Multi‐modal imaging for assessment of tissue‐engineered bone in a critical‐sized calvarial defect mouse model

Tissue‐engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end‐point evaluations requiring the sacrifice of animals at specific time points. Due to differences in animal response to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non‐invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each animal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical‐sized calvarial defect mouse model. Osteogenesis of the TEB was monitored through signal variation, using magnetic resonance imaging (MRI) and near‐infrared (NIR) optical imaging with IRDye® 800CW BoneTag^TM (800CW BT, a bone‐specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the control (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a signal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi‐modal non‐invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright © 2015 John Wiley & Sons, Ltd.     

PECAM‐1‐targeted micron‐sized particles of iron oxide as MRI contrast agent for detection of vascular remodeling after cerebral ischemia

An increasing amount of studies have provided evidence for vascular remodeling, for example, angiogenesis, after cerebral ischemia, which may play a significant role in post‐stroke brain plasticity and recovery. Molecular imaging can provide unique in vivo whole‐brain information on alterations in the expression of specific endothelial markers. A possible target for molecular magnetic resonance imaging (MRI) of post‐stroke (neo)vascularization is platelet endothelial cell adhesion molecule‐1 (PECAM‐1). Here we describe significantly increased PECAM‐1 mRNA levels in ipsilesional brain tissue at 6 h, 24 h and 3 days after transient middle cerebral artery occlusion in mice, and elevated PECAM‐1 staining throughout the lesion at 3, 7 and 21 days post‐stroke. The potential of micron‐sized particles of iron oxide (MPIO) conjugated with PECAM‐1‐targeted antibodies, that is, αPECAM‐1‐MPIO, to expose stroke‐induced PECAM‐1 upregulation with molecular MRI was assessed. In vitro studies demonstrated that PECAM‐1‐expressing brain endothelial cells could be effectively labeled with αPECAM‐1‐MPIO, giving rise to a fourfold increase in MRI relaxation rate R₂. Injection of near‐infrared fluorescent dye‐labeled αPECAM‐1 showed target specificity and dose efficiency of the antibody for detection of brain endothelial cells at 3 days post‐stroke. However, in vivo molecular MRI at 3 and 7 days after stroke revealed no αPECAM‐1‐MPIO‐based contrast enhancement, which was corroborated by the absence of αPECAM‐1‐MPIO in post mortem brain tissue. This indicates that this molecular MRI approach, which has been proven successful for in vivo detection of other types of cell adhesion molecules, is not invariably effective for MRI‐based assessment of stroke‐induced alterations in expression of cerebrovascular markers. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo and ex vivo 19‐fluorine magnetic resonance imaging and spectroscopy of beta‐cells and pancreatic islets using GLUT‐2 specific contrast agents

The assessment of the β‐cell mass in experimental models of diabetes and ultimately in patients is a hallmark to understand the relationship between reduced β‐cell mass/function and the onset of diabetes. It has been shown before that the GLUT‐2 transporter is highly expressed in both β‐cells and hepatocytes and that D‐mannoheptulose (DMH) has high uptake specificity for the GLUT‐2 transporter. As 19‐fluorine MRI has emerged as a new alternative method for MRI cell tracking because it provides potential non‐invasive localization and quantification of labeled cells, the purpose of this project is to validate β‐cell and pancreatic islet imaging by using fluorinated, GLUT‐2 targeting mannoheptulose derivatives (¹⁹FMH) both in vivo and ex vivo. In this study, we confirmed that, similar to DMH, ¹⁹FMHs inhibit insulin secretion and increase the blood glucose level in mice temporarily (approximately two hours). We were able to assess the distribution of ¹⁹FMHs in vivo with a temporal resolution of about 20 minutes, which showed a quick removal of ¹⁹FMH from the circulation (within two hours). Ex vivo MR spectroscopy confirmed a preferential uptake of ¹⁹FMH in tissue with high expression of the GLUT‐2 transporter, such as liver, endocrine pancreas and kidney. No indication of further metabolism was found. In summary, ¹⁹FMHs are potentially suitable for visualizing and tracking of GLUT‐2 expressed cells. However, current bottlenecks of this technique related to the quick clearance of the compound and relative low sensitivity of ¹⁹F MRI need to be overcome. Copyright © 2016 John Wiley & Sons, Ltd.     

