The In Vivo Biological Fate of Protein Corona: A Comparative PET Study of the Fate of Soft and Hard Protein Corona in Healthy Animal Models.

Radiolabeling and nuclear imaging techniques are used to investigate the biodistribution patterns of the soft and hard protein corona around poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) after administration to healthy mice. Soft and hard protein coronas of 131I-labeled BSA or 131I-labeled serum are formed on PLGA NPs functionalized with either polyehtylenimine (PEI) or bovine serum albumin (BSA). The exchangeability of hard and soft corona is assessed in vitro by gamma counting exposing PLGA NPs with corona to non-labeled BSA, serum, or simulated body fluid. PEI PLGA NPs form larger and more stable coronas than BSA PLGA NPs. Soft coronas are more exchangeable than hard ones. The in vivo fate of PEI PLGA NPs coated with preformed 18F-labeled BSA hard and soft coronas is assessed by positron emission tomography (PET) following intravenous administration. While the soft corona shows a biodistribution similar to free 18F BSA with high activity in blood and kidney, the hard corona follows patterns characteristic of nanoparticles, accumulating in the lungs, liver, and spleen. These results show that in vivo fates of soft and hard corona are different, and that soft corona is more easily exchanged with proteins from the body, while hard corona is largely retained on the nanoparticle surface.

Synthesis and PET Imaging Biodistribution Studies of Radiolabeled Iododiflunisal, a Transthyretin Tetramer Stabilizer, Candidate Drug for Alzheimer's Disease.

The small-molecule iododiflunisal (IDIF) is a transthyretin (TTR) tetramer stabilizer and acts as a chaperone of the TTR-Amyloid beta interaction. Oral administration of IDIF improves Alzheimer's Disease (AD)-like pathology in mice, although the mechanism of action and pharmacokinetics remain unknown. Radiolabeling IDIF with positron or gamma emitters may aid in the in vivo evaluation of IDIF using non-invasive nuclear imaging techniques. In this work, we report an isotopic exchange reaction to obtain IDIF radiolabeled with 18F. [19F/18F]exchange reaction over IDIF in dimethyl sulfoxide at 160 °C resulted in the formation of [18F]IDIF in 7 ± 3% radiochemical yield in a 20 min reaction time, with a final radiochemical purity of >99%. Biodistribution studies after intravenous administration of [18F]IDIF in wild-type mice using positron emission tomography (PET) imaging showed capacity to cross the blood-brain barrier (ca. 1% of injected dose per gram of tissue in the brain at t > 10 min post administration), rapid accumulation in the liver, long circulation time, and progressive elimination via urine. Our results open opportunities for future studies in larger animal species or human subjects.

Positron Emission Tomography Studies of the Biodistribution, Translocation, and Fate of Poly Allyl Amine-Based Carriers for Sirna Delivery by Systemic and Intratumoral Administration.

Polyamine-based vectors offer many advantages for gene therapy, but they are hampered by a limited knowledge on their biological fate and efficacy for nucleic acid delivery. The 18 F radiolabeled siRNA is complexed with poly(allyl amine) hydrochloride (PAH), PEGylated PAH (PAHPEG ), or oleic acid-modified PAH (PAHOleic ) to form polyplexes, and injected them intravenously into healthy rodents. The biodistribution patterns obtained by positron emission tomography (PET) imaging vary according to the polymer used for complexation. Free siRNA is quickly eliminated through the bladder. PAH and oleic acid modify PAH polyplexes accumulate in the lungs and liver. No elimination through the bladder is observed for PAH and PAHOleic within 2 h after administration. PAHPEG polyplexes accumulate in kidneys and are eliminated through the bladder. Polyplexes prepared with 18 F-labeled oleic acid-modified PAH and non-labeled siRNA show similar biodistribution to those prepared with labeled siRNA, but with more accumulation in the lungs due to the presence of non-complexed polymer. Intravenous administration of PAHOleic polyplexes in tumor models results in a limited availability of siRNA. When PAHOleic polyplexes are administered intratumorally in tumor bearing rodents, ≈40% of the radioactivity is retained in the tumor after 180 min while free siRNA is completely eliminated.

