by using αvß3-targeted Gd-DOTA-PE nanoparticles (n = 4) or nontargeted Gd-DOTA-PE nanoparticles (n = 4). Data were analyzed with analysis of variance and nonparametric statistics. RESULTS: At 3.0 T, Gd-DTPA-BOA nanoparticles had an ionic r1 of 10.3 L · mmol(-1) · sec(-1) and a particulate r1 of 927000 L · mmol(-1) · sec(-1). Gd-DOTA-PE nanoparticles had an ionic r1 of 13.3 L · mmol(-1) · sec(-1) and a particulate r1 of 1 197000 L · mmol(-1) · sec(-1). Neovascular contrast enhancement in Vx2 tumors (at 14 days) was 5.4% ± 1.06 of the surface volume with αvß3-targeted Gd-DOTA-PE nanoparticles and 3.0% ± 0.3 with αvß3-targeted Gd-DTPA-BOA nanoparticles (P = .03). MR neovascular contrast maps of tumors 8, 14, and 16 days after implantation revealed temporally consistent and progressive surface enhancement (1.0% ± 0.3, 4.5% ± 0.9, and 9.3% ± 1.4, respectively; P = .0008), with similar time-dependent changes observed among individual animals. CONCLUSION: Temporal-spatial patterns of angiogenesis for individual animals were followed to monitor longitudinal tumor progression. Neovasculature enhancement was dependent on the relaxivity of the targeted agent.

Sensitive biological detection with a soluble and stable polymeric paramagnetic nanocluster.

We describe the design, synthesis, and biological characterization of manganese oxocluster-based "single molecule magnets (SMMs)". We demonstrate that polymeric micellar nanoparticles can serve as a carrier and help to stabilize delicate SMM molecules from breaking down easily and thus prevent their property loss. Concentrating thousands of Mn-clusters per micelle provided a high ionic and per-particle relaxivity allowing sensitive MR imaging in vivo. This reports one of the earliest examples of in vivo imaging of a rationally designed polymeric micelle that features SMM.

MR angiogenesis imaging with Robo4- vs. alphaVbeta3-targeted nanoparticles in a B16/F10 mouse melanoma model.

The primary objective of this study was to utilize MR molecular imaging to compare the 3-dimensional spatial distribution of Robo4 and α(V)ß(3)-integrin as biosignatures of angiogenesis, in a rapidly growing, syngeneic tumor. B16-F10 melanoma-bearing mice were imaged with magnetic resonance (MR; 3.0 T) 11 d postimplantation before and after intravenous administration of either Robo4- or α(V)ß(3)-targeted paramagnetic nanoparticles. The percentage of MR signal-enhanced voxels throughout the tumor volume was low and increased in animals receiving α(V)ß(3)- and Robo4-targeted nanoparticles. Neovascular signal enhancement was predominantly associated with the tumor periphery (i.e., outer 50% of volume). Microscopic examination of tumors coexposed to the Robo4- and α(V)ß(3)-targeted nanoparticles corroborated the MR angiogenesis mapping results and further revealed that Robo4 expression generally colocalized with α(V)ß(3)-integrin. Robo4- and α(V)ß(3)-targeted nanoparticles were compared to irrelevant or nontargeted control groups in all modalities. These results suggest that α(V)ß(3)-integrin and Robo4 are useful biomarkers for noninvasive MR molecular imaging in syngeneic mouse tumors, but α(V)ß(3)-integrin expression was more detectable by MR at 3.0 T than Robo4. Noninvasive, neovascular assessments of the MR signal of Robo4, particularly combined with α(V)ß(3)-integrin expression, may help define tumor character prior to and following cancer therapy.

Manganese-based MRI contrast agents: past, present and future.

Paramagnetic and superparamagnetic metals are used as contrast materials for magnetic resonance (MR) based techniques. Lanthanide metal gadolinium (Gd) has been the most widely explored, predominant paramagnetic contrast agent until the discovery and association of the metal with nephrogenic systemic fibrosis (NSF), a rare but serious side effects in patients with renal or kidney problems. Manganese was one of the earliest reported examples of paramagnetic contrast material for MRI because of its efficient positive contrast enhancement. In this review, manganese based contrast agent approaches are discussed with a particular emphasis on their synthetic approaches. Both small molecules based typical blood pool contrast agents and more recently developed novel nanometer sized materials are reviewed focusing on a number of successful molecular imaging examples.

Angiogenesis imaging with vascular-constrained particles: the why and how.

