How transfer rates generate Gd‐BOPTA concentrations in rat liver compartments: implications for clinical liver imaging with hepatobiliary contrast agents

Following the injection of hepatobiliary contrast agents, MRI detects all molecules included in a region of interest but cannot estimate true concentrations in sinusoids, interstitium, hepatocytes or bile canaliculi. The aim of the study was to measure true concentrations in hepatocytes and to show how transfer rates across sinusoidal and canalicular membranes generate these concentrations. We perfused livers isolated from normal rats with 200 μM Gd‐DTPA and Gd‐BOPTA and measured clearances from sinusoids to liver and from hepatocytes to bile canaliculi or back to interstitium. We detected Gd‐BOPTA with a gamma probe and determined true concentrations in each liver compartment knowing their liver volumes. No pharmacokinetic modelling was applied. Gd‐BOPTA clearance from sinusoids to liver (2.5 ± 0.4 mL/min) was 50 times higher than that of Gd‐DTPA (0.05 ± 0.02 mL/min) when portal flow rate was 30 mL/min (p < 0.0001). Gd‐BOPTA clearance from sinusoids to liver was always superior to hepatocyte clearance, and hepatocyte Gd‐BOPTA concentrations measured by the probe increased over time. Gd‐BOPTA concentrations reached 439 ± 83 μM in hepatocytes and 15 × 700 ± 3100 μM in bile canaliculi, while concentrations in sinusoids were 200 μM. Gd‐BOPTA true concentrations in hepatocytes depend on the simultaneous clearances from sinusoids to hepatocytes and from hepatocytes to bile canaliculi and back to sinusoids. The study better defines how signal intensities are generated when hepatobiliary contrast agents are injected in clinical imaging. Copyright © 2016 John Wiley & Sons, Ltd.     

A physiologically based pharmacokinetic model of vascular–extravascular exchanges during liver carcinogenesis: application to MRI contrast agents

The extraction of physiological parameters by non‐invasive imaging techniques such as dynamic magnetic resonance imaging (MRI) or positron emission tomography requires a knowledge of molecular distribution and exchange between microvascularization and extravascular tissues. These phenomena not only depend on the physicochemical characteristics of the injected molecules but also the pathophysiological state of the targeted organ. We developed a five‐compartment physiologically based pharmacokinetic model focused on hepatic carcinogenesis and MRI contrast agents. This model includes physical characteristics of the contrast agent, dual specific liver supply, microvessel wall properties and transport parameters that are compatible with hepatocarcinoma development. The evolution of concentrations in the five compartments showed significant differences in the distribution of three molecules (differentiated by their diameters and diffusion coefficients ranging, respectively, from 0.9 to 62 nm and from 68.10^?9 to 47.10^?7 cm² s^?1) in simulated regeneration nodules and dysplastic nodules, as well as in medium‐ and poorly differentiated hepatocarcinoma. These results are in agreement with known vascular modifications such as arterialization that occur during hepatocarcinogenesis. This model can be used to study the pharmacokinetics of contrast agents and consequently to extract parameters that are characteristic of the tumor development (like permeability), after fitting simulated to in vivo data. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis and evaluation of a polydisulfide with Gd–DOTA monoamide side chains as a biodegradable macromolecular contrast agent for MR blood pool imaging

Macromolecular Gd(III)‐based contrast agents are effective for contrast‐enhanced blood pool and cancer MRI in preclinical studies. However, their clinical applications are impeded by potential safety concerns associated with slow excretion and prolonged retention of these agents in the body. To minimize the safety concerns of macromolecular Gd contrast agents, we have developed biodegradable macromolecular Gd contrast agents based on polydisulfide Gd(III) complexes. In this study, we designed and synthesized a new generation of the polydisulfide Gd(III) complexes containing a macrocyclic Gd(III) chelate, Gd–DOTA monoamide, to improve the in vivo kinetic inertness of the Gd(III) chelates. (N₆‐Lysyl)lysine‐(Gd–DOTA) monoamide and 3‐(2‐carboxyethyldisulfanyl)propanoic acid copolymers (GODC) were synthesized by copolymerization of (N₆‐lysyl)lysine DOTA monoamide and dithiobis(succinimidylpropionate), followed by complexation with Gd(OAc)₃. The GODC had an apparent molecular weight of 26.4 kDa and T₁ relaxivity of 8.25 m m ^?1 s^?1 per Gd at 1.5 T. The polymer chains of GODC were readily cleaved by l ‐cysteine and the chelates had high kinetic stability against transmetallation in the presence of an endogenous metal ion Zn²⁺. In vivo MRI study showed that GODC produced strong and prolonged contrast enhancement in the vasculature and tumor periphery of mice with breast tumor xenografts. GODC is a promising biodegradable macromolecular MRI contrast agent with high kinetic stability for MR blood pool imaging. Copyright © 2013 John Wiley & Sons, Ltd.     