Hindered diffusion of MRI contrast agents in rat brain extracellular micro‐environment assessed by acquisition of dynamic T1 and T2 maps

The knowledge of brain tissues characteristics (such as extracellular space and tortuosity) represents valuable information for the design of optimal MR probes for specific biomarkers targeting. This work proposes a methodology based on dynamic acquisition of relaxation time maps to quantify in vivo MRI contrast agent concentration after intra‐cerebral injection in rat brain. It was applied to estimate the hindered diffusion in brain tissues of five contrast agents with different hydrodynamic diameters (Dotarem® ≈ 1 nm, P846 ≈ 4 nm, P792 ≈ 7 nm, P904 ≈ 22 nm and Gd‐based emulsion ≈ 170 nm). In vivo apparent diffusion coefficients were compared with those estimated in an obstacle‐free medium to determine brain extracellular space and tortuosity. At a 2 h imaging timescale, all contrast agents except the Gd‐based emulsion exhibited significant diffusion through brain tissues, with characteristic times compatible with MR molecular imaging (<70 min to diffuse between two capillaries). In conclusion, our experiments indicate that MRI contrast agents with sizes up to 22 nm can be used to perform molecular imaging on intra‐cerebral biomarkers. Our quantification methodology allows a precise estimation of apparent diffusion coefficients, which is helpful to calibrate optimal timing between contrast agent injection and MRI observation for molecular imaging studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Use of perfluorocarbon nanoparticles for non‐invasive multimodal cell tracking of human pancreatic islets

In vivo imaging of engraftment and immunorejection of transplanted islets is critical for further clinical development, with ¹H MR imaging of superparamagnetic iron oxide‐labeled cells being the current premier modality. Using perfluorocarbon nanoparticles, we present here a strategy for non‐invasive imaging of cells using other modalities. To this end, human cadaveric islets were labeled with rhodamine‐perfluorooctylbromide (PFOB) nanoparticles, rhodamine‐perfluoropolyether (PFPE) nanoparticles or Feridex® as control and tested in vitro for cell viability and c‐peptide secretion for 1 week. ¹⁹F MRI, computed tomography (CT) and ultrasound (US) imaging was performed on labeled cell phantoms and on cells following transplantation beneath the kidney capsule of mice and rabbits. PFOB and PFPE‐labeling did not reduce human islet viability or glucose responsiveness as compared with unlabeled cells or SPIO‐labeled cells. PFOB‐ and PFPE‐labeled islets were effectively fluorinated for visualization by ¹⁹F MRI. PFOB‐labeled islets were acoustically reflective for detection by US imaging and became sufficiently brominated to become radiopaque allowing visualization with CT. Thus, perfluorocarbon nanoparticles are multimodal cellular contrast agents that may find applications in real‐time targeted delivery and imaging of transplanted human islets or other cells in a clinically applicable manner using MRI, US or CT imaging. Copyright © 2011 John Wiley & Sons, Ltd.     

Design of ProCAs (Protein‐Based Gd3+ MRI Contrast Agents) with High Dose Efficiency and Capability for Molecular Imaging of Cancer Biomarkers

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics, providing high resolution, three‐dimensional images noninvasively. MRI contrast agents are designed to improve the contrast and sensitivity of MRI. However, current clinically used MRI contrast agents have relaxivities far below the theoretical upper limit, which largely prevent advancing molecular imaging of biomarkers with desired sensitivity and specificity. This review describes current progress in the development of a new class of protein‐based MRI contrast agents (ProCAs) with high relaxivity using protein design to optimize the parameters that govern relaxivity. Further, engineering with targeting moiety allows these contrast agents to be applicable for molecular imaging of prostate cancer biomarkers by MRI. The developed protein‐based contrast agents also exhibit additional in vitro and in vivo advantages for molecular imaging of disease biomarkers, such as high metal‐binding stability and selectivity, reduced toxicity, proper blood circulation time, and higher permeability in tumor tissue in addition to improved relaxivities.     