Angiogenesis is a keystone in the treatment of cancer and potentially many other diseases. In cancer, first-generation antiangiogenic therapeutic approaches have demonstrated survival benefit in subsets of patients, but their high cost and notable adverse side effect risk have fueled alternative development efforts to personalize patient selection and reduce off-target effects. In parallel, rapid advances in cost-effective genomic profiling and sensitive early detection of high-risk biomarkers for cancer, atherosclerosis, and other angiogenesis-related pathologies will challenge the medical imaging community to identify, characterize, and risk stratify patients early in the natural history of these disease processes. Conventional diagnostic imaging techniques were not intended for such sensitive and specific detection, which has led to the emergence of novel noninvasive biomedical imaging approaches. The overall intent of molecular imaging is to achieve greater quantitative characterization of pathologies based on microanatomical, biochemical, or functional assessments; in many approaches, the capacity to deliver effective therapy, e.g., antiangiogenic therapy, can be combined. Agents with both diagnostic and therapy attributes have acquired the moniker "theranostics." This review will explore biomedical imaging options being pursued to better segment and treat patients with angiogenesis-influenced disease using vascular-constrained contrast platform technologies.

Nanotherapy delivery of c-myc inhibitor targets Protumor Macrophages and preserves Antitumor Macrophages in Breast Cancer.

Tumor-associated macrophages (TAMs) enhance tumor growth in mice and are correlated with a worse prognosis for breast cancer patients. While early therapies sought to deplete all macrophages, current therapeutics aim to reprogram pro-tumor macrophages (M2) and preserve those necessary for anti-tumor immune responses (M1). Recent studies have shown that c-MYC (MYC) is induced in M2 macrophages in vitro and in vivo where it regulates the expression of tumor-promoting genes. In a myeloid lineage MYC KO mouse model, MYC had important roles in macrophage maturation and function leading to reduced tumor growth. We therefore hypothesized that targeted delivery of a MYC inhibitor to established M2 TAMs could reduce polarization toward an M2 phenotype in breast cancer models. Methods: In this study, we developed a MYC inhibitor prodrug (MI3-PD) for encapsulation within perfluorocarbon nanoparticles, which can deliver drugs directly to the cytosol of the target cell through a phagocytosis independent mechanism. We have previously shown that M2-like TAMs express significant levels of the vitronectin receptor, integrin ß3, and in vivo targeting and therapeutic potential was evaluated using αvß3 integrin targeted rhodamine-labeled nanoparticles (NP) or integrin αvß3-MI3-PD nanoparticles. Results: We observed that rhodamine, delivered by αvß3-rhodamine NP, was incorporated into M2 tumor promoting macrophages through both phagocytosis-independent and dependent mechanisms, while NP uptake in tumor suppressing M1 macrophages was almost exclusively through phagocytosis. In a mouse model of breast cancer (4T1-GFP-FL), M2-like TAMs were significantly reduced with αvß3-MI3-PD NP treatment. To validate this effect was independent of drug delivery to tumor cells and was specific to the MYC inhibitor, mice with integrin ß3 knock out tumors (PyMT-Bo1 ß3KO) were treated with αvß3-NP or αvß3-MI3-PD NP. M2 macrophages were significantly reduced with αvß3-MI3-PD nanoparticle therapy but not αvß3-NP treatment. Conclusion: These data suggest αvß3-NP-mediated drug delivery of a c-MYC inhibitor can reduce protumor M2-like macrophages while preserving antitumor M1-like macrophages in breast cancer.

Three-dimensional MR mapping of angiogenesis with alpha5beta1(alpha nu beta3)-targeted theranostic nanoparticles in the MDA-MB-435 xenograft mouse model.

Our objectives were 1) to characterize angiogenesis in the MDA-MB-435 xenograft mouse model with three-dimensional (3D) MR molecular imaging using alpha(5)beta(1)(RGD)- or irrelevant RGS-targeted paramagnetic nanoparticles and 2) to use MR molecular imaging to assess the antiangiogenic effectiveness of alpha(5)beta(1)(alpha(nu)beta(3))- vs. alpha(nu)beta(3)-targeted fumagillin (50 mug/kg) nanoparticles. Tumor-bearing mice were imaged with MR before and after administration of either alpha(5)beta(1)(RGD) or irrelevant RGS-paramagnetic nanoparticles. In experiment 2, mice received saline or alpha(5)beta(1)(alpha(nu)beta(3))- or alpha(nu)beta(3)-targeted fumagillin nanoparticles on days 7, 11, 15, and 19 posttumor implant. On day 22, MRI was performed using alpha(5)beta(1)(alpha(nu)beta(3))-targeted paramagnetic nanoparticles to monitor the antiangiogenic response. 3D reconstructions of alpha(5)beta(1)(RGD)-signal enhancement revealed a sparse, asymmetrical pattern of angiogenesis along the tumor periphery, which occupied <2.0% tumor surface area. alpha(5)beta(1)-targeted rhodamine nanoparticles colocalized with FITC-lectin corroborated the peripheral neovascular signal. alpha(5)beta(1)(alpha(nu)beta(3))-fumagillin nanoparticles decreased neovasculature to negligible levels relative to control; alpha(nu)beta(3)-targeted fumagillin nanoparticles were less effective (P>0.05). Reduction of angiogenesis in MDA-MB-435 tumors from low to negligible levels did not decrease tumor volume. MR molecular imaging may be useful for characterizing tumors with sparse neovasculature that are unlikely to have a reduced growth response to targeted antiangiogenic therapy.

