Abstract/Video opens by clicking at the talk title.
Abstract/Video opens by clicking at the talk title.
|10:00 a.m.||PS 14-01||
Introductory Lecture (#673)
1 Weizmann Institute of Science, Rehovot, Israel
|10:18 a.m.||PS 14-02||
19F MR-based Quantitative Method for Determination of Ca(II) Using Lanthanide Complexes (#118)
Giuseppe Gambino1, Tanja Gambino1, Rolf Pohmann2, Goran Angelovski1
1 Max Planck Institute for Biological Cybernetics, Magnetic Resonance Neuroimgaing Contrast Agents - Dep. of Physiology of Cognitive Processes, Tübingen, Baden-Württemberg, Germany
Determining non-invasively the absolute quantification of endogenous ions would be a substantial step forward in the investigation of numerous biological disorders and metabolic functions. Here we present a method for assessing extracellular Ca(II) concentration using 19F chemical shift imaging (CSI), which holds great potential for molecular imaging applications. Specifically, the Ca(II)-responsive and lanthanide-based 19F MRI probe LnL, that undergoes a strong and highly specific modulation of its signal upon the coordination of Ca(II) ions, was developed and its performance is presented.
19F NMR Ca(II) titrations were performed at 7T and 25 °C using a 50 mM CaCl2 solution and solutions of DyL or YL with starting [Ln(III)] = 5.0 mM in HEPES (pH 7.4). A coaxial capillary, containing 100 mM NaF was used as reference. 19F NMR spectra were acquired after every CaCl2 addition. The ratiometric 19F NMR titrations were performed on three solutions containing DyL mixed with YL in different ratios (1:1, 7:3, 9:1, [DyL] = 2.0 mM) in HEPES (pH 7.4) with CaCl2. MRI measurements were performed at 7T and room temperature. The samples 1-6 contained 5 mM DyL + 0.5 mM YL in HEPES (50 mM) at pH 7.4, mixed with 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 equivalents of CaCl2. 19F MR images were acquired using 2D CSI pulse sequence with total acquisition time = 60 min 44 sec.
The 19F NMR spectra of DyL and YL displayed a single resonance at -72.7 and -70.4 ppm respectively, which is a very advantageous and desired, but unusual, property of most of Ln(III) probes. The obtained SNR of the 19F NMR signal undergoes a massive 10-fold decrease upon Ca(II) coordination in the case of DyL, while remaining unchanged for YL. By having Ca(II)-independent or -dependent 19F NMR signals for YL and DyL, respectively, we were able to quantify variations in [Ca(II)] by using the signal of the former as the internal reference for changes in signal of the latter probe. To do so, we titrated a mixture of DyL and YL in three different ratios with CaCl2, maintaining constant [DyL] (Fig.1). To further explore the potential of this method, we performed 19F CSI measurements in a 7T MRI scanner. After data analysis, we were able to obtain a voxel-wise map of the absolute [Ca(II)] expressed in mM, which was in excellent agreement with the actual [Ca(II)] in each sample (Fig.2).
Here we present a very promising ratiometric method for the absolute quantification of [Ca(II)] by means of 19F CSI. This method tackles common drawbacks associated to the application of MRI responsive probes and resolves them very effectively. This new approach offers very good perspectives for performing quantitative molecular imaging studies as well as development of further methods for different ionic and molecular targets.
AcknowledgmentThe financial support of the German Research Foundation (DFG, grant AN 716/7-1), the German Federal Ministry of Education and Research (BMBF, e:Med program: FKZ: 01ZX1503) and the German Academic Exchange Service (DAAD, Ph.D. fellowship to T.G.) is gratefully acknowledged.
19F NMR titration experiments with DyL and YL
a) 19F NMR spectra of YL (left) and DyL (right) in presence of increasing [Ca2+] (from 0 to 2.0 equiv.) measured at 7T and 25 °C ; b) Signal intensity values for ADy (blue area in the panel a left) and AY (red area in panel a right) plotted as a function of increasing [Ca2+]; c) Average values for ADy/AY plotted against the normalized [Ca2+] for a set of three samples containing DyL and YL in a ratio of 50:50, 75:25 and 90:10 ([Dy] = 2.0 mM), measured at 7T and 25 °C.
