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|12:30 p.m.||PS 02-01||
This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.
|12:48 p.m.||PS 02-02||
Molecular engineering of paramagnetic doped-nanofluorides for sensitive in vivo 19F-MRI (#212)
Dana Cohen1, Reut Mashiach1, Lothar Houben2, Hyla Allouche-Arnon1, Amnon Bar-Shir1
1 Weizmann Institute of Science, Organic Chemistry, Rehovot, Israel
Inorganic nanofluorides allow scientists to synthesize them with variable contents and design them with varied sizes and shapes [1, 2]. Recently CaF2 nanocrystals (NCs) showed their ability to be used in 19F-MRI studies. However, their relatively long T1 make them far from ideal 19F-MRI tracers. Here we show that engineered paramagnetic-lanthanide doped CaF2 NCs result in 100 times shorter T1 (from 10 sec to 0.1 sec) relaxation times of the fluoride within the NC core, resulting in 5-fold increase in 19F-MRI SNR for improved in vivo molecular imaging.
Using the solvothermal synthetic approach small CaF2 NCs were fabricated with the addition of paramagnetic lanthanide (Ln3+) dopants to obtain Ln:CaF2 NCs. The produced Ln:CaF2 NCs were fully characterized using NMR for T1 and T2 characterization, Transmission Electron Microcopy (TEM) for NCs morphology evaluation along dopant quantification, and Dynamic Light Scattering (DLS) to evaluate their monodispersity in solution. MRI experiments were performed on 15.2T horizontal scanner (Biospec, Bruker) using UTE-3D sequence for 19F images with TR= 4 msec, TE= 8 µsec, FOV=2x2cm2 , matrix size = 32x32 and RARE sequence for 1H images with TR= 1000 msec, TE= 20 msec, FOV=2x2cm2 and matrix size = 128x128.
Figure 1 shows the high-resolution 19F-NMR spectra of the fabricated Ln3+:CaF2 NCs in solution with a clear 19F-NMR line broadening observed for the most paramagnetic lanthanide used (Gd3+). Interestingly, the addition of low amounts of Sm3+ (up to 2% as determined by TEM-EDS, Fig. 1 inset), which is the Ln3+ having the lowest paramagnetic relaxation enhancement (PRE), led to a dramatic shortening of the T1 of water-soluble CaF2, from 10 sec for CaF2 NCs to 0.1 sec for Sm:CaF2 NCs, a factor of 100 (Fig. 1b). Since T1 is the limiting factor for 19F-MRI scan time, the use of nanofluorides having extremely short T1 (0.1 sec) results in a 5-fold increase in SNR for 19F-MRI (Fig. 2). Figure 2a clearly shows the improved 19F-MRI sensitivity obtained by the paramagnetic dopant (Sm3+, as compared to La3+ diamagnetic dopant) allowing fast in vivo 19F-MRI acquisitions or alternatively, a more reliable data obtained from larger number of signal averaging at the given experiment time (Fig. 2b).
We have demonstrated the ability to engineer paramagnetic doped CaF2 NCs as superior tracers for 19F-MRI. Using paramagnetic dopants that resulted in a 100-fold shorter T1 of the 19F-spins in the CaF2 NCs (T1=0.1 sec), which is comparable to the T1 values of perfluorocarbon nanoemulsions, makes CaF2 NCs legitimate sensitive nanotracers for wide range of in vivo 19F-MRI applications.
1. Mai, H.-X., et al., High-Quality Sodium Rare-Earth Fluoride Nanocrystals: Controlled Synthesis and Optical Properties. Journal of the American Chemical Society, 2006.
2. Chen, G., et al., (α-NaYbF4:Tm3+)/CaF2 Core/Shell Nanoparticles with Efficient Near-Infrared to Near-Infrared Upconversion for High-Contrast Deep Tissue Bioimaging. ACS Nano, 2012.
