Claire Bernhard

Ultrasmall Rigid Particles as Multimodal Probes for Medical Applications

Ultrasmall but multifunctional: Rigid imaging particles that are smaller than 5 nm in size can be obtained in a top-down process starting from a core–shell structure (core=gadolinium oxide; shell=polysiloxane). They represent the first multifunctional silica-based particles that are sufficiently small to escape hepatic clearance and enable animal imaging by four complementary techniques

Synthesis and photodynamics of fluorescent blue BODIPY-porphyrin tweezers linked by triazole rings.

Novel zinc porphyrin tweezers in which two zinc porphyrins were connected with π-conjugated boron dipyrromethenes (BDP meso-Por(2) and BDP β-Por(2)) through triazole rings were synthesized to investigate the photoinduced energy transfer and electron transfer. The UV-vis spectrum of BDP β-Por(2) which has less bulky substituents than BDP meso-Por(2) exhibits splitting of the Soret band as a result of the interaction between porphyrins of BDP β-Por(2) in the excited state. Such interaction between porphyrins of both BDP β-Por(2) and BDP meso-Por(2) is dominant at room temperature, while the coordination of the nitrogen atoms of the triazole rings to the zinc ions of the porphyrins occurs at low temperature. The conformational change of the BDP-porphyrin composites was confirmed by the changes in UV-vis and fluorescence spectra depending on temperature. Photodynamics of BDP meso-Por(2) and BDP β-Por(2) has also been investigated by laser flash photolysis. Efficient singlet-singlet energy transfer from the ZnP to the π-conjugated BDP moiety of both BDP meso-Por(2) and BDP β-Por(2) occurred in opposite direction as compared to energy transfer from conventional BDP to ZnP due to the π-conjugation in nonpolar toluene. In polar benzonitrile, however, additional electron transfer occurred along with energy transfer.

DOTAGA-anhydride: a valuable building block for the preparation of DOTA-like chelating agents.

A DOTA derivative that contains an anhydride group was readily synthesized by reacting DOTAGA with acetic anhydride and its reactivity was investigated. Opening the anhydride with propylamine led to the selective formation of one of two possible regioisomers. The structure of the obtained isomer was unambiguously determined by 1D and 2D NMR experiments, including COSY, HMBC, and NOESY techniques. This bifunctional chelating agent offers a convenient and attractive approach for labeling biomolecules and, more generally, for the synthesis of a large range of DOTA derivatives. The scope of the reaction was extended to prepare DOTA-like compounds that contained various functional groups, such as isothiocyanate, thiol, ester, and amino acid moieties. This versatile building block was also used for the synthesis of a bimodal tag for SPECT or PET/optical imaging.

Thermal and Chemical Stability of Thiol Bonding on Gold Nanostars.

The stability of thiol bonding on the surface of star-shaped gold nanoparticles was studied as a function of temperature in water and in a set of biologically relevant conditions. The stability was evaluated by monitoring the release of a model fluorescent dye, Bodipy-thiol (BDP-SH), from gold nanostars (GNSs) cocoated with poly(ethylene glycol) thiol (PEG-SH). The increase in the BDP-SH fluorescence emission, quenched when bound to the GNSs, was exploited to this purpose. A maximum 15% dye release in aqueous solution was found when the bulk temperature of gold nanostars solutions was increased to T = 42 °C, the maximum physiological temperature. This fraction reduces 3-5% for temperatures lower than 40 °C. Similar results were found when the temperature increase was obtained by laser excitation of the near-infrared (NIR) localized surface plasmon resonance of the GNSs, which are photothermally responsive. Besides the direct impact of temperature, an increased BDP-SH release was observed upon changing the chemical composition of the solvent from pure water to phosphate-buffered saline and culture media solutions. Moreover, also a significant fraction of PEG-SH was released from the GNS surface due to the increase in temperature. We monitored it with a different approach, that is, by using a coating of α-mercapto-ω-amino PEG labeled with tetramethylrhodamine isothiocyanate on the amino group, that after heating was separated from GNS by ultracentrifugation and the released PEG was determined by spectrofluorimetric techniques on the supernatant solution. These results suggest some specific limitations in the use of the gold-thiolate bond for coating of nanomaterials with organic compounds in biological environments. These limitations come from the duration and the intensity of the thermal treatment and from the medium composition and could also be exploited in biological media to modulate the in vivo release of drugs.

DMAP‐BODIPY Alkynes: A Convenient Tool for Labeling Biomolecules for Bimodal PET–Optical Imaging

Several new boron dipyrromethene/N,N-dimethylaminopyridine (BODIPY-DMAP) assemblies were synthesized as precursors for bimodal imaging probes (optical imaging, OI/positron emission tomography, PET). The photophysical properties of the new compounds were also studied. The first proof-of-concept was obtained with the preparation of several new BODIPY-labeled bombesins and evaluation of the affinity for bombesin receptors by using a competition binding assay. Fluorination reactions were investigated on DMAP-BODIPY precursors as well as on DMAP-BODIPY-labeled bombesins. Chemical modifications on the BODIPY core were also performed to obtain luminescent dyes emitting in the therapeutic window (650-900 nm), suitable for in vivo imaging, making these compounds promising precursors for PET/optical dual-modality imaging agents.