19F CSI for quantification of calcium concentration
a) Normalized CSI image of ADy/AY in phantom tubes 1-6, containing 5 mM DyL + 0.5 mM YL in HEPES (50 mM) at pH 7.4, mixed with 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 equivalents of CaCl2, respectively; b) Quantitative [Ca2+] map obtained by fitting the ratiometric data.
Keywords: 19F, calcium, MRI, ratiometry, MRS
|10:30 a.m.||PS 14-03||
Multimodal detection of atherosclerosis using phospholipid-coated nanomicelles (#378)
Maria Muñoz Hernando1, 2, Paula Nogales1, Jacob F. Bentzon1, Fernando Herranz2
1 Centro Nacional de Investigaciones Cardiovasculares (CNIC), Experimental Pathology of atherosclerosis, Madrid, Spain
Atherosclerosis and its clinical complications are major constraints to living long and healthy lives. Therefore, tools capable of measuring disease activity are necessary. During this work, several types of phospholipid coated iron oxide nanomicelles (IONM) were synthesized as potential contrast agents for atherosclerosis. On the one hand, the enzyme driven accumulation of Phosphatidylcholine (PC)1 and Sphingomyelin (SPH) IONM in atherosclerotic plaque was exploited. And on the other hand, the use of Edelfosine (EDF), a phospholipid shown to bind lipid rafts2, was used.
PC-, SPH- and EDF-IONM, for T2-MRI were synthesized in a two-step nanoemulsion protocol. Firstly, hydrophobic oleic-acid-coated Fe3O4 NPs were prepared by thermal decomposition. Subsequently, using a nanoemulsion method, physiologically viable nanoparticles were made by encapsulation into micelles composed of amphiphilic PC, SPH or EDF molecules. The same protocol was used for the preparation of nanomicelles for fluorescence-based imaging except that 1mg of the fluorophore DilC18(5) was added during the nanoemulsion step.
Hydrodynamic size distributions measured by dynamic light scattering (DLS) showed a narrow size distribution for all samples. Furthermore, relaxometric values yielded r2/r1 ratios suitable for T2-weighted contrast. PC-IONM and SPH-IONM showed aggregation when incubated with their specific enzymes, PC-PLC and Sphingomyelinase, respectively. DLS showed an abrupt increase in their hydrodynamic size and core aggregation was observed by TEM imaging. In addition, EDF-IONM TEM images showed a typical nanomicelle structure composed of several hydrophobic nanoparticles.
We have fully characterized PC-, SPH- and EDF-IONM. Furthermore, in vivo T2-MRI with PC-IONM have shown the selective accumulation of this probe in the atherosclerotic plaque. In addition, ex vivo fluorescence imaging and histology have shown the selective accumulation of PC-, SPH- and EDF-IONM in the atherosclerotic plaque, making them a suitable potential probe for pathophysiology and activity characterization.
The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)
 Mollinedo, F.; Gajate, C.; Morales, A. I.; Del Canto-Jañez, E.; Justies, N.; Collía, F.; Rivas, J. V.; Modolell, M.; Iglesias, A. Novel Anti-Inflammatory Action of Edelfosine Lacking Toxicity with Protective Effect in Experimental Colitis. J. Pharmacol. Exp. Ther. 2009, 329 (2), 439–449. https://doi.org/10.1124/jpet.108.148254.
 Lechuga-Vieco, A. V.; Groult, H.; Pellico, J.; Mateo, J.; Enríquez, J. A.; Ruiz-Cabello, J.; Herranz, F. Protein Corona and Phospholipase Activity Drive Selective Accumulation of Nanomicelles in Atherosclerotic Plaques. Nanomedicine Nanotechnology, Biol. Med. 2018, 14 (3), 643–650. https://doi.org/10.1016/j.nano.2017.12.021
PC-IONM accumulation in the aorta
(a) In vivo MRI of the aorta an ApoE−/− mouse before and 24 h after injection of PC-IONM. NM accumulation denoted by white dot; (b) Histogram showing relative MRI signal change in aortic plaque areas identified in circular ROIs surrounding the lumen; (c) Ex vivo fluorescence image of C57BL/6 (control) and ApoE−/− mice aortas extracted 24 h after injection of Dil-PC-IONM.
