15th European Molecular Imaging Meeting
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MRI | Probe Chemistry

Session chair: Andreas Maurer (Tuebingen, Germany); Dominik von Elverfeldt (Freiburg, Germany)
 
Shortcut: PW26
Date: Friday, 27 August, 2021, 10:45 a.m. - 12:15 p.m.
Session type: Poster

Contents

Click at talk title to open the abstract

800

In vivo animal study of the excretion time of a macrocyclic Gadolinium Based Contrast Agent and the specific retention in bladder, spleen and bone

Chiara Furlan1, Enza Di Gregorio1, Fiammetta Gatto1, Valeria Bitonto1, Silvio Aime1, Eliana Gianolio1

1 University of Turin, Molecular Biotechnology Center, Turin, Italy

Introduction

Gadolinium based contrast agents (GBCAs) are commonly employed at clinical settings to add relevant information to the anatomical resolution of the magnetic resonance images.1 In recent years, concern on the use of GBCAs has raised as it has been found that tiny amount of Gd can be retained in brain and other tissues, also in patients without renal dysfunctions.2-5 Whereas much work has been carried out to investigate the issue of Gd-retention in the brain, the aim of this work is to bring the attention to tissues less considered in the past such as bladder, spleen and bones.

Methods

In order to evaluate the amount of Gd retained in the tissues, mice were administered with 20 doses of 0.6 mmol Gadoteridol/kg over a period of 4 weeks. The sacrifice time was set at 4 different time points (4, 15, 30 and 90 days) after the last injection. After sacrifice, urine, tissues and organs were collected. One tibia was weighted, mineralized and Gd quantified through ICP-MS. The other tibia was handled in order to separate bone matrix (compact bone and periosteum) and bone marrow and Gd quantified separately. Spleen was processed as well, to measure the amount of Gd in the splenocytes and in its fibrous part. In bladder, beside ICP-MS total Gd quantification, UPLC-MS was performed to study the chemical form of the retained metal.

Results/Discussion

The quantification of Gd in the bladder showed the highest amount of metal retained, especially at the shortest times, among all the investigated organs. Urine samples were also analysed to determine the Gd concentration and the rate of elimination over time. Urine Gd concentration rapidly decreased over time to suggest that most of the administered GBCA is correctly excreted through the renal route (Figure 1A). In addition, the amount of Gd found in the spleen decreased over time, and, quite surprisingly, the metal found in the fibrous part was higher than that found in the splenocytes (Figure 1B). The quantitative analysis of the whole tibia showed a constant quantity of metal retained until 90 days after the last administration. The separate analysis of bone marrow and bone matrix revealed that most of Gd was retained constantly by the bone matrix, while a very low, and time decreasing, amount of metal was found in the bone marrow (Figure 1C).

Conclusions

Our results point out that bladder could be an extremely specific organ for the metal retention. This result is likely related to the passage and stasis of urines, which contain a very large amount of GBCA starting from the hours immediately after the administration.  The analysis on bones showed a very quick deposition of Gd: as a matter of fact, the excretion rate is very low, in particular in the bone matrix, where most of Gd is retained.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Yousaf T, Dervenoulas G, Politis M. Advances in MRI Methodology. In: International Review of Neurobiology.Vol 141. Academic Press Inc.; 2018:31–76.
[2] 2. Errante Y, Cirimele V, Mallio CA, et al. Progressive increase of T1 signal intensity of the dentate nucleus on unenhanced magnetic resonance images is associated with cumulative doses of intravenously administered gadodiamide in patients with normal renal function, suggesting dechelation. Invest. Radiol. 2014;49(10):685–690.
[3] Kanda T, Ishii K, Kawaguchi H, Kitajima K, Takenaka D. High signal intensity in the dentate nucleus and globus pallidus on unenhanced T1-weighted MR images: Relationship with increasing cumulative dose of a gadoliniumbased contrast material. Radiology. 2014;270(3):834-841. 
[4] Schlemm L, Chien C, Bellmann-Strobl J, et al. Gadopentetate but not gadobutrol accumulates in the dentate nucleus of multiple sclerosis patients. Mult Scler. 2017;23(7):963-972.
[5] Di Gregorio E, Ferrauto G, Furlan C, et al. The issue of gadolinium retained in tissues: insights on the role of metal complex stability by comparing metal uptake in murine tissues upon the concomitant administration of lanthanum- and gadolinium-diethylentriamminopentaacetate. Invest Radiol. 2018;53:167-172.
Figure 1
Amount of Gd 4, 15, 30, and 90 days after the last administration of Gadoteridol (20 doses of 0.6 mmol Gadoteridol/kg over a period of 4 weeks) in: A) Bladder and Urine; B) Spleen and its fibrous part; C) Bone and bone matrix.
Keywords: Gadolinium, Retention, Bladder, Spleen, Bone
801