How morphology influences relaxivity – comparative study of superparamagnetic iron oxide–polymer hybrid nanostructures

Superparamagnetic iron oxides (SPIOs) are widely used in MRI as T₂ contrast agents, and interest is still growing. Here, the T₂ relaxivity of three different SPIO–polymer hybrid morphologies, i.e. homogeneously distributed iron oxide within a polymer matrix, Janus‐like nanoparticles and polymer nanocapsules containing iron oxides, is studied. Making use of calculations based on theory for agglomerated systems, the obtained T₂ values could be predicted for all different morphologies, except for nanocapsules. Nanocapsules, in contrast to full spheres, allow for water exchange between encapsulated water and bulk water, and thus have two contributions to relaxivity. One originates from the capsules acting as a weakly magnetized cluster and the other stems from the individual SPIOs inside the capsule. Therefore, the relaxivities were also computed using an empirical equation found in the literature, which considers water exchange, resulting in a better T₂ forecast for the nanocapsules. The presented study is the first example of a comparison between measured and calculated relaxivities of nanocapsules. Copyright © 2015 John Wiley & Sons, Ltd.     

Assessing cytotoxicity of (iron oxide‐based) nanoparticles: an overview of different methods exemplified with cationic magnetoliposomes

Iron oxide nanoparticles are the most widely used T₂/T₂* contrast agents and for biomedical research purposes, one of the main applications is the in vitro labeling of stem or therapeutic cells, allowing them to be subsequently tracked in vivo upon transplantation. To allow this, the nanoparticles used should not show any sign of cytotoxicity and not affect cellular physiology as this could impede normal cell functionality in vivo or lead to undesired side‐effects. Assessing the biocompatibility of the nanoparticles has proven to be quite a difficult task. In the present work, a small overview of commonly used assays is presented in order to assess several aspects, such as cell viability, induction of reactive oxygen species, nanoparticle uptake, cellular morphology, cellular proliferation, actin cytoskeleton architecture and differentiation of stem cells. The main focus is on comparing the advantages and disadvantages of the different assays, highlighting several common problems and presenting possible solutions to these problems as well as pointing out the high importance of the relationship between intracellular nanoparticle concentration and cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and characterization of a porphyrazine–Gd(III) MRI contrast agent and in vivo imaging of a breast cancer xenograft model

Porphyrazines (Pz), or tetraazaporphyrins, are being studied for their potential use in detection and treatment of cancer. Here, an amphiphilic Cu–Pz–Gd(III) conjugate has been prepared via azide–alkyne Huisgen cycloaddition or ‘click’ chemistry between an azide functionalized Pz and alkyne functionalized DOTA–Gd(III) analog for use as an MRI contrast agent. This agent, Cu–Pz–Gd(III), is synthesized in good yield and exhibits solution‐phase ionic relaxivity (r₁ = 11.5 mm ^?1 s^?1) that is approximately four times higher than that of a clinically used monomeric Gd(III) contrast agent, DOTA–Gd(III). Breast tumor cells (MDA‐MB‐231) associate with Cu–Pz–Gd(III) in vitro, where significant contrast enhancement (9.336 ± 0.335 contrast‐to‐noise ratio) is observed in phantom cell pellet MR images. This novel contrast agent was administered in vivo to an orthotopic breast tumor model in athymic nude mice and MR images were collected. The average T₁ of tumor regions in mice treated with 50 mg kg^?1 Cu–Pz–Gd(III) decreased relative to saline‐treated controls. Furthermore, the decrease in T₁ was persistent relative to mice treated with the monomeric Gd(III) contrast agent. An ex vivo biodistribution study confirmed that Cu–Pz–Gd(III) accumulates in the tumors and is rapidly cleared, primarily through the kidneys. Differential accumulation and T₁ enhancement by Cu–Pz–Gd(III) in the tumor's core relative to the periphery offer preliminary evidence that this agent would find application in the imaging of necrotic tissue. Copyright © 2014 John Wiley & Sons, Ltd.     