Keywords: atherosclerosis, nanomicelles, MRI, Fluorescence imaging, phospolipids
|10:42 a.m.||PS 14-04||
Ligand mediated crystallographic defects in nanofluorides for improved in vivo 19F-MRI sensitivity (#470)
Reut Mashiach1, Hyla Allouche-Arnon1, Dana Cohen1, Lothar Houben2, Amnon Bar-Shir1
1 Weizmann Institute of Science, Organic Chemistry, Rechovot, Israel
Nano-sized inorganic fluoride nanocrystals (NCs) may offer an alternative3 to the commonly used perfluorocarbon nanoemulsions (100-200nm)1,2 where small-sized (<10 nm) nanoformulations are desired for 19F-MRI. However, their long T1 values (10s) require very long acquisition times. Here, we propose a lanthanid-free approach for shortening the T1 of CaF2 NCs for improved 19F-MRI sensitivity. By introducing structural defects in 19F-NCs we were able to shorten their T1 by tenfold, resulting in a dramatic improvement in their SNR and ability to detect them in vivo with short acquisition times.
Oleic acid (OA) and oleyl phosphate (OP) CaF2 NCs were synthesized followed by phospholipid incorporation to obtain water—dispersed NCs that were characterized by TEM. 19F-NMR and 19F-MRI experiments were performed on 9.4T MR spectrometer; 1H-MRI and 19F-MRI: 3D ultrashort TE (3D-UTE) protocol, flip angle 10o, TR/TE=16.8/0.03 ms, FOV=3.2×3.2×3.2 cm3, and NA=1. In vivo MRI experiments (brain injections) were performed 15.2 T MRI scanner; 1H-MRI and 19F-MRI 3D-UTE with a flip angle of 5o, TR/TE=5/0.03 ms, FOV=20×20×20 cm3, matrix=32×32×32, and NA=150. In vivo MRI (muscle injections) were performed on a 9.4T MRI; 1H-MRI RARE and 19F-MRI 3D-UTE protocol with a flip angle of 10o, TR/TE=16.8/0.03 ms, NA=4, 3:30 min scan time.
CaF2 NCs were synthesized with two types of ligands, i.e., oleic acid (CaF2-OA, Fig.1a) and oleyl phosphate (CaF2-OP, Fig.1b). Next, phospholipids incorporation to their coating ligands4 provided NCs with the desired water solubility that resulted in a single 19F NMR signal while in solution (Fig.1c and d). Modifying the head group of the ligand (from OA to OP) resulted in a shorter T1 by an order of magnitude, (11sec to 1.1sec, Fig.1e). These T1 variations are attributed to the differences in crystal morphology (HRTEM, Fig.1a and b in inset). While CaF2-OA presented high crystallinity of a single crystal the CaF2-OP NCs show crystallinity defects, i.e., grain boundaries within the crystal. The shorter T1 results in improved 19F-MRI sensitivity by 4-fold as compared to the longer T1 NCs (Fig.1f). The water-soluble NCs were injected to the mouse brain and muscle demonstrating their biocompatibility and potentiality to be used as paramagnetic-free, sensitive 19F-MRI nanotracers (Fig. 2).
To conclude, we showed here an approach for improving in vivo 19F-MRI sensitivity by shortening the T1 relaxation properties of 10 nm-sized CaF2 NCs by introducing crystal defects as a safe alternative to paramagnetic elements doping. The obtained lanthanide-free nanofluorides emphasizes the versatility and tunability of fluoride-NCs nano-tracers for further in vivo 19F-MRI studies.
 Ahrens, E.T. & Bulte, J.W. Tracking immune cells in vivo using magnetic resonance imaging. Nature Reviews Immunology 13, 755-763 (2013).
 Kislukhin, A.A., et al. Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging. Nature materials (2016).
 Ashur, I., Allouche‐Arnon, H. & Bar‐Shir, A.J.A.C.I.E. Calcium Fluoride Nanocrystals: Tracers for In Vivo 19F Magnetic Resonance Imaging. 57, 7478-7482 (2018).