Minute dosages of alpha(nu)beta3-targeted fumagillin nanoparticles impair Vx-2 tumor angiogenesis and development in rabbits.

Fumagillin suppresses angiogenesis in cancer models and clinical trials, but it is associated with neurotoxicity at systemic doses. In this study, alpha(nu)beta(3)-targeted fumagillin nanoparticles were used to suppress the neovasculature and inhibit Vx-2 adenocarcinoma development using minute drug doses. Tumor-bearing rabbits were treated on days 6, 9, and 12 postimplantation with alpha(nu)beta(3)-targeted fumagillin nanoparticles (30 microg/kg), alpha(nu)beta(3)-targeted nanoparticles without drug, nontargeted fumagillin nanoparticles (30 microg/kg) or saline. On day 16, MRI was performed with alpha(nu)beta(3)-targeted paramagnetic nanoparticles to quantify tumor size and assess neovascularity. Tumor volume was reduced among rabbits receiving alpha(nu)beta(3)-targeted fumagillin nanoparticles (470+/-120 mm(3)) compared with the three control groups: nontargeted fumagillin nanoparticles (1370+/-300 mm(3), P<0.05), alpha(nu)beta(3)-targeted nanoparticles without drug (1080+/-180 mm(3), P<0.05) and saline (980+/-80 mm(3), P<0.05). MR molecular imaging of control rabbits (no fumagillin) revealed a predominant peripheral distribution of neovascularity representing 7.2% of the tumor rim volume, which decreased to 2.8% (P<0.05) with alpha(nu)beta(3)-targeted fumagillin nanoparticle treatment. Microscopically, the tumor parenchyma tended to show T-cell infiltration after targeted fumagillin treatment, which was not appreciated in control animals. These results suggest that alpha(nu)beta(3)-targeted fumagillin nanoparticles could provide a safe and effective means to deliver MetAP2 inhibitors alone or in combination with cytotoxic or immunotherapy.

Local Intratracheal Delivery of Perfluorocarbon Nanoparticles to Lung Cancer Demonstrated with Magnetic Resonance Multimodal Imaging.

Eighty percent of lung cancers originate as subtle premalignant changes in the airway mucosal epithelial layer of bronchi and alveoli, which evolve and penetrate deeper into the parenchyma. Liquid-ventilation, with perfluorocarbons (PFC) was first demonstrated in rodents in 1966 then subsequently applied as lipid-encapsulated PFC emulsions to improve pulmonary function in neonatal infants suffering with respiratory distress syndrome in 1996. Subsequently, PFC nanoparticles (NP) were extensively studied as intravenous (IV) vascular-constrained nanotechnologies for diagnostic imaging and targeted drug delivery applications. Methods: This proof-of-concept study compared intratumoral localization of fluorescent paramagnetic (M) PFC NP in the Vx2 rabbit model using proton (1H) and fluorine (19F) magnetic resonance (MR) imaging (3T) following intratracheal (IT) or IV administration. MRI results were corroborated by fluorescence microscopy. Results: Dynamic 1H-MR and 19F-MR images (3T) obtained over 72 h demonstrated marked and progressive accumulation of M-PFC NP within primary lung Vx2 tumors during the first 12 h post IT administration. Marked 1H and 19F MR signal persisted for over 72 h. In contradistinction, IV M-PFC NP produced a modest transient signal during the initial 2 h post-injection that was consistent circumferential blood pool tumor enhancement. Fluorescence microscopy of excised tumors corroborated the MR results and revealed enormous intratumor NP deposition on day 3 after IT but not IV treatment. Rhodamine-phospholipid incorporated into the PFC nanoparticle surfactant was distributed widely within the tumor on day 3, which is consistent with a hemifusion-based contact drug delivery mechanism previously reported. Fluorescence microscopy also revealed similar high concentrations of M-PFC NP given IT for metastatic Vx2 lung tumors. Biodistribution studies in mice revealed that M-PFC NP given IV distributed into the reticuloendothelial organs, whereas, the same dosage given IT was basically not detected beyond the lung itself. PFC NP given IT did not impact rabbit behavior or impair respiratory function. PFC NP effects on cells in culture were negligible and when given IV or IT no changes in rabbit hematology nor serum clinical chemistry parameters were measured. Conclusion: IT delivery of PFC NP offered unique opportunity to locally deliver PFC NP in high concentrations into lung cancers with minimal extratumor systemic exposure.