Assessment of tumor angiogenesis: dynamic contrast‐enhanced MRI with paramagnetic nanoparticles compared with Gd‐DTPA in a rabbit Vx‐2 tumor model

The purpose of this study was to evaluate the suitability of a macromolecular MRI contrast agent (paramagnetic nanoparticles, PNs) for the characterization of tumor angiogenesis. Our aim was to estimate the permeability of PNs in developing tumor vasculature and compare it with that of a low molecular weight contrast agent (Gd‐DTPA) using dynamic contrast‐enhanced MRI (DCE). Male New Zealand white rabbits (n = 5) underwent DCE MRI 12–14 days after Vx‐2 tumor fragments were implanted into the left hind limb. Each contrast agent (PNs followed by Gd‐DTPA) was evaluated using a DCE protocol and transendothelial transfer coefficient (K_i) maps were calculated using a two‐compartment model. Two regions of interest (ROIs) were located within the tumor core and hindlimb muscle and five ROIs were placed within the tumor rim. Comparisons were performed using repeated measures analysis of variance (ANOVA). The K_i values estimated using PNs were significantly lower than those obtained for Gd‐DTPA (p = 0.018). When PNs and Gd‐DTPA data were analyzed separately, significant differences were identified among tumor rim ROIs for PNs (p < 0.0001), but not for Gd‐DTPA data (p = 0.34). The mean K_i for the tumor rim was significantly greater than that of either the core or the hindlimb muscle for both contrast agents (p < 0.05 for each comparison). In summary, the extravasation of Gd‐DTPA was far greater than that of PNs, suggesting that PNs can reveal regional differences in tumor vascular permeability that are not otherwise apparent with clinical contrast agents such as Gd‐DTPA. These results suggest that PNs show potential for the noninvasive delineation of tumor angiogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

Color Doppler ultrasound of liver lesions: Signal enhancement after intravenous injection of the ultrasound contrast agent Levovist®

Patients with focal liver lesions (hemangioma, focal nodular hyperplasia, adenoma, hepatocellular carcinoma, metastatic lesions, focal fatty lesion) received the ultrasound contrast agent Levovist® (300 mg/mL and 400 mg/mL) intravenously. This ultrasound contrast agent (a suspension of micrometer-sized microparticles of galactose and microscopic gaseous bubbles) can pass through the lungs without impairment. After the administration of Levovist®, increased color flow signals were detected in the liver. Five of 6 patients with metastatic liver lesions showed previously undetected blood flow in the rim of the tumor. In 4 patients with hepatocellular carcinoma, enhanced signal intensity was observed in the vessels of the rim and in 3 of those patients in the center of the tumor. One patient with adenoma and one patient with focal nodular hyperplasia showed signal enhancement in the central area of the tumor. No signal enhancement was observed in hemangiomas, a focal fatty lesion, or in a carcinoid metastatic lesion. Levovist® increased the echointensity of normal and tumor vessels in liver lesions. This new ultrasound contrast agent led to the detection of tumor vessels previously not detectable by conventional color flow imaging. © 1996 John Wiley & Sons, Inc.     

Serine‐ and mannose‐modified liposomal contrast agent for computed tomography: evaluation of the enhancement in rabbit liver VX‐2 tumor model

Kupffer cell imaging is a powerful tool for the detection of liver cancer. This diagnostic procedure depends on the faculty of the reticuloendothelial system (RES) which takes up foreign bodies, including small particles. The current study aimed to develop a novel RES targeting liposomal contrast agent that functionalized with serine or mannose, the moiety specifically binding to a corresponding receptor on phagocytic cells. Liposomes loaded with non‐ionic X‐ray contrast media, Iohexol, were prepared by supercritical carbon dioxide reverse‐phase evaporation method and were intravenously injected to healthy rabbits in order to evaluate the liver parenchymal enhancement in X‐ray computed tomography (CT). From 10 to 40 min after injection, the mean enhancement value of the liver parenchyma approached 45 and 34 Hounsfield units (HU) when serine‐modified iodinated liposomal contrast agent (ILCA) and mannose‐modified ILCA were applied, respectively. The tumor‐to‐liver contrast values were also evaluated after the administration of the prepared ILCA to rabbits with VX‐2 carcinoma. For serine‐modified ILCA, tumor‐to‐liver contrast was 82 HU at 1 min and >24 HU at 10–40 min; for mannose‐modified ILCA, the values were 58 HU at 0.5 min and >21 HU at 10–40 min. These vales estimated from the region of intrest and the imaging figures of liver indicate the potential of ILCA for clinical use. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of dynamic contrast‐enhanced MRI and quantitative SPECT in a rat glioma model