Producing High-Performance Iron Oxide Nanoparticlesfor Magnetic Particle Imaging

Seyed M. Dadfar1, Dennis Pantke1, Volkmar Schulz1, Fabian Kiessling1, Twan Lammers1

1 Faculty of Medicine, RWTH Aachen University, Institute for Experimental Molecular Imaging, Aachen, Germany

Introduction

Over the last decade, superparamagnetic iron oxide nanoparticles (SPION) have been extensively tested for magnetic particle imaging (MPI). MPI is an emerging tomographic imaging technique -currently in the preclinical stage- that enables direct spatial and temporal tracking of SPION in vivo. SPION prepared from thermal decomposition of organometallics in the presence of surfactants are optimal in size, shape, composition and crystallography. After hydrophilization and transfer to aqueous phase, SPION can be used for various biomedical applications, including MPI.

Methods

However, SPION with different properties can be produced via different known conditions suggested in the literature; the existence of impurities in the produced nanoparticles leaves adverse effects, making them unsuitable for biomedical applications. In many cases, the purification procedure outlined in the literature is not well described, or even worse, is not optimized. The current study relates to the development of a purification method during the preparation of SPION, resulting in the production of high-performance nanoparticles for MPI applications. Here, we have used the thermal decomposition of iron precursor technique to produce oleic acid-coated SPION and modified their surface with pluronic F127 to make the nanoparticles suitable for biomedical applications (Figure 1).

Results/Discussion

The prototypic sample is labeled as “PS” (Figure 2A). Analyzing the SNR values of the third harmonic, which contains the strongest signal, showed an increased SNR for the PS sample compared to Resovist® and Perimag® (8.2 times higher than Resovist® and 3.4 times higher than Perimag®). MPI phantom images were acquired with the PS sample as well as Perimag® to assess the image quality that can be obtained with these SPION. The phantom images and line profiles are shown in Figure 2B-C. Observing the images of the E- and V- phantoms, one can clearly see that the PS sample provides better spatial resolution compared to Perimag®. These findings indicate that refining SPION production and purification is important to achieve optimal MPI performance.

Conclusions

The present work seeks to provide alternative methods of purifying precursor as well as oleic acid-coated SPION which could substantially ameliorate the deficiencies of the prior methods. The method proposed in our study is to wash precursor several times with plenty of hot water and ethanol for complete removal of impurities. Also, we established a protocol made of 3 steps for accurate and undoubted purification of the final SPION.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Dadfar, S.M., et al., Iron oxide nanoparticles: Diagnostic, therapeutic and theranostic applications. Advanced drug delivery reviews, 2019. 138: p. 302-325.  
[2] Dadfar, S.M., et al., Size-isolation of superparamagnetic iron oxide nanoparticles improves MRI, MPI and hyperthermia performance. Journal of nanobiotechnology, 2020. 18(1): p. 1-13.
[3] Unni, M., et al., Thermal decomposition synthesis of iron oxide nanoparticles with diminished magnetic dead layer by controlled addition of oxygen. ACS nano, 2017. 11(2): p. 2284-2303.
[4] Cotin, G., et al., Unravelling the thermal decomposition parameters for the synthesis of anisotropic iron oxide nanoparticles. Nanomaterials, 2018. 8(11): p. 881.
Figure 2.

(A) Transmission electron image of the PS sample, prepared by our presented protocol. MPI images reconstructed based on E- and V-shaped phantoms filled with (B) the PS sample and (C) Perimag®. Iron concentration was 2.5 mg/ml.

Figure 1. Schematic overview of the synthesis of hydrophobic SPION by thermal decomposition method.

A. Preparation of iron (III) oleate precursors (P-1 or P-2); B. Synthesis of oleic acid-coated nanoparticles at high temperature from P1 precursor (C-x) and from P-2 precursor (S-x or PS-x); C. Coating oleic acid-coated SPION with Pluronic F127.

Keywords: Superparamagnetic iron oxide nanoparticles, SPION, Magnetic particle imaging, MPI, Thermal decomposition
802

Novel phosphorous-based polymer for 31P-magnetic resonance imaging

Natalia Ziółkowska1, 2, Lucie Woldřichová3, Martin Vít4, 2, David Červený5, 2, Olga Šebestová Janoušková3, Richard Laga3, Daniel Jirák2, 1

1 Charles University, First Faculty of Medicine, Institute of Biophysics and Informatics, Prague, Czech Republic
2 Institute for Clinical and Experimental Medicine, Department of Computed Tomography, Magnetic Resonance Imaging, and Clinical and Experimental Spectroscopy, Prague, Czech Republic
3 Czech Academy of Sciences, Institute of Macromolecular Chemistry, Prague, Czech Republic
4 Technical University of Liberec, Faculty of Mechatronics Informatics and Interdisciplinary Studies, Liberec, Czech Republic
5 Technical University of Liberec, Faculty of Health Studies, Liberec, Czech Republic

Introduction

Phosphorus in vivo MR suffers from high natural signal background. Our metal-free probe, based on phosphorus-containing polymer, carry phosphorothioate group (pTMPC), which is extremely rare in living organisms [1] and ensure chemical shift of the 31P-MR signal from the reference with a phosphoester group (pMPC), which is commonly present in biological phosphorus-containing compounds. As a proof of principle, we visualized polymers at 4.7T scanner using surface 1H/31P custom-made dual coil.