3. Ashur, I., H. Allouche‐Arnon, and A. Bar‐Shir, Calcium Fluoride Nanocrystals: Tracers for In Vivo 19F Magnetic Resonance Imaging. Angewandte Chemie,2018.
a. 19F-NMR of oleate-coated CaF2 NCs with different lanthanide-dopants. Inset- TEM image of Sm:CaF2 and elemental mapping with clear indication of Ca, F and Sm as dopant. b. inversion recovery plot shows significant differences of 19F spins relaxation of PEG-coated CaF2 and PEG-coated Sm:CaF2 in water.
Keywords: Nanocrystals, 19F, MRI, Molecular Imaging
|1:00 p.m.||PS 02-03||
Perfluorocarbon lipid nanoparticles for dual-frequency MR Neuroimaging applications (#308)
Giuseppe Gambino1, Tanja Savić1, Goran Angelovski1
1 Max Planck Institute for Biological Cybernetics, MR Neuroimaging Agents, Tübingen, Baden-Württemberg, Germany
Although BOLD-based fMRI is extensively used to indicate neuronal activity, a great step forward in detecting brain activity directly would be the measurement of [Ca2+] by means of bioresponsive MRI contrast agents, probes able to modulate their signal-enhancement effect in response to [Ca2+] fluctuations. Here we report a lipid nanoparticle (GdPFLNPs) with a surface bearing Ca-responsive Gd3+ complexes (GdL) and a perfluorocarbon core. Such nanoprobe would enable the dual-frequency imaging of [Ca2+], coregistering its 1H MRI Ca-enhanced contrast with the quantitative signal of 19F MRI.
The lipid nanoparticles were obtained through the rehydration of a lipid film (L/DPPC/DSPE-PEG2000 in 20:75:5 ratio) with a suspension of perfluoro-15-crown-5-ether (PFCE) in aqueous HEPES and NaCl. The obtained suspension was sonicated and extruded with 200 nm cutoff filters. Complexation was performed after extrusion in order to decorate with Gd3+ exclusively the ligand molecules exposed on the outer nanoparticle surface. The final nanoparticles were purified by dialysis and ultracentrifugation. The size and shape of the obtained particles were assessed by dynamic light scattering (DLS) and cryo-TEM imaging, the relaxometric properties were characterized by 1H and 19F NMR spectrometry, while the imaging potential was demonstrated by means of 1H and 19F MRI on tube phantoms at 7 T.
The obtained suspension was analyzed by DLS resulting in a polydisperse system, with particle diameter of 78.2 nm and 236.9 nm (PDI 0.30). Cryo-TEM images confirmed the size distribution of the sample and revealed particles of spherical shape with an electron-dense core, characteristic for this kind of system.1 Furthermore, no evident change of size or shape of the particles seems to occur upon binding of Ca2+. The potential of GdPFLPNs as Ca-responsive 1H MRI contrast agents was characterized by means of relaxometric titrations at 25 °C and 7 T (Figure 1), resulting in a significant increase of longitudinal and transverse relaxivities, r1 (+31%) and r2 (+343%), respectively. On the other hand, 19F T1 value (974 ms) did not change throughout the titration. Additionally, 1H MRI on tube phantoms showed the great potential of this system as T2w and T2/T1w Ca-responsive agent, while 19F T1w MRI images of the samples exhibited very high SNR for corresponding low [Gd3+] samples (Figure 2).
In this work we report a nanosized Ca-responsive probe for dual-frequency MRI. The possibility of exploiting its advantageous performances as T2- and T2/T1-sensitive contrast agent, together with the high SNR obtained with quantitative 19F MRI, opens new realms for a wide range of possible future applications, focused on the monitoring and mapping of [Ca2+] in the brain, such as Ca-dependent molecular- and stimuli-coupled functional neuroimaging.
