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.

Angiogenesis and airway reactivity in asthmatic Brown Norway rats.

Expanded and aberrant bronchial vascularity, a prominent feature of the chronic asthmatic airway, might explain persistent airway wall edema and sustained leukocyte recruitment. Since it is well established that there are causal relationships between exposure to house dust mite (HDM) and the development of asthma, determining the effects of HDM in rats, mammals with a bronchial vasculature similar to humans, provides an opportunity to study the effects of bronchial angiogenesis on airway function directly. We studied rats exposed bi-weekly to HDM (Der p 1; 50 µg/challenge by intranasal aspiration, 1, 2, 3 weeks) and measured the time course of appearance of increased blood vessels within the airway wall. Results demonstrated that within 3 weeks of HDM exposure, the number of vessels counted within airway walls of bronchial airways (0.5-3 mm perimeter) increased significantly. These vascular changes were accompanied by increased airway responsiveness to methacholine. A shorter exposure regimen (2 weeks of bi-weekly exposure) was insufficient to cause a significant increase in functional vessels or reactivity. Yet, 19F/1H MR imaging at 3T following αvß3-targeted perfluorocarbon nanoparticle infusion revealed a significant increase in 19F signal in rat airways after 2 weeks of bi-weekly HDM, suggesting earlier activation of the process of neovascularization. Although many antigen-induced mouse models exist, mice lack a bronchial vasculature and consequently lack the requisite human parallels to study bronchial edema. Overall, our results provide an important new model to study the impact of bronchial angiogenesis on chronic inflammation and airways hyperreactivity.

Balanced UTE-SSFP for 19F MR imaging of complex spectra.

PURPOSE: A novel technique for highly sensitive detection of multiresonant fluorine imaging agents was designed and tested with the use of dual-frequency 19F/1H ultrashort echo times (UTE) sampled with a balanced steady-state free precession (SSFP) pulse sequence and three-dimensional (3D) radial readout. METHODS: Feasibility of 3D radial balanced UTE-SSFP imaging was demonstrated for a phantom comprising liquid perfluorooctyl bromide (PFOB). Sensitivity of the pulse sequence was measured and compared with other sequences imaging the PFOB (CF2 )6 line group including UTE radial gradient-echo (GRE) at α = 30°, as well as Cartesian GRE, balanced SSFP, and fast spin-echo (FSE). The PFOB CF3 peak was also sampled with FSE. RESULTS: The proposed balanced UTE-SSFP technique exhibited a relative detection sensitivity of 51 µmolPFOB(-1) min(-1/2) (α = 30°), at least twice that of other sequence types with either 3D radial (UTE GRE: 20 µmolPFOB(-1) min(-1/2) ) or Cartesian k-space filling (GRE: 12 µmolPFOB(-1) min(-1/2) ; FSE: 16 µmolPFOB(-1) min(-1/2) ; balanced SSFP: 23 µmolPFOB(-1) min(-1/2) ). In vivo imaging of angiogenesis-targeted PFOB nanoparticles was demonstrated in a rabbit model of cancer on a clinical 3 Tesla scanner. CONCLUSION: A new dual 19F/1H balanced UTE-SSFP sequence manifests high SNR, with detection sensitivity more than two-fold better than traditional techniques, and alleviates imaging problems caused by dephasing in complex spectra.

Improved quantitative (19) F MR molecular imaging with flip angle calibration and B1 -mapping compensation.

PURPOSE: To improve (19) F flip angle calibration and compensate for B1 inhomogeneities in quantitative (19) F MRI of sparse molecular epitopes with perfluorocarbon (PFC) nanoparticle (NP) emulsion contrast agents. MATERIALS AND METHODS: Flip angle sweep experiments on PFC-NP point source phantoms with three custom-designed (19) F/(1) H dual-tuned coils revealed a difference in required power settings for (19) F and (1) H nuclei, which was used to calculate a calibration ratio specific for each coil. An image-based correction technique was developed using B1 -field mapping on (1) H to correct for (19) F and (1) H images in two phantom experiments. RESULTS: Optimized (19) F peak power differed significantly from that of (1) H power for each coil (P < 0.05). A ratio of (19) F/(1) H power settings yielded a coil-specific and spatially independent calibration value (surface: 1.48 ± 0.06; semicylindrical: 1.71 ± 0.02, single-turn-solenoid: 1.92 ± 0.03). (1) H-image-based B1 correction equalized the signal intensity of (19) F images for two identical (19) F PFC-NP samples placed in different parts of the field, which were offset significantly by ~66% (P < 0.001), before correction. CONCLUSION: (19) F flip angle calibration and B1 -mapping compensations to the (19) F images employing the more abundant (1) H signal as a basis for correction resulted in a significant change in the quantification of sparse (19) F MR signals from targeted PFC NP emulsions.