Methods

Presented polymers pTMPC and pMPC were synthesized by controlled radical polymerization technique of the corresponding zwitterionic monomer. MR imaging and spectroscopy were obtained on 4.7T scanner using 1H/31P RF surface coil. 1H-MR imaging (RARE, TR/TE=2500/12ms, field of view FOV=10cm) was applied for localization of phantom (cP=100 mg mL−1). T1 (single pulse sequence, TR=200–4000ms) and T2 (CPMG spin lock sequence, TE=2–1200ms) phosphorus relaxation times were assessed. CSI sequence (TR=500ms, scan time ST=15min–3h, FOV=3.6cm, resolution 2.25x2.25x3.6mm) was used for 31P-MRI. 1H/31P-MRI overlapping and 31P image processing were obtained using imageJ software. 1H relaxation times were obtained using 1.5T relaxometer (cP=10–100 mg mL−1). Cytotoxicity was tested by AlamarBlue assay.

Results/Discussion

1H-MR T1/T2relaxation times (2510/2067.2ms) were not influenced by the pTMPC polymer. 31P-MR T1/T2 relaxation times (2018.3/119.9ms) were found to be adequate for further MR experiments. 31P-MR spectroscopy showed a chemical shift of 56.07ppm (Figure 1) between the polymer with phosphorothioate and phosphoester group, which made it possible to acquire 31P images from both phantoms separately during one CSI measurement by frequency selection (Figure 2). SNR calculated from 31P spectra results in 13.1–195.1 (ST=2min–3h) for pTMPC probe and 12.5–163.7 for its reference. Imaging analyses results in SNR of 6.3–13.6 (ST=15min–3h) and 3.1–5.4, respectively. Higher SNR in both 31P-MRS/MRI obtained from probe with phosphorothioate group favours it over the reference. Cytotoxicity testing confirms that pTMPC is not significantly influencing cells viability. High phosphorus concentration and SNR within a short scan time is beneficial for further application in various in vivo animal models.

Conclusions

A novel phosphorus-containing pTMPC contrast agent based on chemical-shift for 31P-MR showed high sensitivity and biocompatibility. Large chemical shift from biological 31P signal together with high phosphorus concentration enable clear visualization of presented polymer by 31P-MR. This together with biological properties suggest that metal-free phosphorus probe could serve as an efficient phosphorus contrast agent for in vivo application.

Acknowledgement

The authors acknowledge financial support from the Ministry of Health of the Czech Republic (grant #NU20-08-00095); Charles University, First Faculty of Medicine GA UK No 358119; Institute for Clinical and Experimental Medicine IKEM, IN00023001; Ministry of Education of the Czech Republic through the SGS project No 21332/3012 of the Technical University of Liberec.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Petkowski, JJ, Bains, W, Seager, S. Natural Products Containing ‘Rare’ Organophosphorus Functional Groups, Molecules, 2019, 24(5):866. https://doi.org/10.3390/molecules24050866
Figure 1.

31P-MR spectrum (scan time ST=3h) of pTMPC (δ=27.65ppm) and reference pMPC (δ=-28.43ppm) with a chemical shift difference between their signals Δδ=56.07ppm.

Figure 2.
31P-MR CSI measurement with a reference 1H-MRI of pTMPC and pMPC. (A) 1H-MRI of pTMPC on the left and pMPC on the right with water reference between them. (B, C) 31P-MR CSI measurements (scan time ST=3h) of pTMPC and pMPC, respectivelly. (D) Overlapped 1H/31P-MRI of pTMPC and pMPC; phosphorus signal is highlighted by green color. The scale bar represents 10mm.
Keywords: 31P-MR, MR contrast agent, phosphorus-based polymer
803

Development of bimodal contrast agents for paraCEST and 19F MRI

Pierre Ernotte1, Céline Henoumont1, Sophie Laurent1, 2

1 University of Mons, Mons, Belgium
2 Center for Microscopy and Molecular Imaging (CMMI), Gosselies, Belgium

Introduction

Magnetic Resonance Imaging (MRI) is a widely used imaging technique and it often requires the use of contrast agents to increase its sensitivity. Several classes of contrast agents are under development, as paraCEST and 19F MRI contrast agents. In this work, bimodal agents active in both paraCEST and fluorine MRI are synthesized. The paraCEST complexes synthesized are derivatized by adding fluorine atoms. The interaction between the fluorine atoms and the paramagnetic ions can generate an increase in 19F MRI sensitivity.