PET Imaging of Self-Assembled 18 F-Labelled Pd2 L4 Metallacages for Anticancer Drug Delivery.

To advance the design of self-assembled metallosupramolecular architectures as new generation theranostic agents, the synthesis of 18 F-labelled [Pd2 L4 ]4+ metallacages is reported. Different spectroscopic and bio-analytical methods support the formation of the host-guest cage-cisplatin complex. The biodistribution profiles of one of the cages, alone or encapsulating cisplatin have been studied by PET/CT imaging in healthy mice in vivo, in combination to ICP-MS ex vivo.

Increased uptake of the P2X7 receptor radiotracer 18 F-JNJ-64413739 in the brain and peripheral organs according to the severity of status epilepticus in male mice.

OBJECTIVE: The P2X7 receptor (P2X7R) is an important contributor to neuroinflammation, responding to extracellularly released adenosine triphosphate. Expression of the P2X7R is increased in the brain in experimental and human epilepsy, and genetic or pharmacologic targeting of the receptor can reduce seizure frequency and severity in preclinical models. Experimentally induced seizures also increase levels of the P2X7R in blood. Here, we tested 18 F-JNJ-64413739, a positron emission tomography (PET) P2X7R antagonist, as a potential noninvasive biomarker of seizure-damage and epileptogenesis. METHODS: Status epilepticus was induced via an intra-amygdala microinjection of kainic acid. Static PET studies (30 min duration, initiated 30 min after tracer administration) were conducted 48 h after status epilepticus via an intravenous injection of 18 F-JNJ-64413739. PET images were coregistered with a brain magnetic resonance imaging atlas, tracer uptake was determined in the different brain regions and peripheral organs, and values were correlated to seizure severity during status epilepticus. 18 F-JNJ-64413739 was also applied to ex vivo human brain slices obtained following surgical resection for intractable temporal lobe epilepsy. RESULTS: P2X7R radiotracer uptake correlated strongly with seizure severity during status epilepticus in brain structures including the cerebellum and ipsi- and contralateral cortex, hippocampus, striatum, and thalamus. In addition, a correlation between radiotracer uptake and seizure severity was also evident in peripheral organs such as the heart and the liver. Finally, P2X7R radiotracer uptake was found elevated in brain sections from patients with temporal lobe epilepsy when compared to control. SIGNIFICANCE: Taken together, our data suggest that P2X7R-based PET imaging may help to identify seizure-induced neuropathology and temporal lobe epilepsy patients with increased P2X7R levels possibly benefitting from P2X7R-based treatments.

In Vivo Tracking of the Degradation of Mesoporous Silica through 89 Zr Radio-Labeled Core-Shell Nanoparticles.

While mesoporous silica nanoparticles (MSNs) are extensively studied as high-potential drug delivery platforms, the successful clinical translation of these nanocarriers strongly depends on their biodistribution, biodegradation, and elimination patterns in vivo. Here, a novel method is reported to follow the in vivo degradation of MSNs by tracking a radioactive label embedded in the silica structure. Core-shell silica nanoparticles (NPs) with a dense core and a mesoporous shell are labeled with low quantities of the positron emitter 89 Zr, either in the dense core or in the mesoporous shell. In vivo positron emission tomography imaging and ex vivo organ measurements reveal a remarkable difference in the 89 Zr biodistribution between the shell-labeled and the core-labeled NPs. Release of the radiotracer from shell-labeled NPs is used as a probe of the extent of silica dissolution, and a prompt release of the radioisotope is observed, with partial excretion already in the first 2 h post injection, and a slower accumulation in bones over time. On the other hand, when 89 Zr is embedded in the nanoparticle core, the biodistribution remains largely unchanged during the first 6 h. These findings indicate that MSNs have fast, hour-scale, degradation kinetics in vivo.