Na+/Ca2+‐exchanger‐mediated Mn2+‐enhanced 1H2O MRI in hypoxic,perfused rat myocardium

Paramagnetic Mn²⁺ has emerged in the search for non‐invasive magnetic resonance imaging (MRI) techniques to monitor Ca²⁺ in diagnostic and prognostic cardiovascular disease tests because it both alters MRI contrast and behaves as a Ca²⁺ ‘surrogate’ in vivo. However, the reliance on macroscopically averaged measurements to infer microscopic processes constitutes a major limitation of MRI. This investigation circumvents this limitation and contributes an MRI‐based myocardial Ca²⁺‐transporter assay, which probes the Na⁺/Ca²⁺‐exchanger involvement in Mn²⁺ (and presumably Ca²⁺) transport by virtue of its response to pharmacological inhibition. In the model employed herein, ex vivo arrested rat hearts underwent normoxia and then hypoxia while a constant (hyperkalemic) perfusion minimized flow (and uncontrolled Ca²⁺‐channel) contributions to Mn²⁺‐enhanced MRI measurements. The results (i) demonstrate that Mn²⁺ (and presumably Ca²⁺) accumulates via Na⁺/Ca²⁺‐exchanger‐mediated transport during hyperkalemic hypoxia and further, (ii) implicate hypo‐perfusion (rather than the diminished participation of an isolated sarcolemmal Ca²⁺‐transporter) as the mechanism that underlies the reported reductions of Mn²⁺ accumulation (relative to healthy myocardium) subsequent to myocardial insults in MRI studies. Although myriad studies have employed Mn²⁺‐enhanced MRI in myocardial investigations, this appears to be the first attempt to assay the Na⁺/Ca²⁺‐exchanger with MRI under highly circumscribed conditions. MRI‐based Ca²⁺‐transporter assays, such as the Na⁺/Ca²⁺‐exchanger assay utilized here, will inevitably impact disciplines in the medical sciences and beyond. Copyright © 2007 John Wiley & Sons, Ltd.     

First in vivo MRI assessment of a self‐assembled metallostar compound endowed with a remarkable high field relaxivity

{Fe[Gd₂bpy(DTTA)₂(H₂O)₄]₃}^4? is a self‐assembled, metallostar‐structured potential MRI contrast agent, with six efficiently relaxing Gd³⁺ centres confined into a small molecular space. Its proton relaxivity is particularly remarkable at very high magnetic fields (r₁ = 15.8 mM ^?1 s^?1 at 200 MHz, 37°C, in H₂O). Here we report the first in vivo MRI feasibility study, complemented with dynamic γ scintigraphic imaging and biodistribution experiments using the ¹⁵³Sm‐enriched compound. Comparative MRI studies have been performed at 4.7 T in mice with the metallostar and the small molecular weight contrast agent gadolinium(III)‐1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate ([Gd(DOTA)(H₂O)]^? = GdDOTA). The metallostar was well tolerated by the animals at the concentrations of 0.0500 (high dose) and 0.0125 (low dose) mmol Gd kg^?1 body weight; (BW). The signal enhancement in the inversion recovery fast low angle shot (IR FLASH) images after the high‐dose metallostar injection was considerably higher than after GdDOTA injection (0.1 mmol Gd kg^?1 BW), despite the higher dose of the latter. The high‐dose metallostar injection resulted in a greater drop in the spin‐lattice relaxation time (T₁), as calculated from the inversion recovery true fast imaging with steady‐state precession (IR TrueFISP) data for various tissues, than the GdDOTA or the low dose metallostar injection. In summary, these studies have confirmed that the approximately four times higher relaxivity measured in vitro for the metallostar is retained under in vivo conditions. The pharmacokinetics of the metallostar was found to be similar to that of GdDOTA, involving fast renal clearance, a leakage to the extracellular space in the muscle tissue and no leakage to the brain. As expected on the basis of its moderate molecular weight, the metallostar does not function as a blood pool agent. The dynamic γ scintigraphic studies performed in Wistar rats with the metallostar compound having ¹⁵³Sm enrichment also proved the renal elimination pathway. The biodistribution experiments are in full accordance with the MR and scintigraphic imaging. At 15 min post‐injection the activity is primarily localized in the urine, while at 24 h post‐injection almost all radioactivity is cleared from tissues and organs. Copyright © 2006 John Wiley & Sons, Ltd.     

