Synthesis and in vitro preliminary evaluation of prostate-specific membrane antigen targeted upconversion nanoparticles as a first step towards radio/fluorescence-guided surgery of prostate cancer.

Over the last decade, upconversion nanoparticles (UCNP) have been widely investigated in nanomedicine due to their high potential as imaging agents in the near-infrared (NIR) optical window of biological tissues. Here, we successfully develop active targeted UCNP as potential probes for dual NIR-NIR fluorescence and radioactive-guided surgery of prostate-specific membrane antigen (PSMA)(+) prostate cancers. We designed a one-pot thermolysis synthesis method to obtain oleic acid-coated spherical NaYF4:Yb,Tm@NaYF4 core/shell UCNP with narrow particle size distribution (30.0 ± 0.1 nm, as estimated by SAXS analysis) and efficient upconversion luminescence. Polyethylene glycol (PEG) ligands bearing different anchoring groups (phosphate, bis- and tetra-phosphonate-based) were synthesized and used to hydrophilize the UCNP. DLS studies led to the selection of a tetra-phosphonate PEG(2000) ligand affording water-dispersible UCNP with sustained colloidal stability in several aqueous media. PSMA-targeting ligands (i.e., glutamate-urea-lysine derivatives called KuEs) and fluorescent or radiolabelled prosthetic groups were grafted onto the UCNP surface by strain-promoted azide-alkyne cycloaddition (SPAAC). These UCNP, coated with 10 or 100% surface density of KuE ligands, did not induce cytotoxicity over 24 h incubation in LNCaP-Luc or PC3-Luc prostate cancer cell lines or in human fibroblasts for any of the concentrations evaluated. Competitive binding assays and flow cytometry demonstrated the excellent affinity of UCNP@KuE for PSMA-positive LNCaP-Luc cells compared with non-targeted UCNP@CO2H. Furthermore, the binding of UCNP@KuE to prostate tumour cells was positively correlated with the surface density of PSMA-targeting ligands and maintained after 125I-radiolabelling. Finally, a preliminary biodistribution study in LNCaP-Luc-bearing mice demonstrated the radiochemical stability of non-targeted [125I]UCNP paving the way for future in vivo assessments.

Simultaneous proteoglycans and hypoxia mapping of chondrosarcoma environment by frequency selective CEST MRI.

PURPOSE: To evaluate the relevance of CEST frequency selectivity in simultaneous in vivo imaging of both of chondrosarcoma's phenotypic features, that are, its high proteoglycan concentration and its hypoxic core. METHODS: Swarm rat chondrosarcomas were implanted subcutaneously in NMRI nude mice. When tumors were measurable (12-16 days postoperative), mice were submitted to GAG, guanidyl, and APT CEST imaging. Proteoglycans and hypoxia were assessed in parallel by nuclear imaging exploiting 99m Tc-NTP 15-5 and 18 F-FMISO, respectively. Data were completed by ex vivo analysis of proteoglycans (histology and biochemical assay) and hypoxia (immunofluorescence). RESULTS: Quantitative analysis of GAG CEST evidenced a significantly higher signal for tumor tissues than for muscles. These results were in agreement with nuclear imaging and ex vivo data. For imaging tumoral pH in vivo, the CEST ratio of APT/guanidyl was studied. This highlighted an important heterogeneity inside the tumor. The hypoxic status was confirmed by 18 F-FMISO PET imaging and ex vivo immunofluorescence. CONCLUSION: CEST MRI simultaneously imaged both chondrosarcoma properties during a single experimental run and without the injection of any contrast agent. Both MR and nuclear imaging as well as ex vivo data were in agreement and showed that this chondrosarcoma animal model was rich in proteoglycans. However, even if tumors were lightly hypoxic at the stage studied, acidic areas were highlighted and mapped inside the tumor.

Neuroprotective role of lactate in rat neonatal hypoxia-ischemia.