124 I Radiolabeling of a AuIII -NHC Complex for In Vivo Biodistribution Studies.

AuIII complexes with N-heterocyclic carbene (NHC) ligands have shown remarkable potential as anticancer agents, yet their fate in vivo has not been thoroughly examined and understood. Reported herein is the synthesis of new AuIII -NHC complexes by direct oxidation with radioactive [124 I]I2 as a valuable strategy to monitor the in vivo biodistribution of this class of compounds using positron emission tomography (PET). While in vitro analyses provide direct evidence for the importance of AuIII -to-AuI reduction to achieve full anticancer activity, in vivo studies reveal that a fraction of the AuIII -NHC prodrug is not immediately reduced after administration but able to reach the major organs before metabolic activation.

Immuno-PET Imaging and Pharmacokinetics of an Anti-CEA scFv-based Trimerbody and Its Monomeric Counterpart in Human Gastric Carcinoma-Bearing Mice.

Monoclonal antibodies (mAbs) are currently used as therapeutic agents in different types of cancer. However, mAbs and antibody fragments developed so far show suboptimal properties in terms of circulation time and tumor penetration/retention. Here, we report the radiolabeling, pharmacokinetic evaluation, and determination of tumor targeting capacity of the previously validated anti-CEA MFE23-scFv-based N-terminal trimerbody (MFE23N-trimerbody), and the results are compared to those obtained for the monomeric MFE23-scFv. Dissection and gamma-counting studies performed with the 131I-labeled protein scaffolds in normal mice showed slower blood clearance for the trimerbody, and accumulation in the kidneys, the spleen, and the liver for both species. These, together with a progressive uptake in the small intestine, confirm a combined elimination scheme with hepatobiliary and urinary excretion. Positron emission tomography studies performed in a xenograft mouse model of human gastric adenocarcinoma, generated by subcutaneous administration of CEA-positive human MKN45 cells, showed higher tumor accumulation and tumor-to-muscle (T/M) ratios for 124I-labeled MFE23N-trimerbody than for MFE23-scFv. Specific uptake was not detected with PET imaging in CEA negative xenografts as indicated by low T/M ratios. Our data suggest that engineered intermediate-sized trivalent antibody fragments could be promising candidates for targeted therapy and imaging of CEA-positive tumors.

Gold Nanoparticles as Boron Carriers for Boron Neutron Capture Therapy: Synthesis, Radiolabelling and In vivo Evaluation.

Background: Boron Neutron Capture Therapy (BNCT) is a binary approach to cancer therapy that requires accumulation of boron atoms preferentially in tumour cells. This can be achieved by using nanoparticles as boron carriers and taking advantage of the enhanced permeability and retention (EPR) effect. Here, we present the preparation and characterization of size and shape-tuned gold NPs (AuNPs) stabilised with polyethylene glycol (PEG) and functionalized with the boron-rich anion cobalt bis(dicarbollide), commonly known as COSAN. The resulting NPs were radiolabelled with 124I both at the core and the shell, and were evaluated in vivo in a mouse model of human fibrosarcoma (HT1080 cells) using positron emission tomography (PET). Methods: The thiolated COSAN derivatives for subsequent attachment to the gold surface were synthesized by reaction of COSAN with tetrahydropyran (THP) followed by ring opening using potassium thioacetate (KSAc). Iodination on one of the boron atoms of the cluster was also carried out to enable subsequent radiolabelling of the boron cage. AuNPs grafted with mPEG-SH (5 Kda) and thiolated COSAN were prepared by ligand displacement. Radiolabelling was carried out both at the shell (isotopic exchange) and at the core (anionic absorption) of the NPs using 124I to enable PET imaging. Results: Stable gold nanoparticles simultaneously functionalised with PEG and COSAN (PEG-AuNPs@[4]-) with hydrodynamic diameter of 37.8 ± 0.5 nm, core diameter of 19.2 ± 1.4 nm and ξ-potential of -18.0 ± 0.7 mV were obtained. The presence of the COSAN on the surface of the NPs was confirmed by Raman Spectroscopy and UV-Vis spectrophotometry. PEG-AuNPs@[4]- could be efficiently labelled with 124I both at the core and the shell. Biodistribution studies in a xenograft mouse model of human fibrosarcoma showed major accumulation in liver, lungs and spleen, and poor accumulation in the tumour. The dual labelling approach confirmed the in vivo stability of the PEG-AuNPs@[4]-. Conclusions: PEG stabilized, COSAN-functionalised AuNPs could be synthesized, radiolabelled and evaluated in vivo using PET. The low tumour accumulation in the animal model assayed points to the need of tuning the size and geometry of the gold core for future studies.