Rapamycin nanoparticles target defective autophagy in muscular dystrophy to enhance both strength and cardiac function.

Duchenne muscular dystrophy in boys progresses rapidly to severe impairment of muscle function and death in the second or third decade of life. Current supportive therapy with corticosteroids results in a modest increase in strength as a consequence of a general reduction in inflammation, albeit with potential untoward long-term side effects and ultimate failure of the agent to maintain strength. Here, we demonstrate that alternative approaches that rescue defective autophagy in mdx mice, a model of Duchenne muscular dystrophy, with the use of rapamycin-loaded nanoparticles induce a reproducible increase in both skeletal muscle strength and cardiac contractile performance that is not achievable with conventional oral rapamycin, even in pharmacological doses. This increase in physical performance occurs in both young and adult mice, and, surprisingly, even in aged wild-type mice, which sets the stage for consideration of systemic therapies to facilitate improved cell function by autophagic disposal of toxic byproducts of cell death and regeneration.

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.

Assessing intrarenal nonperfusion and vascular leakage in acute kidney injury with multinuclear (1) H/(19) F MRI and perfluorocarbon nanoparticles.

PURPOSE: We sought to develop a unique sensor-reporter approach for functional kidney imaging that employs circulating perfluorocarbon nanoparticles and multinuclear (1) H/(19) F MRI. METHODS: (19) F spin density weighted and T1 weighted images were used to generate quantitative functional mappings of both healthy and ischemia-reperfusion (acute kidney injury) injured mouse kidneys. (1) H blood-oxygenation-level-dependent (BOLD) MRI was also employed as a supplementary approach to facilitate the comprehensive analysis of renal circulation and its pathological changes in acute kidney injury. RESULTS: Heterogeneous blood volume distributions and intrarenal oxygenation gradients were confirmed in healthy kidneys by (19) F MRI. In a mouse model of acute kidney injury, (19) F MRI, in conjunction with blood-oxygenation-level-dependent MRI, sensitively delineated renal vascular damage and recovery. In the cortico-medullary junction region, we observed 25% lower (19) F signal (P < 0.05) and 70% longer (1) H T2* (P < 0.01) in injured kidneys compared with contralateral kidneys at 24 h after initial ischemia-reperfusion injury. We also detected 71% higher (19) F signal (P < 0.01) and 40% lower (1) H T2* (P < 0.05) in the renal medulla region of injured kidneys compared with contralateral uninjured kidneys. CONCLUSION: Integrated (1) H/(19) F MRI using perfluorocarbon nanoparticles provides a multiparametric readout of regional perfusion defects in acutely injured kidneys.

Programmable nanoparticle functionalization for in vivo targeting.

The emerging demand for programmable functionalization of existing base nanocarriers necessitates development of an efficient approach for cargo loading that avoids nanoparticle redesign for each individual application. Herein, we demonstrate in vivo a postformulation strategy for lipidic nanocarrier functionalization with the use of a linker peptide, which rapidly and stably integrates cargos into lipidic membranes of nanocarriers after simple mixing through a self-assembling process. We exemplified this strategy by generating a VCAM-1-targeted perfluorocarbon nanoparticle for in vivo targeting in atherosclerosis (ApoE-deficient) and breast cancer (STAT-1-deficient) models. In the atherosclerotic model, a 4.1-fold augmentation in binding to affected aortas was observed for targeted vs. nontargeted nanoparticles (P<0.0298). Likewise, in the breast cancer model, a 4.9-fold increase in the nanoparticle signal from tumor vasculature was observed for targeted vs. nontargeted nanoparticles (P<0.0216). In each case, the nanoparticle was registered with fluorine ((19)F) magnetic resonance spectroscopy of the nanoparticle perfluorocarbon core, yielding a quantitative estimate of the number of tissue-bound nanoparticles. Because other common nanocarriers with lipid coatings (e.g., liposomes, micelles, etc.) can employ this strategy, this peptide linker postformulation approach is applicable to more than half of the available nanosystems currently in clinical trials or clinical uses.

