MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninvasively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle-labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro-15-crown-5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+ CD133+ CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC-labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.

Earlier tumor detection can improve 5-year survival of patients, particularly among those presenting with cancers less than 1 cm in diameter. a m b 3 - Targeted 111 In nanoparticles (NP) were developed and studied for detection of tumor angiogenesis. Studies were conducted in New Zealand white rabbits implanted 12 days earlier with Vx-2 tumor. a m b 3 - Targeted 111 In/NP bearing 10 111In/NP vs. 1 111In/NP nuclide payloads were compared to nontargeted radiolabeled control particles. In vivo competitive binding studies were used to assess ligand-targeting specificity. a m b 3 -Integrin-targeted NP with 10 111In/NP provided better (p < 0.05) tumor-to-muscle ratio contrast (6.3 6 0.2) than 1 111In/NP (5.1 6 0.1) or nontargeted particles with 10 111In/NP (3.7 6 0.1) over the initial 2-hr postinjection. At 18 hr, mean tumor activity in rabbits receiving a m b 3 -integrin-targeted NP was 4-fold higher than the nontargeted control. Specificity of the NP for the tumor neovasculature was supported by in vivo competition studies and by fluorescence microscopy of a m b 3 -targeted fluorescent-labeled NP. Biodistribution studies revealed that the primary clearance organ in rabbits as a %ID/g tissue was the spleen. Circulatory half-life (t 1/2b ) was estimated to be 5 hr using a 2-compartment model. a m b 3 - Targeted 111 In perfluorocarbon NP may provide a clinically useful tool for sensitively detecting angiogenesis in nascent tumors, particularly in combination with secondary highresolution imaging modalities, such as MRI. ' 2007 Wiley-Liss, Inc.Key words: indium; angiogenesis; integrin; nanoparticles; Vx2; cancer Data accumulated over the last 25 years in the surveillance, epidemiology and end results cancer registry support the principle that earlier tumor detection improves 5-year survival of patients with either localized or regional invasive breast carcinoma. 1 Improvements in survival were correlated with an overall downward shift in tumor size distribution, with particular advantage noted among patients presenting with cancers less than 1 cm. A widespread desire to detect and treat cancer earlier has spawned interest in molecular imaging and genomic-proteomic technologies, which in combination with new strategies to treat cancer may further improve cancer survival.One approach to identifying small solid tumors has involved early detection of angiogenesis by targeting unique biosignatures of neovascular endothelium, such as a v b 3 -integrin. We have previously demonstrated that paramagnetic perfluorocarbon emulsions targeted to the a v b 3 -integrin can be used to detect the neovasculature of tumors 30 mm 3 at clinical field strengths (1.5 T). Because perfluorocarbon nanoparticles (NP) have a nominal particle size of 250 nm and are constrained within the vasculature, access to a v b 3 -integrin expressed on extravascular macrophages, smooth muscle and other cells is sterically precluded. MRI provides outstanding high-resolution images of even minute tumors enhanced by the bound paramagnetic NP; however, the ...

The ability to specifically deliver therapeutic agents to selected cell types while minimizing systemic toxicity is a principal goal of nanoparticle-based drug delivery approaches. Numerous cellular portals exist for cargo uptake and transport, but after targeting, intact nanoparticles typically are internalized via endocytosis prior to drug release. However, in this work, we show that certain classes of nanoparticles, namely lipid-coated liquid perfluorocarbon emulsions, undergo unique interactions with cells to deliver lipophilic substances to target cells without the need for entire nanoparticle internalization. To define the delivery mechanisms, fluorescently-labeled nanoparticles complexed with alphav beta 3-integrin targeting ligands were incubated with alphav beta 3-integrin expressing cells (C32 melanoma) under selected inhibitory conditions that revealed specific nanoparticle-to-cell interactions. We observed that the predominant mechanism of lipophilic delivery entailed direct delivery of lipophilic substances to the target cell plasma membrane via lipid mixing and subsequent intracellular trafficking through lipid raft-dependent processes. We suggest that local drug delivery to selected cell types could be facilitated by employing targeted nanoparticles designed specifically to utilize alternative membrane transport mechanisms.

Apoptosis is generally believed to be a process that requires several hours, in contrast to non-programmed forms of cell death that can occur in minutes. Our findings challenge the time-consuming nature of apoptosis as we describe the discovery and characterization of a small molecule, named Raptinal, which initiates intrinsic pathway caspase-dependent apoptosis within minutes in multiple cell lines. Comparison to a mechanistically diverse panel of apoptotic stimuli reveals Raptinal-induced apoptosis proceeds with unparalleled speed. The rapid phenotype enabled identification of the critical roles of mitochondrial voltage-dependent anion channel function, mitochondrial membrane potential/coupled respiration, and mitochondrial complex I, III and IV function for apoptosis induction. Use of Raptinal in whole organisms demonstrates its utility to study apoptosis in vivo for a variety of applications. Overall, rapid inducers of apoptosis are powerful tools that will be used in a variety of settings to generate further insight into the apoptotic machinery.

Targeted, liquid perfluorocarbon nanoparticles are effective agents for acoustic contrast enhancement of abundant cellular epitopes (e.g., fibrin in thrombi) and for lower prevalence binding sites, such as integrins associated with tumor neovasculature. In this study, we sought to delineate the quantitative relationship between the extent of contrast enhancement of targeted surfaces and the density (and concentration) of bound perfluorocarbon (PFC) nanoparticles. Two dramatically different substrates were utilized for targeting. In one set of experiments, the surfaces of smooth, flat, avidin-coated agar disks were exposed to biotinylated nanoparticles to yield a thin layer of targeted contrast. For the second set of measurements, we targeted PFC nanoparticles applied in thicker layers to cultured smooth muscle cells expressing the transmembrane glycoprotein "tissue factor" at the cell surface. An acoustic microscope was used to characterize reflectivity for all samples as a function of bound PFC (determined via gas chromatography). We utilized a formulation of low-scattering nanoparticles having oil-based cores to compete against high-scattering PFC nanoparticles for binding, to elucidate the dependence of contrast enhancement on PFC concentration. The relationship between reflectivity enhancement and bound PFC content varied in a curvilinear fashion and exhibited an apparent asymptote (approximately 16 dB and 9 dB enhancement for agar and cell samples, respectively) at the maximum concentrations (approximately 150 microg and approximately 1000 microg PFOB for agar and cell samples, respectively). Samples targeted with only oil-based nanoparticles exhibited mean backscatter values that were nearly identical to untreated samples (<1 dB difference), confirming the oil particles' low-scattering behavior. The results of this study indicate that substantial contrast enhancement with liquid perfluorocarbon nanoparticles can be realized even in cases of partial surface coverage (as might be encountered when targeting sparsely populated epitopes) or when targeting surfaces with locally irregular topography. Furthermore, it may be possible to assess the quantity of bound cellular epitopes through acoustic means.