Methods

DOTAM derivatives were chosen as paraCEST ligands as they are well known to reduce the innersphere water exchange rate. Bis-trifluoro benzylamine was then grafted to the chelate using a lysine derivative. The organic ligands were completely synthesized using cyclen as starting material. The ligands were finally complexed with thulium, ytterbium and europium. To characterize the CEST efficiency, Z-Spectra were recorded using a 679Hz saturation pulse, at 37°C, pH 7,4 and 14 T. To evaluate the efficiency in 19F MRI, 19F relaxation times measurements were performed at 11,75 T, 37°C and at pH 7,4.

Results/Discussion

The ligands were characterized by NMR and ESI-MS (figure 1). The bimodal ligand shows a good water solubility, it was then complexed with several lanthanide ions: thulium, europium and ytterbium. The europium complex shows an expected great CEST signal at around 50ppm, which is due to the coordinated innersphere water molecule. Thulium and ytterbium complexes demonstrate two CEST signals, probably due to the two types of amides present on the chelate. 19F relaxation times measurements were then performed before and after complexation to evidence the influence of the paramagnetic ion on those relaxation times. It was observed that europium has a small impact while ytterbium, and more specifically thulium have a greater influence.

Conclusions

The complexes were successfully synthesized and characterized. The europium bimodal complex exhibits a great CEST effect but has relatively long 19F relaxation times. The thulium and the ytterbium complexes both have a lower CEST effect, but reduce the 19F relaxation times more efficiently. To increase their sensitivity, the grafting on a nanoplatform is being studied.

Acknowledgement

This work was performed with the financial support of the FNRS, the ARC, the Walloon Region (Gadolymph, Holocancer and Interreg projects), the Interuniversity Attraction Poles of the Belgian Federal Science Policy Office and the COST actions. Authors thank the Center for Microscopy and Molecular Imaging (CMMI, supported by European Regional Development Fund and Wallonia).

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation

Figure 1
Structures of the different ligands synthesized
Keywords: ParaCEST, MRI, Fluorine
804

The Formation of Fractal Structure can be Used to Tailor the Features of Nanocarriers for 19F Magnetic Resonance Imaging

Margot Verbeelen2, Alexander H. Staal2, Paul B. White7, Edyta Swider-Cios2, Nicolaas K. van Riessen2, 5, Cyril Cadiou6, Françoise Chuburu6, Francesca Baldelli Bombelli3, Pierangelo Metrangolo3, Mangala Srinivas4, 5, Olga Koshkina1

1 University of Twente, Sustainable Polymer Chemistry, Enschede, Netherlands
2 Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Department of Tumor Immunology, Nijmegen, Netherlands
3 Politecnico di Milano, Laboratory of Supramolecular and Bio-Nanomaterials (SupraBioNano Lab), Department of Chemistry, Materials, and Chemical Engineering ‘‘Giulio Natta”, Milan, Italy
4 Wageningen University, Cell Biology and Immunology, Wageningen, Netherlands
5 Cenya Imaging B.V., Amsterdam, Netherlands
6 Universite de Reims, ICMR Equipe Chimie de Coordination, Reims, France
7 Radboud University, Radboud Institute for Molecules and Materials, Nijmegen, Netherlands

Introduction

Perfluorocarbon (PFC)-loaded nanocarriers are powerful agents for 19F Magnetic Resonance Imaging (19F MRI). Yet, the stabilization of PFC is a major challenge, as PFC are hydrophobic and lipophobic. As a solution, we have introduced PFC-loaded nanocarriers with a unique fractal structure.1-4 Here, we show how the fractal structure can be used to tune the MR properties, highlighting two approaches:
1. Encapsulation of two PFC to track the biodistribution and the degradation of nanocarriers.1
2. Co-loading of PFC and paramagnetic Gd-chelates to modulate the relaxation of both proton and fluorine.5

Methods

Perfluoro-15-crown-5 ether (PFCE)-loaded poly(lactic-co-glycolic acid) (PLGA) nanocarriers were synthesized with a miniemulsion technique.1
Two-color probes were obtained by co-encapsulation of PERFECTA with PFCE.3 For the loading with paramagnetic chelates, we used two hydrophobic Gd-chelates that were mixed with PLGA prior the encapsulation of PFCE. Quantitative NMR spectroscopy, 2D Heteronuclear Overhauser Exchange Spectroscopy (HOESY) and relaxation times measurements were done at Brucker Avance III 400 MHz spectrometer. 19F Solid state NMR (ssNMR) was measured on a Varian VNMRS 850 MHz spectrometer. Small Angle Neutron Scattering (SANS) was measured at sans2D-instrument (ISIS-beamline UK) and d11 instrument (ILL, France). MRI was done at Brucker scanner, 10.7 T.