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.

Application of a hemolysis assay for analysis of complement activation by perfluorocarbon nanoparticles.

Nanoparticles offer new options for medical diagnosis and therapeutics with their capacity to specifically target cells and tissues with imaging agents and/or drug payloads. The unique physical aspects of nanoparticles present new challenges for this promising technology. Studies indicate that nanoparticles often elicit moderate to severe complement activation. Using human in vitro assays that corroborated the mouse in vivo results we previously presented mechanistic studies that define the pathway and key components involved in modulating complement interactions with several gadolinium-functionalized perfluorocarbon nanoparticles (PFOB). Here we employ a modified in vitro hemolysis-based assay developed in conjunction with the mouse in vivo model to broaden our analysis to include PFOBs of varying size, charge and surface chemistry and examine the variations in nanoparticle-mediated complement activity between individuals. This approach may provide the tools for an in-depth structure-activity relationship study that will guide the eventual development of biocompatible nanoparticles. FROM THE CLINICAL EDITOR: Unique physical aspects of nanoparticles may lead to moderate to severe complement activation in vivo, which represents a challenge to clinical applicability. In order to guide the eventual development of biocompatible nanoparticles, this team of authors report a modified in vitro hemolysis-based assay developed in conjunction with their previously presented mouse model to enable in-depth structure-activity relationship studies.

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.

Rapid quantification of oxygen tension in blood flow with a fluorine nanoparticle reporter and a novel blood flow-enhanced-saturation-recovery sequence.

We present a novel blood flow-enhanced-saturation-recovery (BESR) sequence, which allows rapid in vivo T1 measurement of blood for both (1)H and (19)F nuclei. BESR sequence is achieved by combining homogeneous spin preparation and time-of-flight image acquisition and therefore preserves high time efficiency and signal-to-noise ratio for (19)F imaging of circulating perfluorocarbon nanoparticles comprising a perfluoro-15-crown-5-ether core and a lipid monolayer (nominal size = 250 nm). The consistency and accuracy of the BESR sequence for measuring T1 of blood was validated experimentally. With a confirmed linear response feature of (19)F R1 with oxygen tension in both salt solution and blood sample, we demonstrated the feasibility of the BESR sequence to quantitatively determine the oxygen tension within mouse left and right ventricles under both normoxia and hyperoxia conditions. Thus, (19)F BESR MRI of circulating perfluorocarbon nanoparticles represents a new approach to noninvasively evaluate intravascular oxygen tension.

Loss of Consciousness in a 34 Yo Male Related to Marijuana.

Nontraumatic exertional syncope can be an ominous event reflecting profound arterial hypotension, cerebral hypoperfusion, and transient loss-of consciousness that occurs most commonly in patients with underlying cardiovascular disease. In contradistinction, transient loss-of-consciousness in "healthy adults" is typically vasovagal syncope related to exaggerated orthostatic cardiovascular responses attributed to a hyper-reactive autonomic nervous system. In the present report, a 34 yo male presents to the hospital emergency department (ED) for a sudden loss of consciousness and fall ultimately related to cardiac syncope ascribed to chronic recreational marijuana use complicated by coronary vasospasm.

Transferrin receptor in primary and metastatic breast cancer: Evaluation of expression and experimental modulation to improve molecular targeting.

BACKGROUND: Conjugation of transferrin (Tf) to imaging or nanotherapeutic agents is a promising strategy to target breast cancer. Since the efficacy of these biomaterials often depends on the overexpression of the targeted receptor, we set out to survey expression of transferrin receptor (TfR) in primary and metastatic breast cancer samples, including metastases and relapse, and investigate its modulation in experimental models. METHODS: Gene expression was investigated by datamining in twelve publicly-available datasets. Dedicated Tissue microarrays (TMAs) were generated to evaluate matched primary and bone metastases as well as and pre and post chemotherapy tumors from the same patient. TMA were stained with the FDA-approved MRQ-48 antibody against TfR and graded by staining intensity (H-score). Patient-derived xenografts (PDX) and isogenic metastatic mouse models were used to study in vivo TfR expression and uptake of transferrin. RESULTS: TFRC gene and protein expression were high in breast cancer of all subtypes and stages, and in 60-85% of bone metastases. TfR was detectable after neoadjuvant chemotherapy, albeit with some variability. Fluorophore-conjugated transferrin iron chelator deferoxamine (DFO) enhanced TfR uptake in human breast cancer cells in vitro and proved transferrin localization at metastatic sites and correlation of tumor burden relative to untreated tumor mice. CONCLUSIONS: TfR is expressed in breast cancer, primary, metastatic, and after neoadjuvant chemotherapy. Variability in expression of TfR suggests that evaluation of the expression of TfR in individual patients could identify the best candidates for targeting. Further, systemic iron chelation with DFO may upregulate receptor expression and improve uptake of therapeutics or tracers that use transferrin as a homing ligand.