Fluorescence lifetime imaging of activatable target specific molecular probes

In vivo optical imaging using fluorescently labeled self‐quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target‐to‐background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self‐quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2⁺ and BALB/3T3 (HER2^?) cell lines. Changes in fluorescence lifetime correlated with temperature‐ and time‐dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2⁺ and BALB/3T3 (HER2^?)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target‐specific activatable antibody–fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

VCAM‐1‐targeting gold nanoshell probe for photoacoustic imaging of atherosclerotic plaque in mice

The development of molecular probes and novel imaging modalities, allowing better resolution and specificity, is associated with an increased potential for molecular imaging of atherosclerotic plaques especially in basic and pre‐clinical research applications. In that context, a photoacoustic molecular probe based on gold nanoshells targeting VCAM‐1 in mice (immunonanoshells) was designed. The molecular probe was validated in vitro and in vivo, showing no noticeable acute toxic effects. We performed the conjugation of gold nanoshells displaying near‐infrared absorption properties with VCAM‐1 antibody molecules and PEG to increase their biocompatibility. The resulting immunonanoshells obtained under different conditions of conjugation were then assessed for specificity and sensitivity. Photoacoustic tomography was performed to determine the ability to distinguish gold nanoshells from blood both in phantoms and in vivo. Ex vivo optical projection tomography of hearts and aortas from atherosclerotic and control mice confirmed the selective accumulation of the immunonanoshells in atherosclerotic‐prone regions in mice, thus validating the utility of the probe in vivo in small animals for pre‐clinical research. These immunonanoshells represent an adequate mean to target atherosclerotic plaques in small animals, leading to new tools to follow the effect of therapies on the progression or regression of the disease. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a peptide‐functionalized imaging nanoprobe for the targeting of (FXYD2)γa as a highly specific biomarker of pancreatic beta cells

Diabetes is characterized by a progressive decline of the pancreatic beta cell mass (BCM), which is responsible for insufficient insulin secretion and hyperglycaemia. There are currently no reliable methods to measure non‐invasively the BCM in diabetic patients. Our work describes a phage display‐derived peptide (P88) that is highly specific to (FXYD2)γa expressed by human beta cells and is proposed as a molecular vector for the development of functionalized imaging probes. P88 does not bind to the exocrine pancreas and is able to detect down to ~156 human pancreatic islets/mm³ in vitro after conjugation to ultra‐small particles of iron oxide (USPIO), as proven by the R₂ measured on MR images. For in vivo evaluation, MRI studies were carried out on nude mice bearing Capan‐2 tumours that also express (FXYD2)γa. A strong negative contrast was obtained subsequent to the injection of USPIO–P88, but not in negative controls. On human histological sections, USPIO–P88 seems to be specific to pancreatic beta cells, but not to duodenum, stomach or kidney tissues. USPIO–P88 thus represents a novel and promising tool for monitoring pancreatic BCM in diabetic patients. The quantitative correlation between BCM and R₂ remains to be demonstrated in vivo, but the T₂ mapping and the black pixel estimation after USPIO–P88 injection could provide important information for the future pancreatic BCM evaluation by MRI. Copyright © 2015 John Wiley & Sons, Ltd.     