Results/Discussion

The internal structure, and particularly the confinement of PFCE in fractal domains, can be used to tune the 19F MR features of nanocarriers.1-5 The co-encapsulation of PFCE and PERFECTA in fractal nanocarriers leads to a probe that changes the MR properties upon degradation.3 Thus, we have shown that the 19F relaxation times changed upon hydrolysis. Moreover, both PFC showed Nuclear Overhauser Enhancement (NOE) in the intact nanocarriers. Upon the hydrolysis, not NOE could be detected, suggesting that the distance between both PFC increased. Confinement of PFCE along with the encapsulation of paramagnetic chelates in the polymer matrix enables to tune the relaxation of PFCE.5 Interestingly, both T1 and T2 became faster with increasing concentration of Gd-chelate in the polymer matrix. This effect was not detected in conventional core-shell capsules. Moreover, the polymer matrix remained water permeable, uniquely enabling to modulate not only 19F, but also proton signal.

Conclusions

The formation of fractal structure is an efficient approach to design multifunctional 19F MRI probes, as we outlined on two examples. The encapsulation of PFCE and PEFECTA leads to nanocarriers with trackable degradation, which is particularly interesting for the drug delivery. The co-loading of paramagnetic chelates and PFCE in nanocarriers enables to tune the relaxation of PFCE and water protons, for instance, for sensing applications.

Acknowledgement

ERC-2014-StG-336454-CoNQUeST, TTW-NWO open technology grant STW-14716, ERC-2015-PoC-713524-CONQUEST, Alexander von Humboldt Foundation

Disclosure

Mamngala Srinivas and I have the followinf financial interest to disclose regarding the subject matter of this presentation: patent application EP3216464A1, PCT/EP2017/055827, WO2017153605A1, US20190077925A1, pending, first filed on 11.03.2016, liceneced to Cenya Imaging B.V.

References
[1] Koshkina, O.; Lajoinie, G.; Bombelli, F. B.; Swider, E.; Cruz, L. J.; White, P.; Schweins, R.; Dolen, Y.; Dinther, E. v.; Riessen, N. K. v.; Rogers, S. E.; Fokkink, R.; Voets, I. K.; Eck, E. R. H. v.; Heerschap, A.; Versluis, M.; Korte, C. d.; Figdor, C.; Vries, I. J. M. d.; Srinivas, M., Multicore Liquid Perfluorocarbon-loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking. Adv. Funct. Mater. 2019, 29, 1806485.
[2] Staal, A. H. J.; Becker, K.; Tagit, O.; van Riessen, N. K. v.; Koshkina, O.; Veltien, A.; Bouvain, P.; Cortenbach, K.; Scheenen, T.; Flögel, U.; Temme, S.; Srinivas, M., The Biological Half-life of Perfluorocarbon-loaded Nanoparticles is Strongly Influenced by the Ultrastructure of Nanoparticles, Biomaterials, 2020261, 120307
[3] Koshkina, O.; White, P. B.; Staal, A. H. J.; Schweins, R.; Swider, E.; Tirotta, I.; Tinnemans, P.; Fokkink, R.; Veltien, A.; van Riessen, N. K.; van Eck, E. R. H.; Heerschap, A.; Metrangolo, P.; Baldelli Bombelli, F.; Srinivas, M., Nanoparticles for “Two Color” 19F Magnetic Resonance Imaging: Towards Combined Imaging of Biodistribution and Degradation. J. Colloid Interface Sci. 2020, 565, 278-287
[4] Hoogendijk, E.; Swider, E.; Staal, A. H. J.; White, P.; van Riessen, N. K.; Glasser, G.; Lieberwirth, I.; Musyanovych, A.; Serra, C., A.; Srinivas, M.; Koshkina, O., Continuous-flow Production of Perfluorocarbon-loaded Polymeric Nanoparticles: from the Bench to Clinic. ACS Appl. Mater. Interfaces 2020, 12, 49335-49345.
[5] Verbeelen, M.; White, P.; Staal, A.; Swider-Cios, E.; Cortenbach, K.; van Riessen, N.K., Cadiou, C.; Chuburu, F.; Srinivas, M.; Koshkina, O.; Gadolinium chelates strongly affect the 19F-relaxation Rate of Liquid Perfluorocarbon in Multicore Nanoparticles, 2021, in submission.
Exploring the fractal structure to tune the MR performance of nanocarriers

Top: the structure of fractal nanocarriers based on the Small-Angle Neutron Scattering (SANS), and the structures of the components.

Bottom-left: the encapsulation of lipophilic Gd-chelates alters the relaxation of both 19F and 1H nuclei, leading to changed MRI intensities.