 Cormode, D.P., et al. Nanocrystal core high-density lipoproteins: a multimodality contrast agent platform. 8, 3715-3723 (2008).
Characterization of CaF2 NCs
Figure 1. Characterization and MR properties of CaF2 NC coated with different ligands; oleic acid (CaF2-OA) and oleyl phosphate (CaF2-OP). TEM images (a,b respectively), scale bar 50nm, with high resolution TEM in inset. 19F-NMR spectra of CaF2 in water (c,d respectively). T1 relaxation time of CaF2 in water (e). Signal to noise ratio measured from 19F in vitro MRI.
In vitro and In-vivo 19F-MRI studies of CaF2 NCs
Figure 2. (a) In vitro 19F-MRI of CaF2-OP (in pink) and CaF2-OA (in blue) shown as pseudo-color maps overlaid on the 1H-MR image of the phantom containing gelatin and water. (b) In vivo 19F-MRI of CaF2-OP (injected right) and CaF2-OA (injected left) NCs in a mouse brain shown as pseudo-color maps overlaid on the anatomical 1H-MR image. (c) In vivo 19F-MRI of CaF2-OP (inected right) and CaF2-OA (injected left) NCs in a mouse muscle shown as pseudo-color maps overlaid on the anatomical 1H-MR axial image.
Keywords: 19F MRI, Nanoparticles, MRI probes
|10:54 a.m.||PS 14-05||
Multivalent glycan coating of paramagnetic nanofluorides for background-free mapping of inflammation with 19F-MRI (#382)
Dana Cohen1, Reut Mashiach1, Lothar Houben2, Hyla Allouche-Arnon1, Amnon Bar-Shir1
1 Weizmann Institute of Science, Department of Organic Chemistry, Rehovot, Israel
Developing targeted imaging tracers with improved sensitivity and specificity is a central goal of biomedical imaging in general and in mapping inflammation in particular. Here, we show that paramagnetic glyconanofluorides, composed of lactose-coated Sm3+ doped CaF2 nanocrystals (NCs), can function as small-sized (<10 nm) and sensitive nanotracers for targeted in-vivo 19F-MRI. Rationalized coating of the Sm:CaF2 NCs with lactose-based glycans, dramatically enhanced their uptake by active immune cells, allowing background-free in-vivo mapping of inflammatory activity using 19F-MRI.
Small CaF2 NCs were fabricated with the addition of paramagnetic Sm3+ dopant to obtain Sm:CaF2 NCs using the solvothermal synthetic approach. To target Sm:CaF2 uptake into immune cells, the surface of the hydrophobic NCs was coated with phospholipids (PLs), using lactose-modified and fluorescently-labeled PLs for further FACS analysis. These biocompatible NCs were characterized by TEM/EDS elemental mapping and for their T1 relaxation times by 19F-NMR. Mice were immunized by footpad injection of immunogenic emulsion, FACS analysis and MRI data sets were collected 10 days post immunization following subcutaneous injection of the NCs to the footpad. MRI experiments were performed on 15.2T MRI scanner using UTE-3D sequence for 19F images with TR= 4ms, TE= 8µs,FOV=4x2.5cm2,matrix size = 32x32.
Schematic illustration of the synthetic route at which hydrophobic nanofluorides are modified to obtain glyconanofluorides (GNFs) shown in Fig.1a.
The paramagnetic nanofluorides introduced here, offer a small-sized inorganic NC-based approach for the synthesis of glycomimetic nanoformulations, with improved sensitivity and enhanced specificity to immune cells. This imaging platform could be used for non-invasive imaging of immune cells, and provide quantitative in-vivo insights regarding their migration and localization and could be further applied to study inflammation-associated diseases.
Design and Characterization of glycan coated paramagnetic NCs
Figure 1: a. Schematic representation of phospholipid (PL) coating the surface of oleate-coated Sm:CaF2 NCs. b. HR-STEM/HAADF indicates the Sm3+ ions are part of the NC. c. T1 bar-graph of Sm:CaF2, La:CaF2 and CaF2 NCs resulted from inversion recovery experiment. d. 19F-MRI phantoms of PL-coated CaF2, La:CaF2 and Sm:CaF2 NCs and e. SNR column of the studied solutions shown in d.