Non‐invasive in vivo tracking of fibrin degradation by fluorescence imaging

Fibrin‐based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid‐phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non‐invasive molecular imaging systems enable real‐time tracking of processes in the living animal. In this study, a non‐invasive fluorescence based imaging system was applied to follow and quantify site‐specific degradation of fibrin sealant. To enable non‐invasive tracking of fibrin in vivo, fibrin‐matrix was labelled by incorporation of a fluorophore‐conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore‐labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16 days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site. Copyright © 2014 John Wiley & Sons, Ltd.     

Disruptive chemical doping in a ferritin‐based iron oxide nanoparticle to decrease r2 and enhance detection with T1‐weighted MRI

Inorganic doping was used to create flexible, paramagnetic nanoparticle contrast agents for in vivo molecular magnetic resonance imaging (MRI) with low transverse relaxivity (r₂). Most nanoparticle contrast agents formed from superparamagnetic metal oxides are developed with high r₂. While sensitive, they can have limited in vivo detection due to a number of constraints with T₂ or T₂*‐weighted imaging. T₁‐weighted imaging is often preferred for molecular MRI, but most T₁‐shortening agents are small chelates with low metal payload or are nanoparticles that also shorten T₂ and limit the range of concentrations detectable with T₁‐weighting. Here we used tungsten and iron deposition to form doped iron oxide crystals inside the apoferritin cavity to form a WFe nanoparticle with a disordered crystal and un‐coupled atomic magnetic moments. The atomic magnetic moments were thus localized, resulting in a principally paramagnetic nanoparticle. The WFe nanoparticles had no coercivity or saturation magnetization at 5 K and sweeping up to ±20 000 Oe, while native ferritin had a coercivity of 3000 Oe and saturation at ±20 000 Oe. This tungsten–iron crystal paramagnetism resulted in an increased WFe particle longitudinal relaxivity (r₁) of 4870 mm ^?1 s^?1 and a reduced transverse relaxivity (r₂) of 9076 mm ^?1 s^?1 compared with native ferritin. The accumulation of the particles was detected with T₁‐weighted MRI in concentrations from 20 to 400 nm in vivo, both injected in the rat brain and targeted to the rat kidney glomerulus. The WFe apoferritin nanoparticles were not cytotoxic up to 700 nm particle concentrations, making them potentially important for targeted molecular MRI. Copyright © 2014 John Wiley & Sons, Ltd.     

Sensitivity‐enhanced chemical exchange saturation transfer (CEST) MRI with least squares optimization of Carr Purcell Meiboom Gill multi‐echo echo planar imaging

Chemical exchange saturation transfer (CEST) imaging is a novel MRI technique that is sensitive to biomolecules, local pH and temperature, and offers considerable advantages for in vivo applications. However, the magnitude of CEST effect for dilute CEST agents undergoing slow or intermediate chemical exchange is typically small, requiring the use of signal averaging to enhance its sensitivity. Given that T₂‐induced signal loss can be normalized by asymmetry analysis, the magnitude of CEST effect is independent of echo time. Therefore, CEST MRI with multi‐echo echo planar imaging (EPI) readout should yield the same CEST effect as conventional single echo acquisition. Importantly, CEST multi‐echo (CESTme) EPI images can be averaged to enhance CEST MRI sensitivity. The goal of this study was to validate CESTme EPI using a creatine–agarose gel CEST phantom with similar T₂ as biological tissue. Using least‐squares optimization, we found that the sensitivity of CESTme sequence was significantly higher than that obtained by conventional single echo CEST‐EPI acquisition. Specifically, signal‐to‐noise ratio and contrast‐to‐noise ratio from the proposed CESTme EPI were approximately equivalent to that obtained by doubling the number of signal averages of the standard single echo CEST MRI sequence. In summary, our results demonstrated CESTme EPI for sensitivity‐enhanced CEST imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Direct coupling of annexin A5 to VSOP yields small,protein‐covered nanoprobes for MR imaging of apoptosis