Hypoxic-ischemic (HI) encephalopathy remains a major cause of perinatal mortality and chronic disability in newborns worldwide (1-6 for 1000 births). The only current clinical treatment is hypothermia, which is efficient for less than 60% of babies. Mainly considered as a waste product in the past, lactate, in addition to glucose, is increasingly admitted as a supplementary fuel for neurons and, more recently, as a signaling molecule in the brain. Our aim was to investigate the neuroprotective effect of lactate in a neonatal (seven day old) rat model of hypoxia-ischemia. Pups received intra-peritoneal injection(s) of lactate (40 µmol). Size and apparent diffusion coefficients of brain lesions were assessed by magnetic resonance diffusion-weighted imaging. Oxiblot analyses and long-term behavioral studies were also conducted. A single lactate injection induced a 30% reduction in brain lesion volume, indicating a rapid and efficient neuroprotective effect. When oxamate, a lactate dehydrogenase inhibitor, was co-injected with lactate, the neuroprotection was completely abolished, highlighting the role of lactate metabolism in this protection. After three lactate injections (one per day), pups presented the smallest brain lesion volume and a complete recovery of neurological reflexes, sensorimotor capacities and long-term memory, demonstrating that lactate administration is a promising therapy for neonatal HI insult.

Online Quantification of Lactate Concentration in Microdialysate During Cerebral Activation Using 1H-MRS and Sensitive NMR Microcoil.

The dynamic in vivo profiling of lactate is of uppermost importance in both neuroenergetics and neuroprotection fields, considering its central suspected role as a metabolic and signaling molecule. For this purpose, we implemented proton magnetic resonance spectroscopy (1H-MRS) directly on brain microdialysate to monitor online the fluctuation of lactate contents during neuronal stimulation. Brain activation was obtained by right whisker stimulation of rats, which leads to the activation of the left barrel cortex area in which the microdialysis probe was implanted. The experimental protocol relies on the use of dedicated and sensitive home-made NMR microcoil able to perform lactate NMR profiling at submillimolar concentration. The MRS measurements of extracellular lactate concentration were performed inside a pre-clinical MRI scanner allowing simultaneous visualization of the correct location of the microprobe by MRI and detection of metabolites contained in the microdialysis by MRS. A 40% increase in lactate concentration was measured during whisker stimulation in the corresponding barrel cortex. This combination of microdialysis with online MRS/MRI provides a new approach to follow in vivo lactate fluctuations, and can be further implemented in physio-pathological conditions to get new insights on the role of lactate in brain metabolism and signaling.

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation.

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

A neuronal MCT2 knockdown in the rat somatosensory cortex reduces both the NMR lactate signal and the BOLD response during whisker stimulation.

Although several in vitro and ex vivo evidence support the existence of lactate exchange between astrocytes and neurons, a direct demonstration in vivo is still lacking. In the present study, a lentiviral vector carrying a short hairpin RNA (shRNA) was used to downregulate the expression of the monocarboxylate transporter type 2 (MCT2) in neurons of the rat somatosensory cortex (called S1BF) by ~ 25%. After one hour of whisker stimulation, HRMAS 1H-NMR spectroscopy analysis of S1BF perchloric acid extracts showed that while an increase in lactate content is observed in both uninjected and shRNA-control injected extracts, such an effect was abrogated in shMCT2 injected rats. A 13C-incorporation analysis following [1-13C]glucose infusion during the stimulation confirmed that the elevated lactate observed during activation originates from newly synthesized [3-13C]lactate, with blood-derived [1-13C]glucose being the precursor. Moreover, the analysis of the 13C-labeling of glutamate in position C3 and C4 indicates that upon activation, there is an increase in TCA cycle velocity for control rats while a decrease is observed for MCT2 knockdown animals. Using in vivo localized 1H-NMR spectroscopy, an increase in lactate levels is observed in the S1BF area upon whisker stimulation for shRNA-control injected rats but not for MCT2 knockdown animals. Finally, while a robust BOLD fMRI response was evidenced in control rats, it was absent in MCT2 knockdown rats. These data not only demonstrate that glucose-derived lactate is locally produced following neuronal activation but also suggest that its use by neurons via MCT2 is probably essential to maintain synaptic activity within the barrel cortex.