In vivo imaging of Α7 nicotinic receptors as a novel method to monitor neuroinflammation after cerebral ischemia.

In vivo positron emission tomography (PET) imaging of nicotinic acetylcholine receptors (nAChRs) is a promising tool for the imaging evaluation of neurologic and neurodegenerative diseases. However, the role of α7 nAChRs after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. In vivo and ex vivo evaluation of α7 nAChRs expression after transient middle cerebral artery occlusion (MCAO) was carried out using PET imaging with [11 C]NS14492 and immunohistochemistry (IHC). Pharmacological activation of α7 receptors was evaluated with magnetic resonance imaging (MRI), [18 F]DPA-714 PET, IHC, real time polymerase chain reaction (qPCR) and neurofunctional studies. In the ischemic territory, [11 C]NS14492 signal and IHC showed an expression increase of α7 receptors in microglia and astrocytes after cerebral ischemia. The role played by α7 receptors on neuroinflammation was supported by the decrease of [18 F]DPA-714 binding in ischemic rats treated with the α7 agonist PHA 568487 at day 7 after MCAO. Moreover, compared with non-treated MCAO rats, PHA-treated ischemic rats showed a significant reduction of the cerebral infarct volumes and an improvement of the neurologic outcome. PHA treatment significantly reduced the expression of leukocyte infiltration molecules in MCAO rats and in endothelial cells after in vitro ischemia. Despite that, the activation of α7 nAChR had no influence to the blood brain barrier (BBB) permeability measured by MRI. Taken together, these results suggest that the nicotinic α7 nAChRs play a key role in the inflammatory reaction and the leukocyte recruitment following cerebral ischemia in rats.

Biocatalysis in radiochemistry: Enzymatic incorporation of PET radionuclides into molecules of biomedical interest.

Biocatalysis is emerging as a new approach to radiolabel biologically active molecules with short half-lived positron emitters for their use in positron emission tomography. Despite the golden era of biocatalysis in radiochemistry occurred in the 70s and 80s, advances in enzyme engineering during the last decade have significantly enhanced the toolbox of enzymes available for chemical reactions, which may find application in the context of radiochemistry. In the present review, we intend to give an overview of the biocatalytic approaches that have been reported during the last 4 decades for the synthesis of PET radiotracers using nitrogen-13, carbon-11, and fluorine-18.

In vivo PET imaging of the α4ß2 nicotinic acetylcholine receptor as a marker for brain inflammation after cerebral ischemia.

PET imaging of nicotinic acetylcholine receptors (nAChRs) could become an effective tool for the diagnosis and therapy evaluation of neurologic diseases. Despite this, the role of nAChRs α4ß2 receptors after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. To investigate this, we performed in parallel in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 at 1, 3, 7, 14, 21, and 28 d after middle cerebral artery occlusion (MCAO) in rats. In the ischemic territory, PET with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 showed a progressive binding increase from days 3-7, followed by a progressive decrease from days 14-28 after cerebral ischemia onset. Ex vivo immunohistochemistry for the nicotinic α4ß2 receptor and the mitochondrial translocator protein (18 kDa) (TSPO) confirmed the PET findings and demonstrated the overexpression of α4ß2 receptors in both microglia/macrophages and astrocytes from days 7-28 after experimental ischemic stroke. Likewise, the role played by α4ß2 receptors on neuroinflammation was supported by the increase of [(11)C]PK11195 binding in ischemic rats treated with the α4ß2 antagonist dihydro-ß-erythroidine hydrobromide (DHBE) at day 7 after MCAO. Finally, both functional and behavioral testing showed major impaired outcome at day 1 after ischemia onset, followed by a recovery of the sensorimotor function and dexterity from days 21-28 after experimental stroke. Together, these results suggest that the nicotinic α4ß2 receptor could have a key role in the inflammatory reaction underlying cerebral ischemia in rats.