Bottom Right: the encapsulation of PERFECTA and PFCE leads to two-color probes which change their MR properties upon degradation.

Keywords: perfluorocarbons, biodegradable polymers, 19F MRI, ultrasound, fractal structure
805

Glycol chitosan functionalized with a Gd(III)-HPDO3A-like chelate as a redox-responsive probe for MRI

Sergio Padovan1, Carla Carrera1, Valeria Catanzaro3, Cristina Grange2, Malvina Koni2, Giuseppe Digilio3

1 CNR, Institute for Biostructures and Bioimages c/o Molecular Biotechnology Centre, TORINO, Italy
2 University of Turin, Department of Medical Sciences, TORINO, Italy
3 Università del Piemonte Orientale "A. Avogadro", Department of Science and Technologic Innovation, ALESSANDRIA, Italy

Introduction

Cell therapy relies on the transplantation of living cells into damaged organs or tissues. Cells are often encapsulated within alginate-based hydrogels to increase graft retention. However, pericapsular overgrowth of host immune cells may still arise, eventually leading to ischemia/hypoxia and death of therapeutic cells. Non-invasive imaging tools could be of great value to follow-up the function of encapsulated cells grafts over time.[1,2] Here we present redox-responsive MRI contrast agent designed to probe hypoxia within the lumen of cell embedding alginate-based capsules.

Methods

Glycol chitosan (gCHT) was functionalized through a disulfide bond with a Gd(III)-HPDO3A-like chelate [3] as a positive contrast agent for T1-weighted (T1w) MRI. Such a compound (named GdL-SS-gCHT) can be used to label the lumen of alginate/chitosan (AC) capsules. Under healthy (normoxic) conditions, the Gd(III)-based contrast agent is retained into AC capsules. Increasing reducing conditions arising from hypoxia promote the reductive cleavage of the disulfide bond, and the releas of the low molecular weight Gd-HPDO3A-like chelate in the free thiol form (GdL-SH; Scheme 1). The latter compound, owing to its small, hydrophilic and non-ionic nature can diffuse out of alginate capsules. This process can be readily detected by a a decrease of contrast enhancement in T1w-MR images at 7T.

Results/Discussion

Cell free AC capsules labelled with GdL-SS-gCHT were incubated in a medium supplemented with increasing amounts of reduced glutathione (GSH, 0-500 μM) to assess the sensitivity of the system. Capsules incubated with GSH showed a 30% decrease of the MRI signal enhancement (SE), indicating a good stability of the magnetic labelling. The decrease of SE was 70%, 80% and about 100% at GSH levels of 50, 100, and 500 μM, respectively, indicating a very high sensitivity. Next, magnetically labelled AC capsules embedding human fibroblasts at a density of 5x105 cells/mL were incubated under either normoxia or hypoxia and followed by MRI at 7T for 8 days. Cell viability was assessed in parallel by means of the MTT assay. A much steeper time-dependent decrease of SE was observed for hypoxia caspules as compared to normoxia. The loss of signal enhancement was correlated with the loss of cell viability.

Conclusions

GdL-SS-gCHT is a very sensitive probe to follow-up non-invasively the redox microenvironment within cell embedding AC capsules. MRI readings are correlated to the status of cells within capsules. However, optimization of the formulation of cell embedding AC capsules is needed to increase the stability of the labelling.

Acknowledgement

Economic support from University of Piemonte Orientale (Bando FAR 2017) and the Italian Ministry of University and Education (PRIN-2017 n. 2017A2KEPL_002) are gratefully acknowledged.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Naumova AV, Modo M, Moore A, Murry CE,  Frank JA, Nature biotech. 2014, 32, 804-818.
[2] Srivastava AK, Kadayakkara DK, Bar-Shir A, Gilad AA, McMahon MT, Bulte JWM, Disease Models & Mechanisms 2015, 8, 323-336.
[3] Muñoz Úbeda M,  Carniato F, Catanzaro V, Padovan S, Grange C, Porta S, Carrera C, Tei L, Digilio G, Chem. Eur. J. 2016, 22, 7716-7720.
Scheme 1

GdL-SS-gCHT as a MRI redox-responsive agent.