1 - De Vries A., Moonen R., Yildrim M., Langereis S., Lamerichs R., Pikkemaat J.A., Baroni S., Terreno E., Nicolay K., Strijkers G.J., Grüll H., Contrast Media & Mol. Imaging, 2014, 9 83-91.
Keywords: MRI, dual-frequency, fluorine, neuroimaging, contrast agents
|1:12 p.m.||PS 02-04||
Radiolabeled Iron Oxide/Aluminum Hydroxide Nanostructures as New Dual Contrast Agents for Simultaneous PET/MRI (#86)
Sarah Belderbos1, 2, Manuel Antonio González-Gómez3, Yolanda Piñeiro3, Frederik Cleeren2, 4, Jens Wouters1, 2, Bella B. Manshian2, 5, Stefaan J. Soenen2, 5, Christophe M. Deroose2, 6, Willy Gsell1, 2, Guy Bormans2, 4, Jose Rivas3, Uwe Himmelreich1, 2
1 KU Leuven, Biomedical MRI, Department of Imaging and Pathology, Leuven, Belgium
Iron oxide nanoparticles (Fe304 NPs) and PET tracers have shown great potential for cell tracking applications1,2. However, radiolabeling of new contrast agents for simultaneous PET/MRI is often time-consuming3,4. Embedding Fe304 NPs in an aluminum hydroxide (Al(OH)3) shell (Fe3O4@Al(OH)3 NPs), allows direct, fast labeling with sodium fluoride (Na18F), thus enabling NP visualization with PET and MRI, and combining advantages of both modalities5,6. In this study, we aimed to visualize intravenously (IV) injected Na18F-labeled NPs and NP-labeled mouse mesenchymal stem cells (mMSCs) with PET/MRI.
Fe3O4@Al(OH)3 NPs were synthesized by forced chemical hydrolysis of magnetic NPs coated with polyacrylic acid. Labeling efficiency and stability of NPs with Na18F were evaluated with instant thin layer chromatography. In vitro contrast properties of radiolabeled (RL) NPs or RL NPs-labeled mMSCs were evaluated after acquisition of a 1h PET scan and T2*, T2 and T1 maps using a 7T MRI with PET insert (Bruker PCI). For in vivo biodistribution studies, RL NPs or 105 mMSCs labeled with RL NPs were injected IV in C57BL/6 mice. Following scans were acquired: a 1h PET scan simultaneously with dynamic contrast-enhanced (DCE)-MRI, whole-body 3D T2-weighted MRI and parametric T2 maps pre and post injection. After sacrificing the mice, liver and lungs were analyzed using a γ-counter.
Labeling of Fe3O4@Al(OH)3 NPs is fast (>97% Na18F bound after 2’) and stable in Milli-Q and saline (86.6% and 76.9% Na18F bound), while labeling efficiency was lower under other conditions. In vitro, we were able to visualize small amounts of RL NPs/cells on PET and MRI (Fig. 1). After IV injection, most RL NPs accumulated in the liver as seen on PET (increased standardized uptake values; SUV) and MRI (lower T2 values, Fig. 2). Conversely, increased lung SUV values were detected after injection of RL mMSCs when compared to injection of RL NPs. Moreover, increased SUV values in bone and spleen were measured, indicating NP defluorination and clearance of NPs/cells. All findings were confirmed by ex vivo measurements. Dynamic reconstruction of Na18F PET and DCE-MRI scans yielded time-activity curves showing rapid accumulation of NPs after NP/cell injection, which stayed stable over time. While lung SUV increase was transient after NPs administration, it was stable after cell injection.
Using simultaneous PET/MRI, we were able to visualize RL NPs/mMSCs in vitro and in vivo. The high potential of PET/MRI contrast agents is based on 1) detection of cells/NPs by PET in hypointense MR areas, 2) combined advantages of both modalities (i.e. MRI: anatomical information, lasting contrast, PET: high sensitivity/specificity, low background)6–8, providing more data on whole-body NPs/cell distribution compared to single modality imaging.