In vivo FACS and 19F-MRI of inflammation in a mouse model
Figure 2: a. Schematic illustration of the in vivo experimental setup at which both types of nanofluorides, i.e., +Lac, left side or –Lac, right side, were injected to inflamed mice footpads. b. FACS analysis of cells excised from lymph nodes 2 hrs post injections of the nanofluorides described in a. and bar-graphs of fluorescent activity (Rhodamine) excised lymphatic cells (for nanofluorides accumulation). c. from left to right are - 1H-MRI ,19F-MRI and 1H/19F MRI overlay of a representative mouse and 19F-MRI SNR bar-graphs.
Keywords: 19F-MRI, Nanofluorides, Targeting, Inflammation, In-vivo
|11:06 a.m.||PS 14-06||
Perfluorocarbon-loaded Polymeric Nanoparticles for 19F MRI and Multimodal Imaging: From Functional Control to Clinical Use (#98)
Olga Koshkina1, Esmee Hoogendijk2, Edyta Swider-Cios2, Alexander H. J. Staal2, Nicolaas K. van Riessen2, Paul B. White3, Christophe A. Serra4, Andreas Riedinger1, Katharina Landfester1, Mangala Srinivas2
1 Max Planck Institute for Polymer Research, Physical Chemistry of Polymers, Mainz, Germany
Perfluorocarbon (PFC)-loaded nanoparticles (NPs) are powerful imaging agents for 19F MRI and multimodal imaging. Controlled synthesis of PFC-loaded NPs and scaling up the production are essential for clinical translation. However, it remains a challenge, as PFC are both hydrophobic and lipophobic. We present microfluidic miniemulsion synthesis of PFC-loaded poly(lactic-co-glycolic acid) (PLGA) NPs. We further highlight general aspects in design of NPs that affect in vivo biodistribution and imaging performance, focusing on internal structure, surface modification and colloidal stability.1,3
Microfluidic miniemulsion setup consisted of 2 SIMMV2 micromixers (Micro4Industries), flow sonifier (Branson) and 3 HPLC pumps (50 mL/min head). Perfluoro15-crown-5 ether (PFCE) or perfluorooctyl bromide (PFOB) were encapsulated in PLGA at different flow rates. The solvent was evaporated. NPs were washed and freeze-dried.
Microfluidic miniemulsion method allows for continuous-flow production of PFC-PLGA NPs with 30 times higher product yields compared to conventional sonication and negligible variation between fractions (Fig. 1a, b). The properties of NPs can be tuned by adjusting the flow rate and type of the organic solvent, resulting in PFC-loading of 20-60 wt-%. Moreover, encapsulation of additional cargo is feasible. NPs are biocompatible and can be imaged with19F MRI and multimodal imaging including ultrasound (Fig. 1c).2
Miniemulsion technique enables tailoring the properties of multimodal PFC-loaded imaging agents and can be scaled up with microfluidics. Multicore PFCE-NPs display fast clearance in vivo, which is advantageous for clinical translation. Overall, our results demonstrate the necessity to precisely design the chemical properties of PFC-based imaging agents. Thus, our findings will facilitate the clinical translation of 19F MRI agents in the future.
AcknowledgmentERC-2014-StG-336454-CoNQUeST, Max Planck-University of Twente Center for Complex Fluid Dynamics, TTW-NWO open technology grant (STW-14716)
 Koshkina, O., Lajoinie G., Baldelli Bombelli, F., Cruz, J.L., White, P.B., Schweins, R., Dolen, Y., van Dinther, E.,Voets, I.K., Rogers, S.E., van Eck, E., Heerschap, A., Versluis, M., de Korte, C.L., Figdor, C.G., de Vries, I.J.M., Srinivas, M. 2019 "Multicore liquid perfluorocarbon-loaded multimodal nanoparticles for stable ultrasound and 19F MRI applied to in vivo cell tracking." Adv. Funct. Mater. 29, 1806485.
 Koshkina, O., Srinivas, M., de Vries, I. J. M., Figdor, C. G. 2016 "Process for preparation of beads for imaging" Pending patent application, filed March 11.