Cellular Trafficking of Sn-2 Phosphatidylcholine Prodrugs Studied with Fluorescence Lifetime Imaging and Super-resolution Microscopy.

While the in vivo efficacy of Sn-2 phosphatidylcholine prodrugs incorporated into targeted, non-pegylated lipid-encapsulated nanoparticles was demonstrated in prior preclinical studies, the microscopic details of cell prodrug internalization and trafficking events are unknown. Classic fluorescence microscopy, fluorescence lifetime imaging microscopy, and single-molecule super-resolution microscopy were used to investigate the cellular handling of doxorubicin-prodrug and AlexaFluor™-488-prodrug. Sn-2 phosphatidylcholine prodrugs delivered by hemifusion of nanoparticle and cell phospholipid membranes functioned as phosphatidylcholine mimics, circumventing the challenges of endosome sequestration and release. Phosphatidylcholine prodrugs in the outer cell membrane leaflet translocated to the inner membrane leaflet by ATP-dependent and ATP-independent mechanisms and distributed broadly within the cytosolic membranes over the next 12 h. A portion of the phosphatidylcholine prodrug populated vesicle membranes trafficked to the perinuclear Golgi/ER region, where the drug was enzymatically liberated and activated. Native doxorubicin entered the cells, passed rapidly to the nucleus, and bound to dsDNA, whereas DOX was first enzymatically liberated from DOX-prodrug within the cytosol, particularly in the perinuclear region, before binding nuclear dsDNA. Much of DOX-prodrug was initially retained within intracellular membranes. In vitro anti-proliferation effectiveness of the two drug delivery approaches was equivalent at 48 h, suggesting that residual intracellular DOX-prodrug may constitute a slow-release drug reservoir that enhances effectiveness. We have demonstrated that Sn-2 phosphatidylcholine prodrugs function as phosphatidylcholine mimics following reported pathways of phosphatidylcholine distribution and metabolism. Drug complexed to the Sn-2 fatty acid is enzymatically liberated and reactivated over many hours, which may enhance efficacy overtime.

Diagnosis of LVAD Thrombus using a High-Avidity Fibrin-Specific 99mTc Probe.

Treatment of advanced heart failure with implantable LVADs is increasing, driven by profound unmet patient need despite potential serious complications: bleeding, infection, and thrombus. The experimental objective was to develop a sensitive imaging approach to assess early thrombus accumulation in LVADs under operational high flow and high shear rates. Methods: A monomeric bifunctional ligand with a fibrin-specific peptide, a short spacer, and 99mTc chelating amino acid sequence (F1A) was developed and compared to its tetrameric PEG analogue (F4A). Results:99mTc attenuation by LVAD titanium (1 mm) was 23%. 99mTc-F1A affinity to fibrin was Kd ~10 µM, whereas, the bound 99mTc-F4A probe was not displaced by F1A (120,000:1). Human plasma interfered with 99mTc-F1A binding to fibrin clot (p<0.05) in vitro, whereas, 99mTc-F4A targeting was unaffected. The pharmacokinetic half-life of 99mTc-F4A was 28% faster (124±41 min) than 99mTc-F1A (176±26 min) with both being bioeliminated through the urinary system with negligible liver or spleen biodistribution. In mice with carotid thrombus, 99mTc-F4A binding to the injured carotid was much greater (16.3±3.3 %ID/g, p=0.01) than that measured with an irrelevant negative control, 99mTc-I4A (3.4±1.6 %ID/g). In an LVAD mock flow-loop (1:1, PBS:human plasma:heparin) operating at maximal flow rate, 99mTc-F4A bound well to phantom clots in 2 min (p<0.05), whereas 99mTc-F1A had negligible targeting. Excised LVADs from patients undergoing pump exchange or heart transplant were rewired, studied in the mock flow loop, and found to have spatially variable fibrin accumulations in the inlet and outlet cannulas and bearings. Conclusions:99mTc-F4A is a high-avidity prototype probe for characterizing thrombus in LVADs that is anticipated to help optimize anticoagulation, reduce thromboembolic events, and minimize pump exchange.