This work was funded by the European Horizon 2020 ‘PANA’ project under grant agreement 686009 and the KU Leuven program financing ‘In Vivo Molecular Imaging Research’ (IMIR).
Fig. 1: Succesful In Vitro Radiolabeling of Fe3O4@Al(OH)3 Nanostructures
A) Instant thin layer chromatography shows rapid labeling of Fe3O4@Al(OH)3 NPs with Na18F, which is B) stable in Milli-Q and saline, while less stable in media containing phosphates (medium=85% DMEM/15% FBS, 50/50=50% DMEM/50% FBS). C) PET and MRI of (mMSCs labeled with) Fe3O4@Al(OH)3 NPs labeled with estimated amounts of Na18F. Both have excellent contrast properties on PET and MRI.
Fig. 2: In vivo biodistribution of Na18F-labeled Fe3O4@Al(OH)3 NPs or 100,000 mouse mesenchymal stem
A) Representative PET/MRI images of C57BL/6 mice intravenously injected with saline, radiolabeled NPs (0.9 ± 0.2 MBq) or mMSCs labeled with such NPs (0.25 ± 0.08 MBq, 1h cell labeling) and B) corresponding 3D T2 weighted MRI images. Results indicate uptake of RL NPs in the liver (red arrow), while increased SUV was seen in the lungs after the injection of radiolabeled NPs.
Keywords: Radiolabeling, PET/MRI, nanoparticles, cell tracking
|1:24 p.m.||PS 02-05||
Developing new targeted molecular contrast agents for imaging inflammation of vulnerable plaque (#271)
Rhiannon J. Evans1, 2, 3, Javier Hernandez-Gil1, Zahra Mohri2, Kok Y. Chooi2, 5, Begona Lavin-Plaza3, Alkystis Phinikaridou3, Joseph Boyle4, Rob Krams5, René Botnar3, Nicholas J. Long1
1 Imperial College London, Department of Chemistry, London, United Kingdom
Vulnerable plaque rupture is the underlying cause of high cardiovascular disease-related mortality, and is currently challenging to detect. Magnetic Resonance Imaging (MRI) is non-ionising and non-invasive, with excellent soft-tissue contrast. Superparamagnetic iron oxide nanoparticles (SPIONs) are an exciting alternative to gadolinium chelates for MRI contrast. CX3CL1 is overexpressed in vulnerable plaque but not in stable plaque1. The aim of this project is to synthesise a range of nanoparticles targeted to CX3CL1 for vulnerable plaque detection with MRI.
Nanoparticles of four core sizes were synthesised by thermal decomposition and coated with poly(maleicanhydride-alt-1-octadecene), poly(ethyleneimine) or alendronate, then characterised for core size, hydrodynamic size, surface potential and relaxivity. Anti-CX3CL1 targeting antibody was coupled to nanoparticles through carbodiimide coupling chemistry. In vivo studies were performed in ApoE-/- mice on western diet instrumented with a cuff on the left carotid on 3T Phillips Achieva, with control mice ApoE-/- on chow diet with no surgery. Mice were scanned with a novel SPION contrast agent, and an elastin-targeted gadolinium agent. Histological analysis was undertaken to confirm imaging findings through staining for macrophages, CX3CL1, elastin, tropoelastin, and iron.
The lead SPION agent consists of a 10 nm iron oxide core with poly(maleicanhydride-alt-1-octadecene), -36.21 mV, 18.806 mmol-1/s-1. The irregular faceting of the iron oxide core results in high relaxivity compared to a more spherical nanoparticle, and PMAO is a coating which has not yet been widely explored in contrast agent chemistry. It provides a foundation for further functionalisation by surface -COOH groups. The overall properties of the contrast agent are promising, with a negative surface charge and a hydrodynamic size which should maximise circulation time and evade rapid clearance through the renal system or phagocytosis.