Variable antibody-dependent activation of complement by functionalized phospholipid nanoparticle surfaces.

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.

Quantification of water exchange kinetics for targeted PARACEST perfluorocarbon nanoparticles.

PARACEST (PARAmagnetic Chemical Exchange Saturation Transfer) agents offer the ability to generate "contrast on demand", negating the need to image before contrast agent injection. Perfluorocarbon (PFC) nanoparticles can deliver very large payloads of PARACEST agents, lowering the effective detection limit for molecular imaging of sparse biomarkers. Also, the PFC core provides a quantitative (19)F signal for measuring particle binding with high signal intensity and no background signal. (19)F quantization coupled with mathematical modeling of the PARACEST signal showed that incorporating PARACEST chelates onto the nanoparticle surface reduces the bound water lifetime and diminishes the available contrast to noise ratio compared to the parent small molecule PARACEST chelate. PARACEST nanoparticles were targeted to fibrin, an early biomarker for atherosclerotic plaque rupture, and bound to the surface of in vitro clots, yielding a detection limit of 2.30 nM at 11.7T. When the particles bind to a target surface, the image contrast is higher than predicted from phantom experiments, perhaps due to improved water exchange kinetics. We demonstrated that PARACEST PFC nanoparticles can provide two unique signatures, (19)F and PARACEST, for quantitative targeted molecular imaging of fibrin.

Molecular photoacoustic imaging of angiogenesis with integrin-targeted gold nanobeacons.

Photoacoustic tomography (PAT) combines optical and acoustic imaging to generate high-resolution images of microvasculature. Inherent sensitivity to hemoglobin permits PAT to image blood vessels but precludes discriminating neovascular from maturing microvasculature. α(v)ß(3)-Gold nanobeacons (α(v)ß(3)-GNBs) for neovascular molecular PAT were developed, characterized, and demonstrated in vivo using a mouse Matrigel-plug model of angiogenesis. PAT results were microscopically corroborated with fluorescent α(v)ß(3)-GNB localization and supporting immunohistology in Rag1(tm1Mom) Tg(Tie-2-lacZ)182-Sato mice. α(v)ß(3)-GNBs (154 nm) had 10-fold greater contrast than blood on an equivolume basis when imaged at 740 nm to 810 nm in blood. The lowest detectable concentration in buffer was 290 nM at 780 nm. Noninvasive PAT of angiogenesis using a 10-MHz ultrasound receiver with α(v)ß(3)-GNBs produced a 600% increase in signal in a Matrigel-plug mouse model relative to the inherent hemoglobin contrast pretreatment. In addition to increasing the contrast of neovessels detected at baseline, α(v)ß(3)-GNBs allowed visualization of numerous angiogenic sprouts and bridges that were undetectable before contrast injection. Competitive inhibition of α(v)ß(3)-GNBs with α(v)ß(3)-NBs (no gold particles) almost completely blocked contrast enhancement to pretreatment levels, similar to the signal from animals receiving saline only. Consistent with other studies, nontargeted GNBs passively accumulated in the tortuous neovascular but provided less than half of the contrast enhancement of the targeted agent. Microscopic studies revealed that the vascular constrained, rhodamine-labeled α(v)ß(3)-GNBs homed specifically to immature neovasculature (PECAM(+), Tie-2(-)) along the immediate tumor periphery, but not to nearby mature microvasculature (PECAM(+), Tie-2(+)). The combination of PAT and α(v)ß(3)-GNBs offered sensitive and specific discrimination and quantification of angiogenesis in vivo, which may be clinically applicable to a variety of highly prevalent diseases, including cancer and cardiovascular disease.