Endothelial alpha(v)beta3 integrin-targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis.

OBJECTIVE: Angiogenic expansion of the vasa vasorum is a well-known feature of progressive atherosclerosis, suggesting that antiangiogenic therapies may stabilize or regress plaques. Alpha(v)beta3 integrin-targeted paramagnetic nanoparticles were prepared for noninvasive assessment of angiogenesis in early atherosclerosis, for site-specific delivery of antiangiogenic drug, and for quantitative follow-up of response. METHODS AND RESULTS: Expression of alpha(v)beta3 integrin by vasa vasorum was imaged at 1.5 T in cholesterol-fed rabbit aortas using integrin-targeted paramagnetic nanoparticles that incorporated fumagillin at 0 microg/kg or 30 microg/kg. Both formulations produced similar MRI signal enhancement (16.7%+/-1.1%) when integrated across all aortic slices from the renal arteries to the diaphragm. Seven days after this single treatment, integrin-targeted paramagnetic nanoparticles were readministered and showed decreased MRI enhancement among fumagillin-treated rabbits (2.9%+/-1.6%) but not in untreated rabbits (18.1%+/-2.1%). In a third group of rabbits, nontargeted fumagillin nanoparticles did not alter vascular alpha(v)beta3-integrin expression (12.4%+/-0.9%; P>0.05) versus the no-drug control. In a second study focused on microscopic changes, fewer microvessels in the fumagillin-treated rabbit aorta were counted compared with control rabbits. CONCLUSIONS: This study illustrates the potential of combined molecular imaging and drug delivery with targeted nanoparticles to noninvasively define atherosclerotic burden, to deliver effective targeted drug at a fraction of previous levels, and to quantify local response to treatment.

Anti-angiogenic Nanotherapy Inhibits Airway Remodeling and Hyper-responsiveness of Dust Mite Triggered Asthma in the Brown Norway Rat.

Although angiogenesis is a hallmark feature of asthmatic inflammatory responses, therapeutic anti-angiogenesis interventions have received little attention. Objective: Assess the effectiveness of anti-angiogenic Sn2 lipase-labile prodrugs delivered via αvß3-micellar nanotherapy to suppress microvascular expansion, bronchial remodeling, and airway hyper-responsiveness in Brown Norway rats exposed to serial house dust mite (HDM) inhalation challenges. Results: Anti-neovascular effectiveness of αvß3-mixed micelles incorporating docetaxel-prodrug (Dxtl-PD) or fumagillin-prodrug (Fum-PD) were shown to robustly suppress neovascular expansion (p<0.01) in the upper airways/bronchi of HDM rats using simultaneous 19F/1H MR neovascular imaging, which was corroborated by adjunctive fluorescent microscopy. Micelles without a drug payload (αvß3-No-Drug) served as a carrier-only control. Morphometric measurements of HDM rat airway size (perimeter) and vessel number at 21d revealed classic vascular expansion in control rats but less vascularity (p<0.001) after the anti-angiogenic nanotherapies. CD31 RNA expression independently corroborated the decrease in airway microvasculature. Methacholine (MCh) induced respiratory system resistance (Rrs) was high in the HDM rats receiving αvß3-No-Drug micelles while αvß3-Dxtl-PD or αvß3-Fum-PD micelles markedly and equivalently attenuated airway hyper-responsiveness and improved airway compliance. Total inflammatory BAL cells among HDM challenged rats did not differ with treatment, but αvß3+ macrophages/monocytes were significantly reduced by both nanotherapies (p<0.001), most notably by the αvß3-Dxtl-PD micelles. Additionally, αvß3-Dxtl-PD decreased BAL eosinophil and αvß3+ CD45+ leukocytes relative to αvß3-No-Drug micelles, whereas αvß3-Fum-PD micelles did not. Conclusion: These results demonstrate the potential of targeted anti-angiogenesis nanotherapy to ameliorate the inflammatory hallmarks of asthma in a clinically relevant rodent model.

Bone-Induced Expression of Integrin ß3 Enables Targeted Nanotherapy of Breast Cancer Metastases.

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin ß3 (ß3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, ß3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, ß3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFß signaling through SMAD2/SMAD3 was necessary for breast cancer induction of ß3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvß3 (αvß3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvß3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvß3 at that metastatic site. Cancer Res; 77(22); 6299-312. ©2017 AACR.