In vivo results show that SPIONs accumulate well in plaque with a significant difference between the instrumented diseased vessel and both the unmodified vessel in diseased animals, and healthy control mice. Proof-of-concept scans indicated that the contrast agent was still in circulation 24 hours post-injection and did not show elevated signal in the liver.
This work presents a new MRI contrast agent for atherosclerosis which uses an under-explored surface ligand, demonstrates promising properties for in vivo behaviour, is still in circulation 24 hours post-injection with limited liver uptake, and shows good accumulation in plaque. Further work for this project includes in vitro studies and radiolabelling of the nanoparticle for biodistribution studies.
1. Cheng, C. (2007). Journal of Clinical Investigation, 117 (3), 616-626
This project is funded as part of the EPSRC Centre for Doctoral Training in Medical Imaging at Imperial College London and King’s College London.
Illustration of in vivo results demonstrating SPION uptake
1) Lumen area vs slice number 2) MRI images of ESMA signal superimposed on angiogram, anatomical cross-section of cuff, transverse slice above aortic arch showing signal voids arising from SPION uptake 3) Plaque volume as measured by SPIONs and ESMA above and below the cuff 4) Plaque volume as measured by SPIONs in the LCA and RCA of diseased and healthy animals 5) Similar graph using ESMA
Keywords: iron oxide nanoparticles, MRI, atherosclerosis, vulnerable plaque
|1:36 p.m.||PS 02-06||
Giant liposomes as a new versatile Molecular Imaging platform (#165)
Martina Tripepi1, Giuseppe Ferrauto1, Paolo O. Bennardi1, Marta Gai1, Daniela Delli Castelli1, Silvio Aime1
1 University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy
The last ten years have witnessed the development of a large variety of nanosystems useful as therapeutic and diagnostic carriers, for applications in Molecular Imaging1. The huge contrast agents' payload delivered at a specific target from a nano-carrier drastically increase the sensitivity of the imaging techniques. Moving from nano- to micro- systems the volume of the vesicles would enormously increase, thus pushing further the sensitivity limits of imaging diagnostic modalities. For this reason, we chose to explore the potential of giant liposomes (GUVs) as a multi-modal imaging platform.
Giant liposomes have been prepared according to an optimisation of the “gentle swelling” method reported elsewhere2, in order to design the most suitable probe for Molecular Imaging purposes. The hydration solution consisted of a paramagnetic shift reagent (e.g. Tm-, Gd-, Dy-HPDO3A) and/or a fluorescent dye (carboxyfluorescein) while the lipidic membrane contained DPPC and a targeted or fluorescent phospholipid when appropriated. T1, T2 and CEST characterization has been performed at different magnetic fields. Folate receptor-based targeting experiments on IGROV-1 cells have been achieved and monitored with fluorescence microscopy. Moreover, a carboxyfluorescein-containing formulation was evaluated as an extremely sensitive liposome-based signal amplification microsystem.
GUVs have been successfully prepared and underwent dimensional, T1, T2, HR-NMR (Fig. 1) and CEST characterization. The use of micrometric vesicles (ca. 1.5 µm), endowed with a mean diameter ten times greater than nanosized ones (ca. 150 nm), lead to increase the inner water volume of one thousand times. Consequently, T2 and CEST sensitivity per particle has been increased of three orders of magnitude. In cellulo and in vivo toxicity has been evaluated and the same very low toxicity as nanosized liposomes has been found. When functionalized with targeting moieties on the external surface, fluorescent GUVs are not internalized into cells even if they can bind cells membranes, as shown in the case of folate receptors on IGROV-1 cells (Fig. 2). Particularly interesting is the use of carboxyfluorescein-loaded GUVs for applications based on the evaluation of the content release. Indeed, in this way the detection sensitivity reaches values as high as tens of attomolar of vesicles in solution.