Keywords: alginate, chitosan, gadolinium, redox-responsive probe, cell encapsulation
807

Nanosized free radicals for the use as contrast and hyperpolarization agents in ultralow-field and high-field MRI

Paul Fehling1, Aleksandra Pavicevic7, Andrej Korenic6, Sergey Dobrynin2, Denis Morozov2, Yuliya Polienko2, Yulia Khoroshunova2, 3, Jörn Engelmann1, Kai Buckenmaier1, Klaus Scheffler1, 4, Goran Angelovski1, 5, Igor Kirilyuk2, Milos Mojovic7, Pavle Andjus6, Yulia Borozdina1

1 Max Planck Institute for Biological Cybernetics, Tübingen, Germany
2 N.N. Vorozhtsov Institute of Organic Chemistry, Novosibirsk, Russian Federation
3 Novosibirsk State University, Novosibirsk, Russian Federation
4 University of Tübingen, Tübingen, Germany
5 Chinese Academy of Sciences, Center for Excellencein Brain Science and Intelligence Technology, Shanghai, China
6 University of Belgrade, The Centre for Laser Microscopy, Faculty of Biology, Belgrade, Serbia
7 University of Belgrade, Faculty of Physical Cehmistry, Belgrade, Serbia

Introduction

Overhauser MRI is a technique, which could enable in vivo magnetic resonance experiments at low (<0.5 T) and ultralow (<10 mT) fields. The higher spin order of electrons is transferred to e.g. protons. Enhancement factors >100 can be achieved. This technique requires a stable free electron source in mM concentrations, usually in the form of free radicals. To enhance the stability of free radicals one can use nanosized carrier molecules. Here, we present cyclodextrines as carriers for nitroxide free radicals. The Overhauser DNP performance as well as toxicity and stability are tested.

Methods

We tested biotin, avidin, dendrimers, liposomes and cyclodextrines as carriers for different nitroxide free radicals.

To assess the ODNP performance, the maximum enhancement Emax and the RF power P1/2, needed to reach Emax/2, were measured at 2 mM concentration (a reasonable concentration for in vivo experiments) in a homemade ULF MRI system.

The stability of the selected nitroxides in the aforementioned carriers was tested in ascorbic acid solution and whole blood using EPR and ULF NMR spectroscopy. Cell viability was monitored on rat astrocyte cell cultures using the MTT assay and propidium iodide (PI) staining. Results obtained from probes with and without carriers were compared for commercially available nitroxides 3CP, 3CxP and TEMPOL and synthesized nitroxides.

Results/Discussion

Except for cyclodextrines all other carrier systems showed poor Overhauser DNP properties with nitroxides embedded into them. An increase of spectral line broadening, or lower tumbling rates of the larger carriers seem to be the reasons for the significant drop in ODNP performance.

Cyclodextrines with nitroxides showed a reasonable enhancement with improved water solubility enabling the use of lipophilic radicals. A stability improvement of up to 30% in the presence of ascorbic acid was measured via ULF NMR spectroscopy.

Some nitroxides with γ-cyclodextrin showed reduction in cell viability experiments as seen by PI staining and a decrease in metabolic activity as revealed by the MTT assay, however, these effects were ascribed mostly to the vehicle itself.

Conclusions

Even though most carrier systems decrease the ODNP efficiency, cyclodextrine-based radicals seem to be a promising candidate for future ultralow field Overhauser MRI in vivo experiments and high field T1 contrast agents. They show improved stability compared to nitroxides without carrier systems.

Further investigations should show, if only lipophilic nitroxides benefit from being embedded into cyclodextrine.

Acknowledgement

This work was supported with ERA.Net RUS+ project ST2017-382: NanoHyperRadicals (including RFBR 18-53-76003-ERA-A).

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

Keywords: Hyperpolarization, Overhauser, Radicals
808

A novel manganese-based T1 contrast agent obtained with a single-pot reaction: blood-pool imaging

Anbu Sellamuthu1, 7, Sabrina H. L. Hoffmann3, Fabio Carniato4, Lawrence Kenning2, Thomas W. Price5, Timothy J. Prior7, Mauro Botta4, Andre F. Martins6, 3, Graeme J. Stasiuk5, 1

1 University of Hull, Department of Biomedical Sciences, Hull, United Kingdom
2 Hull Royal Infirmary Hospital NHS Trust, MRI centre, Hull, United Kingdom
3 Eberhard Karls University Tübingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Germany
4 Università del Piemonte Orientale ‘A. Avogadro’, Dipartimento di Scienze e Innovazione Tecnologica, Alessandria, Italy
5 King’s College London, Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
6 University of Tuebingen, Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, Tübingen, Germany
7 University of Hull, Department of Chemistry, Hull, United Kingdom

Introduction

Currently, gadolinium-based T1 contrast agents (GdCAs) are considered the gold standard for MRI.1 However, due to poor stability in acyclic GdCAs, has led to free-Gd3+ toxic effects,2 many researchers started developing Gd3+-free CAs. Especially, manganese-based MnCAs perceived as safer CAs3 to GdCAs since it is biogenic with near equivalent MRI contrasting abilities. Despite this, only MnCl2 (LumenHanceTM) is used for animal MRI studies.4 Therefore, we have developed a novel and incredibly stable MnCA (MnLMe) and studied its relaxometry properties in vitro and in vivo.5

Methods

We designed and synthesized a new pentadentate Schiff base type Mn2+ complex MnLMe (Fig. 1) by an innovative single-pot template strategy.5 We have studied its structural and physicochemical properties using conventional analytical techniques. The relaxometry properties (longitudinal relaxation times (T1), water proton relaxation rates (r1 = 1/T1) and proton relaxivity enhancement (PRE)) and chelation stability of the MnLMe were ascertained by 1H-NMR (JEOL-400 MHz) and 1H-NMRD methods. Phantom and murine MR images were acquired on a 7 T preclinical MR scanner (Bruker BioSpec 70/30, Bruker BioSpin, Ettlingen, Germany) using an 86-mm diameter 1H transceiver volume coil (Bruker). T1-weighted pre- and post-contrast in vivo MR images were acquired with a 3D FLASH-sequence strategy.