Annexin A5 (Anx) has been extensively used for imaging apoptosis by single‐photon emission computed tomography, positron emission tomography, optical imaging and MRI. Recently we introduced ultrasmall Anx–VSOP (very small iron oxide particles) – the smallest high‐relaxivity probe for MRI of apoptosis. Here we present a simplified method for the direct coupling of Anx to VSOP, which resulted in nanoparticles that are nearly completely covered with human Anx. These superparamagnetic nanoparticles are only 14.4 ± 2.3 nm in diameter and have higher T₂* relaxivity. Compared with existing probes, the small size and the Anx shielding provide prerequisites for good biocompatibility and bioavailability in target tissues. In vitro characterization showed specific binding of Anx–VSOP to apoptotic cells, which led to a signal loss in T₂*‐weighted MR measurements, while control probe M1324‐VSOP produced no such change. Exploratory MRI was done in vivo in a cardiac model of ischemia–reperfusion damage illustrating the potential of the probe for future studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Nature‐inspired nanoformulations for contrast‐enhanced in vivo MR imaging of macrophages

Magnetic resonance imaging (MRI) of macrophages in atherosclerosis requires the use of contrast‐enhancing agents. Reconstituted lipoprotein particles that mimic native high‐density lipoproteins (HDL) are a versatile delivery platform for Gd‐based contrast agents (GBCA) but require targeting moieties to direct the particles to macrophages. In this study, a naturally occurring methionine oxidation in the major HDL protein, apolipoprotein (apo) A‐I, was exploited as a novel way to target HDL to macrophages. We also tested if fully functional GBCA–HDL can be generated using synthetic apo A‐I peptides. The fluorescence and MRI studies reveal that specific oxidation of apo A‐I or its peptides increases the in vitro macrophage uptake of GBCA–HDL by 2–3 times. The in vivo imaging studies using an apo E‐deficient mouse model of atherosclerosis and a 3.0 T MRI system demonstrate that this modification significantly improves atherosclerotic plaque detection using GBCA–HDL. At 24 h post‐injection of 0.05 mmol Gd kg^?1 GBCA–HDL containing oxidized apo A‐I or its peptides, the atherosclerotic wall/muscle normalized enhancement ratios were 90 and 120%, respectively, while those of GBCA–HDL containing their unmodified counterparts were 35 and 45%, respectively. Confocal fluorescence microscopy confirms the accumulation of GBCA–HDL containing oxidized apo A‐I or its peptides in intraplaque macrophages. Together, the results of this study confirm the hypothesis that specific oxidation of apo A‐I targets GBCA–HDL to macrophages in vitro and in vivo. Furthermore, our observation that synthetic peptides can functionally replace the native apo A‐I protein in HDL further encourages the development of these contrast agents for macrophage imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Self‐illuminating in vivo lymphatic imaging using a bioluminescence resonance energy transfer quantum dot nano‐particle

Autofluorescence arising from normal tissues can compromise the sensitivity and specificity of in vivo fluorescence imaging by lowering the target‐to‐background signal ratio. Since bioluminescence resonance energy transfer quantum dot (BRET‐QDot) nano‐particles can self‐illuminate in near‐infrared in the presence of the substrate, coelenterazine, without irradiating excitation lights, imaging using BRET‐QDots does not produce any autofluorescence. In this study, we applied this BRET‐QDot nano‐particle to the in vivo lymphatic imaging in mice in order to compare with BRET, fluorescence or bioluminescence lymphatic imaging. BRET‐QDot655, in which QDot655 is contained as a core, was injected at different sites (e.g. chin, ear, forepaws and hind paws) in mice followed by the intravenous coelenterazine injection, and then bioluminescence and fluorescence imaging were serially performed. In all mice, each lymphatic basin was clearly visualized in the BRET imaging with minimal background signals. The BRET signal in the lymph nodes lasted at least 30 min after coelenterazine injections. Furthermore, the BRET signal demonstrated better quantification than the fluorescence signal emitting from QDot655, the core of this BRET particle. These advantages of BRET‐QDot allowed us to perform real‐time, quantitative lymphatic imaging without image processing. BRET‐Qdots have the potential to be a robust nano‐material platform for developing optical molecular imaging probes. Copyright © 2010 John Wiley & Sons, Ltd.     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.     