A new versatile Molecular Imaging microsystem has been developed by using giant liposomes as carriers of paramagnetic shift reagents or fluorescent dyes. It showed to be the most sensitive CEST agent ever reported in literature and appeared to be a good candidate for targeting experiments as it does not internalize into cells. It also showed to be a promising signal amplification tool in the attomolar range of detection.
1. Lammers T. et al, Chem. Rev. 2015; 115, 19, 10907-10937
2. Reeves J.P. et al, J. Cell. Physiol 1968; 73: 49-60
Fig. 1: HR-NMR and fluorescence microscopy characterization of a spherical CEST giant liposome.
HR-NMR spectrum, A) shows the sharp signal of the bulk water and the signal of intraliposomal water which is shifted and enlarged due to the presence of a paramagnetic shift reagent. In B) a fluorescence microscopy image of the same spherical giant liposome is reported.
Fig. 2: Binding of folate-targeted giant liposomes on IGROV-1 cells.
Fluorescent microscopy images of the binding of folate-targeted fluorescent giant liposomes carrying rhodamine in membrane (red) and carboxyfluorescein inside (green) on IGROV-1 ovaric cancer cells (blue) overexpressing folate receptors. IGROV-1 were incubated with targeted giant liposomes for 30 minutes at 37°C and then washed several times with PBS.
Keywords: Giant liposomes, CEST, Fluorescence, Microsystem, Folate receptor
|1:48 p.m.||PS 02-07||
19F Hot Spot MRI of Fluorocapsules Containing Human Islet Cells for Treatment of Type I Diabetes (#194)
Dian Arifin1, Genaro Juarez-Paredes1, Paul de Vos2, Jeff Bulte1
1 Johns Hopkins University School of Medicine, Dept. of Radiology, Baltimore, Maryland, United States of America
Despite promising preclinical studies, clinical trials of encapsulated pancreatic islets for treatment of Type I diabetes have lacked long-term efficacy1-4. Persistent challenges include low or short-term islet survival in vivo and the lack of suitable means to probe the fate of implanted microcapsules after transplantation. We have developed novel mixed-alginate gradient (MAG) immunoprotective microcapsules with a liquid core, which facilitates rapid diffusion of oxygen and nutrients into the capsules, and loaded them with the 19F tracer CS-1000 in order to visualize them with 19F MRI.
MAG “fluoro”capsules were synthesized using a homemade electrostatic generator. Human islets were obtained from donor cadavers. Viability of islets was assayed using Propidium Iodide/Newport Green (dead/live) staining. Varying amounts of CS-1000 were co-encapsulated to optimize MRI sensitivity, mechanical strength, and morphology of the fluorocapsules. To determine the number of encapsulated 19F atoms, MAG fluorocapsules were ruptured with 0.5 M EDTA, pH=8.0 and then measured using a 400 MHz Varian NMR. MAG fluorocapsules (without islets) were transplanted intraperitoneally (IP, 300 fluorocapsules) or subcutaneously (SC, 50 fluorocapsules) into C57BL/6 mice. MRI was performed using a 17.6 T vertical bore scanner and a dual-tunable 19F/1H birdcage resonator.
The viability of human islets encapsulated inside novel MAG microcapsules (without CS-1000) was ~25% higher than that of islets encapsulated inside traditional capsules with a homogeneous alginate content gradient (Fig. 1a,b). MAG fluorocapsules contained 4-6 X 1016 19F atoms per fluorocapsule, which could be clearly visualized as hot spots on 19F MRI when transplanted IP or SC into mice (Fig. 2).
The ability to use 19F MRI to visualize MAG fluorocapsules may enable physicians to observe fluorocapsules during infusion, to confirm implantation at the correct target site, and to non-invasively monitor the dispersion of fluorocapsules without the complication of background artifacts as typically encountered in 1H MRI, in particular for the abdominal region.
Keywords: Diabetes, Cell therapy, 19F MRI, Microcapsule