Pharmacokinetic investigation of sildenafil using positron emission tomography and determination of its effect on cerebrospinal fluid cGMP levels.

Sildenafil (Viagra) is a selective inhibitor of phosphodiesterase type 5 (PDE5), which degrades cyclic guanosine monophosphate to the linear nucleotide. Sildenafil is acutely used in erectile dysfunction and chronically in pulmonary hypertension. Evidence in the last decade shows that sildenafil may have potential as a therapeutic option for Alzheimer's disease or other neurodegenerative disorders. The purpose of this work was to explore whether sildenafil crosses the blood-brain barrier. Pharmacokinetic properties of sildenafil in rodents were investigated using (11) C-radiolabeling followed by in vivo positron emission tomography (PET) and ex vivo tissue dissection and gamma counting. PET results in rats suggest penetration into the central nervous system. Ex vivo data in perfused animals suggest that trapping of [(11) C]sildenafil within the cerebral vascular endothelium limits accumulation in the central nervous system parenchyma. Peroral sildenafil administration to Macaca fascicularis and subsequent chemical analysis of plasma and cerebrospinal fluid (CSF) using liquid chromatography coupled with tandem mass spectrometry showed that drug content in the CSF was high enough to achieve PDE5 inhibition, which was also demonstrated by the significant increases in CSF cyclic guanosine monophosphate levels. Central actions of sildenafil include both relaxation of the cerebral vasculature and inhibition of PDE5 in neurons and glia. This central action of sildenafil may underlie its efficacy in neuroprotection models, and may justify the continued search for a PDE5 ligand suitable for PET imaging. Sildenafil interacts with phosphodiesterase type 5 (PDE5) expressed in the endothelium and/or smooth muscle cells of brain vessels and also crosses the blood-brain barrier to interact with PDE5 expressed in brain cells. At therapeutic doses, the concentration of sildenafil in the cerebrospinal fluid (CSF) is high enough to inhibit PDE5 in the neural cells (neurons and glia). In turn, the concentration of cGMP likely increases in parenchymal cells and, as shown in this report, in the CSF. Read the Editorial Highlight for this article on page 220. Cover Image for this issue: doi: 10.1111/jnc.13302.

In vivo imaging of system xc- as a novel approach to monitor multiple sclerosis.

PURPOSE: Glutamate excitotoxicity contributes to oligodendroglial and axonal damage in multiple sclerosis pathology. Extracellular glutamate concentration in the brain is controlled by cystine/glutamate antiporter (system xc-), a membrane antiporter that imports cystine and releases glutamate. Despite this, the system xc(-) activity and its connection to the inflammatory reaction in multiple sclerosis (MS) is largely unknown. METHODS: Longitudinal in vivo magnetic resonance (MRI) and positron emission tomography (PET) imaging studies with 2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG), [(11)C]-(R)-(1-(2-chlorophenyl)-N-methyl-N-1(1-methylpropyl)-3-isoquinolinecarboxamide ([(11)C]PK11195) and (4S)-4-(3-(18)F-fluoropropyl)-L-glutamate ([(18)F]FSPG) were carried out during the course of experimental autoimmune encephalomyelitis (EAE) induction in rats. RESULTS: [(18)F]FSPG showed a significant increase of system xc(-) function in the lumbar section of the spinal cord at 14 days post immunization (dpi) that stands in agreement with the neurological symptoms and ventricle edema formation at this time point. Likewise, [(18)F]FDG did not show significant changes in glucose metabolism throughout central nervous system and [(11)C]PK11195 evidenced a significant increase of microglial/macrophage activation in spinal cord and cerebellum 2 weeks after EAE induction. Therefore, [(18)F]FSPG showed a major capacity to discriminate regions of the central nervous system affected by the MS in comparison to [(18)F]FDG and [(11)C]PK11195. Additionally, clodronate-treated rats showed a depletion in microglial population and [(18)F]FSPG PET signal in spinal cord confirming a link between neuroinflammatory reaction and cystine/glutamate antiporter activity in EAE rats. CONCLUSIONS: Altogether, these results suggest that in vivo PET imaging of system xc(-) could become a valuable tool for the diagnosis and treatment evaluation of MS.