Intracerebral synthesis of glutamine from hyperpolarized glutamate.

PURPOSE: Changes in glutamate (Glu) levels occur in a number of neurodegenerative diseases. We proposed the use of 13 C spectroscopy and the highly amplified signal generated by hyperpolarization to achieve spatial and temporal resolutions adequate for in vivo studies of Glu metabolism in the healthy rat brain. Thus, we investigated uptake of hyperpolarized [1-13C ]Glu after a temporary blood-brain barrier (BBB) disruption protocol and its conversion to glutamine (Gln) in the brain. METHODS: [1-13 C]Glu was hyperpolarized using the dynamic nuclear polarization process. A temporary BBB disruption using mannitol allowed hyperpolarized [1-13 C]Glu to reach the brain. Then, hyperpolarized [1-13 C]Glu brain metabolism was observed in vivo by MR spectroscopy experiments at 3T. Products synthesized from [1-13 C]Glu were assigned via liquid chromatography-mass spectrometry. RESULTS: Hyperpolarized [1-13 C]Glu reached 20% ± 2.3% polarization after 90 min. After validation of the BBB disruption protocol, hyperpolarized [1-13 C]Glu (175.4 ppm) was detected inside the rat brain, and the formation of [1-13 C]Gln at 174.9 ppm was also observed. CONCLUSION: The Gln synthesis from hyperpolarized [1-13 C]Glu can be monitored in vivo in the healthy rat brain after opening the BBB. Magn Reson Med 78:1296-1305, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Proton MR Spectroscopy for Diagnosis and Evaluation of Treatment Efficacy in Parkinson Disease.

PURPOSE: To assess the neurochemical profile in the putamen of patients with parkinsonian syndromes undergoing L-3,4-dihydroxyphenylalanine (L-DOPA) treatment (drug-on) or after withdrawal of L-DOPA medication (drug-off) compared with healthy volunteers to identify dopaminergic therapy-sensitive biomarkers of Parkinson disease. MATERIALS AND METHODS: The local institutional review board approved the study, and all participants gave informed consent. A short echo-time (29 msec) single-voxel (1-cm(3)) proton (hydrogen 1 [(1)H]) magnetic resonance (MR) spectroscopic approach was used at 3 T to explore the metabolic profile in the putamen of patients with Parkinson disease. Spectra obtained from 20 healthy volunteers were blindly compared with spectra obtained from 20 patients with parkinsonian syndromes in drug-on and drug-off conditions in a randomized permuted block study to assess the accuracy of diagnostic biomarkers for Parkinson disease and efficacy of L-DOPA therapy. The statistical tests were two sided, with a type-I error set at α of .05. Random-effects models were used to compare healthy subjects and patients with parkinsonian syndromes in drug-on or drug-off conditions. RESULTS: Measured concentrations of putaminal total N-acetylaspartate (tNAA) (8.1 ± 0.2 vs 9.4 ± 0.4; P < .01), total creatine (tCr) (7.5 ± 0.2 vs 8.3 ± 0.3; P < .01), and myo-inositol (m-Ins) (3.8 ± 0.3 vs 5.6 ± 0.4; P < .001) were significantly lower in patients with parkinsonian syndromes in drug-off condition than in healthy volunteers. Moreover, L-DOPA therapy restored tNAA (9.1 ± 0.4 vs 8.1 ± 0.2; P < .01) and tCr (8.1 ± 0.3 vs 7.5 ± 0.2; P < .01) levels, whereas m-Ins levels remained unchanged. The combined glutamate and glutamine and choline showed no changes in drug-off or drug-on condition compared with those in control subjects. CONCLUSION: tNAA, tCr, and m-Ins were identified as putative biomarkers of Parkinson disease in the putamen of patients. tNAA and tCr levels are responsive to L-DOPA therapy.