Pharmacokinetic modeling of dynamic contrast‐enhanced (DCE) MRI data provides measures of the extracellular‐extravascular volume fraction (v_e) and the volume transfer constant (K^trans) in a given tissue. These parameter estimates may be biased, however, by confounding issues such as contrast agent and tissue water dynamics, or assumptions of vascularization and perfusion made by the commonly used model. In contrast to MRI, radiotracer imaging with SPECT is insensitive to water dynamics. A quantitative dual‐isotope SPECT technique was developed to obtain an estimate of v_e in a rat glioma model for comparison with the corresponding estimates obtained using DCE‐MRI with a vascular input function and reference region model. Both DCE‐MRI methods produced consistently larger estimates of v_e in comparison to the SPECT estimates, and several experimental sources were postulated to contribute to these differences. Copyright © 2012 John Wiley & Sons, Ltd.     

Pharmacokinetics of Gd(DO3A‐Lys) and MR imaging studies in an orthotopic U87MG glioma tumor model

Pharmacokinetics of Gd(DO3A‐Lys), a macrocyclic gadolinium‐based magnetic resonance imaging (MRI) contrast agent functionalized with a lysine derivative, was studied in Wistar rats. Kinetic data were fitted using a two‐compartment model and revealed Gd(DO3A‐Lys) to have a distribution half‐life, t_1/2(α), of 1.3 min, an elimination half‐life, t_1/2(β), of 24.9 min and a large volume of distribution, V_D, of 0.49 L/kg indicative of the agent being able to rapidly distribute into tissues and organs. Contrast‐enhanced magnetic resonance angiography (CE‐MRA) in an orthotopic U87MG glioma mouse model demonstrated considerable enhancement of both the tumor and surrounding vasculature after intravenous administration of Gd(DO3A‐Lys). Applying dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) in the glioma of different sizes further showed distinct uptake characteristics and patterns of enhancement, which suggests the potential for differentiating changes at different stages of tumor growth. Our results indicate that Gd(DO3A‐Lys) could be a promising candidate for glioma MR imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Alterations in high-density lipoprotein metabolism and reverse cholesterol transport in insulin resistance and type 2 diabetes mellitus: role of lipolytic enzymes, lecithin:cholesterol acyltransferase and lipid transfer proteins

Insulin resistance and type 2 diabetes mellitus are generally accompanied by low HDL cholesterol and high plasma triglycerides, which are major cardiovascular risk factors. This review describes abnormalities in HDL metabolism and reverse cholesterol transport, i.e. the transport of cholesterol from peripheral cells back to the liver for metabolism and biliary excretion, in insulin resistance and type 2 diabetes mellitus. Several enzymes including lipoprotein lipase (LPL), hepatic lipase (HL) and lecithin: cholesterol acyltransferase (LCAT), as well as cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), participate in HDL metabolism and remodelling. Lipoprotein lipase hydrolyses lipoprotein triglycerides, thus providing lipids for HDL formation. Hepatic lipase reduces HDL particle size by hydrolysing its triglycerides and phospholipids. A decreased postheparin plasma LPL/HL ratio is a determinant of low HDL2 cholesterol in insulin resistance. The esterification of free cholesterol by LCAT increases HDL particle size. Plasma cholesterol esterification is unaltered or increased in type 2 diabetes mellitus, probably depending on the extent of triglyceride elevation. Subsequent CETP action results in transfer of cholesteryl esters from HDL towards triglyceride-rich lipoproteins, and is involved in decreasing HDL size. An increased plasma cholesteryl ester transfer is frequently observed in insulin-resistant conditions, and is considered to be a determinant of low HDL cholesterol. Phospholipid transfer protein generates small pre beta-HDL particles that are initial acceptors of cell-derived cholesterol. Its activity in plasma is elevated in insulin resistance and type 2 diabetes mellitus in association with high plasma triglycerides and obesity. In insulin resistance, the ability of plasma to promote cellular cholesterol efflux may be maintained consequent to increases in PLTP activity and pre beta-HDL. However, cellular cholesterol efflux to diabetic plasma is probably impaired. Besides, cellular abnormalities that are in part related to impaired actions of ATP binding cassette transporter 1 and scavenger receptor class B type I are likely to result in diminished cellular cholesterol efflux in the diabetic state. Whether hepatic metabolism of HDL-derived cholesterol and subsequent hepatobiliary transport is altered in insulin resistance and type 2 diabetes mellitus is unknown. Specific CETP inhibitors have been developed that exert major HDL cholesterol-raising effects in humans and retard atherosclerosis in animals. As an increased CETP-mediated cholesteryl ester transfer represents a plausible metabolic intermediate between high triglycerides and low HDL cholesterol, studies are warranted to evaluate the effects of these agents in insulin resistance- and diabetes-associated dyslipidaemia.     