Efficient Enzymatic Preparation of (13) N-Labelled Amino Acids: Towards Multipurpose Synthetic Systems.

Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents.

Functional Single-Chain Polymer Nanoparticles: Targeting and Imaging Pancreatic Tumors in Vivo.

The development of tools for the early diagnosis of pancreatic adenocarcinoma is an urgent need in order to increase treatment success rate and reduce patient mortality. Here, we present a modular nanosystem platform integrating soft nanoparticles with a targeting peptide and an active imaging agent for diagnostics. Biocompatible single-chain polymer nanoparticles (SCPNs) based on poly(methacrylic acid) were prepared and functionalized with the somatostatin analogue PTR86 as the targeting moiety, since somatostatin receptors are overexpressed in pancreatic cancer. The gamma emitter 67Ga was incorporated by chelation and allowed in vivo investigation of the pharmacokinetic properties of the nanoparticles using single photon emission computerized tomography (SPECT). The resulting engineered nanosystem was tested in a xenograph mouse model of human pancreatic adenocarcinoma. Imaging results demonstrate that accumulation of targeted SCPNs in the tumor is higher than that observed for nontargeted nanoparticles due to improved retention in this tissue.

Novel 18F labeling strategy for polyester-based NPs for in vivo PET-CT imaging.

Drug-loaded nanocarriers and nanoparticulate systems used for drug release require a careful in vivo evaluation in terms of physicochemical and pharmacokinetic properties. Nuclear imaging techniques such as positron emission tomography (PET) are ideal and noninvasive tools to investigate the biodistribution and biological fate of the nanostructures, but the incorporation of a positron emitter is required. Here we describe a novel approach for the (18)F-radiolabeling of polyester-based nanoparticles. Our approach relies on the preparation of the radiolabeled active agent 4-[(18)F]fluorobenzyl-2-bromoacetamide ([(18)F]FBBA), which is subsequently coupled to block copolymers under mild conditions. The labeled block copolymers are ultimately incorporated as constituent elements of the NPs by using a modified nano coprecipitation method. This strategy has been applied in the current work to the preparation of peptide-functionalized NPs with potential applications in drug delivery. According to the measurements of particle size and zeta potential, the radiolabeling process did not result in a statistically significant alteration of the physicochemical properties of the NPs. Moreover, radiochemical stability studies showed no detachment of the radioactivity from NPs even at 12 h after preparation. The radiolabeled NPs enabled the in vivo quantification of the biodistribution data in rats using a combination of imaging techniques, namely, PET and computerized tomography (CT). Low accumulation of the nanoparticles in the liver and their elimination mainly via urine was found. The different biodistribution pattern obtained for the "free" radiolabeled polymer suggests chemical and radiochemical integrity of the NPs under investigation. The strategy reported here may be applied to any polymeric NPs containing polymers bearing a nucleophile, and hence our novel strategy may find application for the in vivo and noninvasive investigation of a wide range of NPs.

Synthesis and 11C-Radiolabelling of 2-Carboranyl Benzothiazoles.