Dual labeling of lipopolysaccharides for SPECT-CT imaging and fluorescence microscopy.

Lipopolysaccharides (LPS) or endotoxins are amphipathic, pro-inflammatory components of the outer membrane of Gram-negative bacteria. In the host, LPS can trigger a systemic inflammatory response syndrome. To bring insight into in vivo tissue distribution and cellular uptake of LPS, dual labeling was performed with a bimodal molecular probe designed for fluorescence and nuclear imaging. LPS were labeled with DOTA-Bodipy-NCS, and pro-inflammatory properties were controlled after each labeling step. LPS were then radiolabeled with (111)In and subsequently injected intravenously into wild-type, C57B16 mice, and their in vivo behavior was followed by single photon emission computed tomography coupled with X-ray computed tomography (SPECT-CT) and fluorescence microscopy. Time course of liver uptake of radiolabeled LPS ((111)In-DOTA-Bodipy-LPS) was visualized over a 24-h period in the whole animal by SPECT-CT. In complementary histological analyses with fluorescent microscopy, the bulk of injected (111)In-DOTA-Bodipy-LPS was found to localize early within the liver. Serum kinetics of unlabeled and DOTA-Bodipy-labeled LPS in mouse plasma were similar as ascertained by direct quantitation of β-hydroxymyristate, and DOTA-Bodipy-LPS was found to retain the potent, pro-inflammatory property of the unlabeled molecule as assessed by serum cytokine assays. It is concluded that the dual labeling process, involving the formation of covalent bonds between a DOTA-Bodipy-NCS probe and LPS molecules is relevant for imaging and kinetic analysis of LPS biodistribution, both in vivo and ex vivo. Data of the present study come in direct and visual support of a lipopolysaccharide transport through which pro-inflammatory LPS can be transported from the periphery to the liver for detoxification. The (111)In-DOTA-Bodipy-LPS probe arises here as a relevant tool to identify key components of LPS detoxification in vivo.

Optical Method for Predicting the Composition of Self-Assembled Monolayers of Mixed Thiols on Surfaces Coated with Silver Nanoparticles

With a simple optical method, based on UV-vis absorption spectra on glass slides, it is possible to predict the composition of self-assembled monolayers of mixed thiols, grafted on monolayers of silver nanoparticles. Glass slides are modified with the layer-by-layer technique, first forming a monolayer of mercaptopropyltrimethoxysilane, then grafting a monolayer of silver nanoparticles on it. These surfaces are further coated by single or mixed thiol monolayers, by dipping the slides in toluene solutions of the chosen thiols. Exchange constants are calculated for the competitive deposition between the colorless 1-dodecanethiol or PEG5000 thiol and BDP-SH, with the latter being a thiol-bearing molecule containing the strongly absorbing BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) moiety, synthesized on purpose. The constants are calculated by determining the fraction of BDP-SH deposited on the surface from a solution with a given molar fraction, directly measuring the absorption spectra of BDP-SH on the slides. Then, the exchange constant for the competitive deposition between 1-dodecanethiol and PEG5000 thiol is calculated by combining their exchange constants with BDP-SH. This allows to predict the fraction of the two colorless thiols coating the silver nanoparticles slides obtained from a toluene solution with a given molar fraction, for example, of PEG5000 thiol. The correctness of the calculated surface fraction is verified by studying the coating competition between 1-dodecanethiol and a PEG5000 thiol remotely modified with a strongly absorbing fluorescein fragment.

Straightforward synthesis of bis-tetraazacycloalkanes: towards new potential CXCR4 antagonists?

We report herein an efficient and general method for the synthesis of new bismacrocyclic compounds, structural analogues of biscyclam AMD3100, in which the two macrocycles are linked together through carbon atoms of the cycles. Several representatives of this new class of biscyclic derivatives were prepared by reacting C-aminomethyl-13aneN4 with aromatic dialdehydes. Preliminary in vitro studies were performed to evaluate the affinity of these compounds towards the co-receptor CXCR4.

Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Dual-labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.