Visualization of delayed release of compounds from pH‐sensitive capsules in vitro and in vivo in a hamster model

Delayed controlled release is an innovative strategy to locally administer therapeutic compounds (e.g. chemotherapeutics, antibodies etc.). This would improve efficiency and reduce side effects compared with systemic administration. To enable the evaluation of the efficacy of controlled release strategies both in vitro and in vivo, we investigated the release of contrast agents (¹⁹ F‐FDG and BaSO₄) to the intestinal tract from capsules coated with pH‐sensitive polymers (EUDRAGIT L‐100) by using two complementary techniques, i.e. ¹⁹ F magnetic resonance imaging (MRI) and computed tomography (CT). Using in vitro ¹⁹ F‐MRI, we were able to non‐destructively and dynamically establish a time window of 2 h during which the capsules are resistant to low pH. With ¹⁹ F‐MRI, we could establish the exact time point when the capsules became water permeable, before physical degradation of the capsule. This was complemented by CT imaging, which provided longitudinal information on physical degradation of the capsule at low pH that was only seen after 230 min. After oral administration to hamsters, ¹⁹ F‐MRI visualized the early event whereby the capsule becomes water permeable after 2 h. Additionally, using CT, the integrity and location (stomach and small intestines) of the capsule after administration could be monitored. In conclusion, we propose combined ¹⁹ F‐MRI and CT to non‐invasively visualize the different temporal and spatial events regarding the release of compounds, both in an in vitro setting and in the gastrointestinal tract of small animal models. This multimodal imaging approach will enable the in vitro and in vivo evaluation of further technical improvements to controlled release strategies. Copyright © 2015 John Wiley & Sons, Ltd.     

Surrogate MR markers of response to chemo‐ or radiotherapy in association with co‐treatments: a retrospective analysis of multi‐modal studies

The study of magnetic resonance (MR) markers over the past decade has provided evidence that the tumor microenvironnement and hemodynamics play a major role in determining tumor response to therapy. The aim of the present work is to predict and monitor the efficacy of co‐treatments to radio‐ and chemotherapy by noninvasive MR imaging. Ten different co‐treatments were involved in this retrospective analysis of our previously published data, including NO‐mediated co‐treatments (insulin and isosorbide dinitrate), anti‐inflammatory drugs (hydrocortisone, NS‐398), anti‐angiogenic agents (thalidomide, SU5416 and ZD6474), a vasoactive agent (xanthinol nicotinate), botulinum toxin and carbogen breathing. Dynamic contrast enhanced (DCE) MRI, intrinsic susceptibility‐weighted (BOLD) MRI and electronic paramagnetic resonance (EPR) oximetry all reflect tumor microenvironment hemodynamic variables that are known to influence tumor response. Eight MR‐derived parameters (markers) were tested for their ability to predict therapeutic outcome (factor of increase in regrowth delay) in experimental tumor models (TLT and FSaII) after radiation therapy and/or chemotherapy with cyclophosphamide, namely tumor pO₂ and O₂ consumption rate (using EPR oximetry); tumor blood flow and permeability, i.e. V _p, K _trans, K _ep and percentage of perfused vessels (using DCE‐MRI); and BOLD signal intensity and R ₂* (using functional MRI). This multi‐modal comparison of co‐treatment efficacy points out the limitations of each MR marker and identifies in vivo pO₂ as a relevant endpoint for radiation therapy. DCE parameters (V _p and K _ep) were identified as a relevant endpoints for cyclophosphamide chemotherapy in our tumor models. This study helps qualify relevant imaging endpoints in the preclinical setting of cancer therapy. Copyright © 2010 John Wiley & Sons, Ltd.