Mushroom body‐preferential expression of proteins/genes involved in endoplasmic reticulum Ca2+‐transport in the worker honeybee (Apis mellifera L.) brain

To identify the molecular characteristics specific to the mushroom body (MB, a higher processing centre) neurones in the honeybee brain, we previously used proteomics to identify proteins that are preferentially expressed in these MBs. Here we continued our proteomic analysis to show that reticulocalbin, which is involved in endoplasmic reticulum (ER) Ca²⁺ transport, is also preferentially expressed in the MBs in the honeybee brain. Gene expression analysis revealed that reticulocalbin is preferentially expressed in the large‐type Kenyon cells, which are MB‐intrinsic neurones. In addition, the gene for the ryanodine receptor, which is also involved in ER Ca²⁺ transport, was also preferentially expressed in the large‐type Kenyon cells. In contrast, the expression of three other ER‐related genes, protein disulphide isomerase, sec61 and erp60, was not enriched in the MBs. These findings further support the notion that the function of ER Ca²⁺‐signalling, but not the mere intracellular density of ER, is specifically enhanced in the large‐type Kenyon cells in the honeybee brain.     

Bioinspired hybrid eumelanin–TiO2 antimicrobial nanostructures: the key role of organo–inorganic frameworks in tuning eumelanin's biocide action mechanism through membrane interaction

Intrinsic biocide efficacy of eumelanins can be markedly enhanced through a templated formation in the presence of a TiO₂-sol, leading to hybrid TiO₂–melanin nanostructures. However, mechanisms and processes behind biocide activity still remain poorly understood. This paper discloses the fundamental mechanism of action of these systems providing mechanistic information on their peculiar interaction with Escherichia coli strains. To this purpose biocide characterization is combined with Electron Paramagnetic Resonance (EPR) spectroscopy to investigate radical species produced by the hybrids as well as their interactions with Gram(−) external bacterial membranes. Experimental results indicate that TiO₂ mediated eumelanin polymerization leads to a peculiar mechanism of action of hybrid nanostructures, whose strong interactions with bacterial membranes enhance the action of reactive oxygen species (ROS) produced by eumelanin degradation itself, also concurring with the final biocide action. These findings provide strategic information for the development of eumelanin-based systems with enhanced activity against drug-resistant strains.

Hybrid TiO₂/eumelanin nanostructures showed a peculiar biocide mechanism against Gram(−) bacteria, based on the ROS action, produced by eumelanin degradation under visible light irradiation, and the interactions with external bacterial membranes.     

In vitro and in vivo characterization of several functionalized ultrasmall particles of iron oxide,vectorized against amyloid plaques and potentially able to cross the blood–brain barrier: toward earlier diagnosis of Alzheimer's disease by molecular imaging

Alzheimer's disease (AD) is a neurodegenerative disorder most often diagnosed 10 years after its onset and development. It is characterized by the accumulation of amyloid‐β peptide (ABP) into amyloid plaques between nerve cells, which produces a massive local neurodegeneration. Molecular magnetic resonance imaging allows diagnosis of AD by showing ABP accumulation in the brain. The ultrasmall particles of iron oxide (USPIO) derivatives proposed in the present work were functionalized with peptides that present an affinity for ABP, independently of its state of aggregation. Their nanomolar K_d* confirms the high affinity of our vectorized contrast agents (VCA) for ABP and therefore their high labeling potential, specificity and sensitivity. Their lack of toxicity has been demonstrated, both by in vitro studies using the MTT method on several cell types, and by in vivo investigations with assessment of renal and hepatic biomarkers and by histopathology evaluation. The results of biodistribution studies corroborated by MRI demonstrate that USPIO‐PHO (USPIO coupled to peptide C‐IPLPFYN‐C) are able to cross the blood–brain barrier without any facilitating strategy, and accumulates in the brain 90 min after its injection in NMRI mice. None of the USPIO derivatives were found in any organs one week after administration. To conclude, USPIO‐PHO seems to have a genuine potential for labeling amyloid plaques in the brain; it has a nanomolar binding affinity, no toxic effects, and its elimination half‐life is about 3 h. Further tests will be made on transgenic mice, aimed at confirming the potential of early AD diagnosis using our VCA. Copyright © 2014 John Wiley & Sons, Ltd.