MA-NOTA: a new chelator for efficient Cu-64 antibody radiolabeling

Hypothesis: We assessed in in vitro and in vivo models of ovarian cancer the therapeutic efficacy of 16F12 mAbs directed against Mullerian Inhibiting Substance type II receptor (MISRII) radiolabeled with 213Bi Methods: In vitro, both direct and bystander cytotoxic effects were measured using clonogenic assay and standard medium transfer protocol. Typically, Clonogenic survival was assessed in SK-OV-3 donor cells expressing MISRII and exposed for 90 min to 0.06-0.5MBq/mL of 16F12 213Bi-mAbs. Bystander cytotoxicity was measured in recipient cells grown in non-radioactive culture medium preconditioned for 2 hours in the presence of donor cells. DNA double strand breaks (DSBs) were measured in both donor and recipients cells using immunofluorescent detection of gamma-H2AX and of 53BP1. In vivo we explored in athymic nude mice bearing intraperitoneal (IP) MISRII-expressing AN3CA tumor the therapeutic efficacy of brief-intraperitoneal radioimmunotherapy (BIP-RIT, 12.95 37 MBq; 37MBq/mg) or of intraperitoneal RIT (IP-RIT; 2.96-12.95 MBq; 37MBq/mg) using 213Bi-16F12. BIP-RIT mimics hyperthermic intraperitoneal chemotherapy as used in clinic. It consists of intraperitoneal injection of high activities of radiolabeled mAbs followed 30 min later by wash of the peritoneal cavity with saline solution to remove unbound radioactivity. The biodistribution of radiolabeled antibodies following IP-RIT (12.95 MBq; 37MBq/mg) or BIP-RIT (37 MBq; 37MBq/mg) was assessed. Results: In vitro we showed in donor cells a strong direct cytotoxicity of 16F12 213Bi-mAbs. A significant bystander cytotoxicity was also measured in recipient cells. Genotoxic effects were also demonstrated as measured by the formation of DNA DSBs in both donor and recipient cells. In vivo, results of biodistribution indicated that tumour uptake of 213Bi-16F12 during BIP RIT was higher than after IP RIT. The tumour-to-blood uptake ratio was 9 versus 3, respectively, one hour post RIT while it decreased down to 3 and 1, respectively, three hours post-RIT. Finally, a similar delay in tumor growth was observed in mice treated with 12.95 MBq of 213Bi-16F12 following IP-RIT or treated with 37 MBq using BIP-RIT. Conclusions: We confirmed in vitro the therapeutic efficacy of newly developed 16F12 213Bi-mAbs. in vivo results indicate that similar therapeutic efficacy and lower toxicity could be obtained with BIP-RIT compared with IP-RIT. BIP-RIT could be a new tool in the therapy of peritoneal carcinomatosis. URI: Authors: LADJOHOUNLOU Riad PICHARD Alexandre DEHAYES E BOUDOUSQ Vincent BRUCHERTSEIFER Frank MORGENSTERN Alfred NAVARRO-TEULON Isabelle POUGET Jean-Pierre Publication Year: 2016 Science Areas: Health and consumer protection [1]European Journal of Nuclear Medicine and Molecular Imaging Volume 43, Supplement 1 10.1007/s00259-016-3484-4 This supplement was not sponsored by outside commercial interests. It was funded entirely by the association’s own resources. ABSTRACT DOI 10.1007/s00259-016-3484-4 Eur J Nucl Med Mol Imaging (2016) 43 (Suppl 1):S1–S734Background: In the context of the EORTC LungTech trial, a QA procedure including a PET/CT credentialing has been developed. This procedure will ultimately allow us to pool data from 23 institutions with the overall goal of investigating the impact of tumour motion on quantification. As no standardized procedure exists under respiratory conditions, we investigated the variability of 14 SUV metrics to assess their robustness over respiratory noise. Methods: The customized CIRS-008A phantom was scanned at 13 institutions. This phantom consists of a 18 cm long body, a rod attached to a motion actuator, and a sphere of either 1.5 or 2.5cm diameters. Body, rods and spheres were filled with homogeneous 18FDG solutions representative of activity concentrations in mediastinum, lung and tumour for a 70kg patient. Three respiratory patterns with peak-to-peak amplitudes and periods of 15mm/3sec, 15mm/6sec and 25mm/4sec were tested. Prior to scanning in respiratory condition, a 3D static PET/CT was acquired as reference. During motion, images were acquired using 3D or 4D gated PET(average image) according to institutional settings. 14 SUV(mean) metrics were obtained per acquisition varying VOI/ ROI shape and location. Three ROIs and three VOIs with respective radii of 0.5, 0.6 and 0.8cm were investigated. These ROIs/VOIs were first centred on the maximum activity voxel; a second analysis was made changing the location from the voxel to the region (ROI5voxels) or the volume (VOI7voxels) with the maximum value. Two additional VOIs were defined as 3D isocontours respectively at 70% and 50% of the maximum voxel value. The SUV metrics were normalized by the corresponding 3D static SUV. Converting to recovery coefficients (RC) allowed us to pool data from all institutions, while maintaining focus solely on motion. For each RC from each motion setting we calculated the mean over institutions, we then looked at the standard deviation (Sd) and spread of each averaged RC over each motion setting (formula [1], [2], Figure1). Results: For the institutions visited we found that RCVOI70% and RCVOI50%, yielded over the 14 metrics the lowest variability to motion with Sd of 0.04 and 0.03 respectively. The RCs based on ROIs/VOIs centered on a single voxel were less impacted by motion (Sd: 0.08) compared to region RCs (Sd: 0.14). The averaged Sd over the RCs based on VOIs and ROIs was 0.12 and 0.11 respectively. Conclusion: Quantification over breathing types depends on ROI/VOI definition. Variables based on SUV max thresholds were found the most robust against respiratory noise.