Dicarba-closo-dodecaboranes, commonly known as carboranes, possess unique physico-chemical properties and can be used as hydrophobic moieties during the design of new drugs or radiotracers. In this work, we report the synthesis of two analogues of 2-(4-aminophenyl)benzothiazole (a compound that was found to elicit pronounced inhibitory effects against certain breast cancer cell lines in vitro) in which the phenyl ring has been substituted by a m-carborane cage. Two different synthetic strategies have been used. For the preparation of 1-(9-amino-1,7-dicarba-closo-dodecaboran-1-yl)-benzo-thiazole, the benzothiazole group was first introduced on one of the cluster carbon atoms of m-carborane and the amine group was further attached in three steps. For the synthesis of 1-(9-amino-1,7-dicarba-closo-dodecaboran-1-yl)-6-hydroxybenzothiazole, iodination was performed before introducing the benzothiazole group, and the amino group was subsequently introduced in six steps. Both compounds were radiolabelled with carbon-11 using [11C]CH3OTf as the labelling agent. Radiolabelling yields and radiochemical purities achieved should enable subsequent in vitro and in vivo investigations.

P2X4 receptors control the fate and survival of activated microglia.

Microglia, the resident immune cells of the central nervous system, responds to brain disarrangements by becoming activated to contend with brain damage. Here we show that the expression of P2X4 receptors is upregulated in inflammatory foci and in activated microglia in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) as well as in the optic nerve of multiple sclerosis patients. To study the role of P2X4 receptors in microgliosis, we activated microglia with LPS in vitro and in vivo. We observed that P2X4 receptor activity in vitro was increased in LPS-activated microglia as assessed by patch-clamp recordings. In addition, P2X4 receptor blockade significantly reduced microglial membrane ruffling, TNFα secretion and morphological changes, as well as LPS-induced microglial cell death. Accordingly, neuroinflammation provoked by LPS injection in vivo induced a rapid microglial loss in the spinal cord that was totally prevented or potentiated by P2X4 receptor blockade or facilitation, respectively. Within the brain, microglia in the hippocampal dentate gyrus showed particular vulnerability to LPS-induced neuroinflammation. Thus, microglia processes in this region retracted as early as 2 h after injection of LPS and died around 24 h later, two features which were prevented by blocking P2X4 receptors. Together, these data suggest that P2X4 receptors contribute to controlling the fate of activated microglia and its survival.

68Ga-labeled gold glyconanoparticles for exploring blood-brain barrier permeability: preparation, biodistribution studies, and improved brain uptake via neuropeptide conjugation.

New tools and techniques to improve brain visualization and assess drug permeability across the blood-brain barrier (BBB) are critically needed. Positron emission tomography (PET) is a highly sensitive, noninvasive technique that allows the evaluation of the BBB permeability under normal and disease-state conditions. In this work, we have developed the synthesis of novel water-soluble and biocompatible glucose-coated gold nanoparticles (GNPs) carrying BBB-permeable neuropeptides and a chelator of the positron emitter (68)Ga as a PET reporter for in vivo tracking biodistribution. The small GNPs (2 nm) are stabilized and solubilized by a glucose conjugate. A NOTA ligand is the chelating agent for the (68)Ga, and two related opioid peptides are used as targeting ligands for improving BBB crossing. The radioactive labeling of the GNPs is completed in 30 min at 70 °C followed by purification via centrifugal filtration. As a proof of principle, a biodistribution study in rats is performed for the different (68)Ga-GNPs. The accumulation of radioactivity in different organs after intravenous administration is measured by whole body PET imaging and gamma counter measurements of selected organs. The biodistribution of the (68)Ga-GNPs varies depending on the ligands, as GNPs with the same gold core size show different distribution profiles. One of the targeted (68)Ga-GNPs improves BBB crossing near 3-fold (0.020 ± 0.0050% ID/g) compared to nontargeted GNPs (0.0073 ± 0.0024% ID/g) as measured by dissection and tissue counting.