Results/Discussion

MnLMe shows relaxivity (r1) of 4.2 and 4.9 mM-1 s-1 at 400 MHz (9.4 T) and 64 MHz (1.5 T), respectively. It also displays optimized r1 at both medium (20 and 64 MHz) and high magnetic fields (300 and 400 MHz) and an enhanced r1b = 21.1 mM-1 s-1 (20 MHz, 298 K, pH 7.4) upon binding to BSA (Ka = 4.2 × 103 M-1). Fig. 2 shows the pre- and post-contrast coronal MRIs. Elimination is observed through the liver, kidneys, and bladder. The enhanced r1 when binding with BSA and in vivo MRI and fast renal/hepatic clearance suggest that the capability of MnLMe is comparable to the clinically available Magnevist.

Conclusions

This is the first example to date of a template to MRI contrast agent synthesis. The MnLMe is stable at neutral pH conditions when stressed with other chelators and endogenous metal ions. MnLMe shows delayed clearance from the blood as observed by the continuous high ΔSI on the heart's left ventricle. This data indicates that MnLMe interacts with albumin proteins throughout the imaging window and shows potential for an MRI blood pool agent.

Acknowledgement

SA gratefully acknowledges the Depts. of Chemistry and Biomedical Sciences, the University of Hull, to provide laboratory space and instruments access to this work. The work has also been partially supported by the RSC Research Fund grant (RF19-7464). GJS would like to thank the MRC for funding (MR/T002573/1). AFM gratefully acknowledges the funding and support by the Swiss Werner Siemens Foundation and the Faculty of Medicine at the University of Tuebingen (Nachwuchsgruppen-Programm). The work was also funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2180 – 390900677. MB and FC are grateful to Università del Piemonte Orientale per financial support (FARC 2019).

Disclosure

a) I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Matsumura, T, Hayakawa, M, Shimada, F, Yabuki, M, Dohanish, S, Palkowitsch, P, Yoshikawa, K. 2013, ‘Safety of gadopentetate dimeglumine after 120 million administrations over 25 years of clinical use’, Magn.Reson. Med. Sci. 12(4), 297.
[2] (a) Clough, TJ, Jiang, L, Wong, K-L, Long, NJ, 2019, ‘Ligand design strategies to increase stability of gadolinium-based magnetic resonance imaging contrast agents’, Nature Commun. 10, 1420. (b) Thakral, C, Alhariri, J, Abraham, J L, 2007, Long-term retention of gadolinium in tissues from nephrogenic systemic fibrosis patient after multiple gadolinium-enhanced MRI scans: case report and implications, Contrast Media Mol. Imaging, 2, 199.
[3] Gale, EM, Wey, HY, Ramsay, I, Yen, YF, Sosnovik, DE, Caravan, P, 2018, ‘A Manganese-based Alternative to Gadolinium: Contrast-enhanced MR Angiography, Excretion, Pharmacokinetics, and Metabolism’, Radiology, 286, 865.
[4] Liu, H, Zhang, X-A, 2018, Encyclopedia of Inorganic and Bioinorganic Chemistry, John Wiley & Sons, Ltd. DOI: 10.1002/9781119951438.eibc2626
[5] Anbu, S, Hoffmann, SHL, Carniato, F, Kenning, L, Price, TW, Prior, TJ, Botta, M, Martins, AF, Stasiuk, GJ, 2021, ‘A Single-Pot Template Reaction Towards a Manganese-Based T1 Contrast Agent’, Angew. Chem. Int. Ed. 60, 10736–10744.
Figure 1

Molecular structure of MnLMe, temperature dependent 17O NMR transverse relaxation rates of MnLMe free (blue diamonds) and bound to BSA (red circles) (A), and 1/T1 NMRD profiles measured at 298 K of MnLMe free (black) and bound to BSA (blue) (B).

Figure 2

T1-weighted MR images illustrate contrast enhancement in the kidneys, liver, heart, and bladder after application of MnLMe at a clinical dose in comparison to pre-contrast imaging.

Keywords: Mn(II)-based T1 contrast, Blood Pool Agents, Fast Hepatic Clearance