 Staal, A.H.J., Becker, K., Tagit, O., Cortenbach, K., van Riessen, N. K., Koshkina, O., Veltien, A., Bouvain, P., Scheenen, T., Flögel, U., Temme, S., Srinivas, M. 2019 "Single resonance, disassembling 19F magnetic resonance imaging nanoparticles for clinical applications with fast local and systemic clearance" Nat. Commun, under consideration.
 Jacoby, C., Temme, S., Mayenfels, F., Benoit, N., Krafft, M.P., Schubert, R., Schrader, J., Flögel, U. 2014 "Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half-lives and sensitivity" NMR Biomed. 27, 261-71.
Multimodal PFCE-PLGA nanoparticles: from the bench towards the clinics
(a) Microfluidic production of PFC-loaded NPs with structures of components (left), the photograph of microfluidic chip (slit interdigital micromixer, middle) and schematic of multicore PFCE-PLGA NPs (right) (b) Characterization of NPs produced during a long run. Left: DLS and NMR of NP fractions Right: SEM image of NPs. (c) In vitro imaging shows that nanoparticles can be imaged with ultrasound and confocal microscopy (NPs red, after cell uptake, scale bar 25 µm). (d) In vivo 19F MRI of PFCE-PLGA NPs. NPs were first found in the liver and spleen and cleared after 2 weeks. N=10, 11.7 T.
Keywords: 19F MRI, multimodal imaging, microfluidics, polymeric nanoparticles
|11:18 a.m.||PS 14-07||
Novel quadrupolar peaks based contrast agents for monitoring tissue implants (#373)
Valeria Bitonto1, Simona Baroni1, Rachele Stefania1, Maria Rosaria Ruggiero1, Enza Di Gregorio1, Giampaolo Placidi1, Lionel C. Broche2, David Lurie2, Silvio Aime1, Simonetta Geninatti Crich1
1 University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy
This study aims at developing an innovative class of MRI contrast agents for Fast Field Cycling-MRI applications. They represent a completely new class of MRI contrast agents that display remarkable relaxation effects on tissue water protons. Their detection requires the acquisition of images at variable magnetic field strength as provided by Fast Field Cycling MRI (FFC-MRI) scanners. FFC is an innovative technology that allows detecting the quadrupolar cross-relaxation, appearing as peaks (QPs) in the 1/T1 dispersion profile completely invisible to conventional (fixed-field) MRI1.
PLGA scaffolds were prepared by dissolving PLGA conjugated with polyhistidine (n=15) in tetraglycol with glucose to create porous scaffolds. The mixture was then injected into PBS, where it precipitated by phase inversion to form a solid scaffold as the tetraglycol diffused into the PBS and allowed the porogen to be leached from the scaffold to form a porous structure. MC3T3-E1 cells (subclone 4), a pre-osteoblastic cell line derived from mouse calvaria, were seeded and cultured inside the porous scaffolds in vitro to investigate their ability to adhere to and proliferate on this material. NMRD profiles were acquired on a Fast-Field Cycling relaxometer (SmartTracer , Stelar S.r.l., Mede (PV)) with a microcoil (diameter 6 mm).
PLGA scaffolds containing poly-Histidine of different sizes show QP at 1.35 MHz due to the 14N nuclear quadrupole resonance frequency of the imidazole groups present on the polymeric chains. This QP falls at a frequency well distinguishable from the endogenous ones and therefore it may be used as a new class af frequency-encoded specific sensor.
In this study we exploited this technique for in vivo study of tissue implants. In fact, to date there is an almost complete lack of methods for the rapid, non-invasive and repeated monitoring of tissue implants and new methods are needed to monitor cell status and polymer degradation under physiological conditions (temperature, saline, pH, enzymes etc.) thus allowing the physician to control, in real time, the transplanted scaffold status.
 C. Gösweiner et al. Tuning Nuclear Quadrupole Resonance: A Novel Approach for the Design of Frequency-Selective MRI Contrast Agents, Phys. Rev. X 8, 021076. 1-20 (2018)
 S. Geninatti Crich, S. Aime, R. Stefania, S. Baroni, M.R. Ruggiero, L. Broche, D. Lurie "Nuovi agenti di contrasto per risonanza magnetica per immagini" , Patent number: 102019000007647 (2019)
Keywords: NMRD, contrast agents, tissue implants