15th European Molecular Imaging Meeting
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MRI | Probe Chemistry

Session chair: Monique Bernsen (Rotterdam, Netherlands); Zoltan Kovacs (Dallas, USA)
 
Shortcut: PW25
Date: Friday, 28 August, 2020, 12:00 p.m. - 1:30 p.m.
Session type: Poster

Contents

Abstract/Video opens by clicking at the talk title.

711

LDL mediated delivery of Paclitaxel and MRI contrast agent for Image-guided Drug Delivery

Rachele Stefania1, Sahar Rakhshan1, Diego Alberti1, Silvio Aime1, Simonetta Geninatti Crich1

1 University of Turin, Molecular Imaging Center-Dept of Molecular Biotechnologies & Health Sciences, Torino, Italy

Introduction

Image-guided drug delivery refers to the combination of drug targeting and imaging and can be used for different purposes, e.g. for monitoring biodistribution, drug release and drug efficacy. Low density lipoproteins (LDL) have been used for the encapsulation of Paclitaxel (PTX), an antitumor drug, and an amphiphilic contrast agent Gd-AAZTA-C17 to obtain new LDL nanoparticles namely PTX-LDL-Gd. Mesothelioma and melanoma cell lines that overexpress LDL receptors were used to explore non-invasive imaging measurement of the biodistribution and pharmacokinetics of these drug delivery systems

Methods

PTX-LDL-Gd were prepared by incubating Gd-AAZTAC17 and PTX with native human LDL at 37°C for 4 hours using respectively a 300:1 and 700:1 molar ratio of drug/LDL. The final Gd concentration was determined by 1 H NMR T1 measurement of the mineralized solution while the PTX entrapped in LDL was measured by RP-HPLC in PBS pH 7.4 and 37°C. Then the cytotoxicity of LDL-PTX-Gd were evaluated in the AB22 and B16-F10 cells by MTT method and compared with and free PTX. In vitro cellular uptake experiments on the same cell lines were performed by incubating different amount of LDL-PTX-Gd and by measuring the amount of Gd uptake in cells by ICP-MS analysis. T1 weighted MR images of an agar phantom containing B16-F10 cells labeled with LDL-PTX-Gd were acquired at 7 T on a Bruker Avance300 spectrometer

Results/Discussion

The PTX-LDL-Gd have particle size of 34 nm and displayed r1= 24.0 s-1mmolGd-1 (0.5 T, 25°C). The HPLC analysis showed an encapsulation efficiency of 10% of PTX while ICP-MS analysis showed an encapsulation of paramagnetic contrast agents around 25%. The MTT assay experiments demonstrated that LDL-PTX-Gd has a similar cytotoxic effect against AB22 cells as free PTX, whereas against B16-F10 cells the LDL-PTX-Gd shower a greater cytotoxicity. In particular, in B16-F10 cells we observed a dramatic reduction of cell viability at 0.5 µM PTX concentration (about 50 %) when PTX is administered to cells loaded into LDL, whereas the viability remains relatively high (>80%) when the incubation was carried out in the presence of the same concentration of free PTX. These results are in agreement with the higher expression of LDLR on this cell line. MR image of an agar phantom with B16-F10 labeled with LDL-PTX-Gd showed the well detectable signal intensity (SI) enhancement of cells

Conclusions

In conclusion, LDLs are selectively taken-up by melanoma cells and can be successfully exploited for the selective delivery of paclitaxel and MRI imaging agents. This is an important advantage to obtain a more selective biodistribution of the drug delivered thus reducing the side effects. In the next step, the drug and imaging agent biodistribution will be evaluated in T1-weighted MRI images

References
[1] Geninatti‐Crich, S., Alberti, D., Szabo, I., Deagostino, A., Toppino, A., Barge, A., ... & Stella, S. (2011). MRI‐guided neutron capture therapy by use of a dual gadolinium/boron agent targeted at tumour cells through upregulated low‐density lipoprotein transporters. Chemistry–A European Journal, 17(30), 8479-8486.
[2] Geninatti -Crich, S., Lanzardo, S., Alberti, D., Belfiore, S., Ciampa, A., Giovenzana, G. B., ... & Aime, S. (2007). Magnetic resonance imaging detection of tumor cells by targeting low-density lipoprotein receptors with Gd-loaded low-density lipoprotein particles. Neoplasia (New York, NY), 9(12), 1046.
Low density lipoprotein as a carrier for both Paclitaxel and GD-AAZTA-C17

Low-density lipoprotein (LDL) carries cholesteryl esters to peripheral cells. It contains one major apolipoprotein that allows LDL to bind to the LDL receptors and it is internalized by cells through a receptor mediated endocytosis. LDL is the endogenous carrier of cholesterol. In this work LDL were used as a vehicle for targeting antitumor PTX to cancer cells and for direct MR imaging and quantification of drug delivery as a vehicle of Gd-AAZTAC17

Keywords: Image-guided drug delivery, LDL, Paclitaxel, Gd-AAZTAC17
712

Novel contrast agents based on pyclen derivative manganese complexes to be incorporated inside polymersomes.

Marie Devreux1, 2, Céline Henoumont1, Fabienne Dioury3, Olivier Sandre2, Sophie Laurent1, 4

1 University of Mons, General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, Mons, Belgium
2 University of Bordeaux, CNRS, Bordeaux INP, ENSCBP, Laboratory of Organic Polymer Chemistry (LCPO), Pessac, France
3 Conservatoire national des arts et métiers (CNAM), CMGPCE Laboratory, EA 7341, Paris, France
4 Center for microscopy and molecular imaging (CMMI), Charleroi, Belgium

Introduction

In medicine, magnetic resonance imaging (MRI) has a leading place in the diagnosis setting. The commercially available contrast agents are based on gadolinium complexes. Recently, it has been shown that gadolinium can lead, mainly for patients with renal dysfunctions, to nephrogenic systemic fibrosis. It is thus interesting to develop an efficient contrast agent based on another paramagnetic ion such as manganese. Complexes based on pyclen appear very interesting and their incorporation inside polymersomes is envisaged in order to achieve better relaxivities.

Methods

The synthesis of the two complexes starts by a macrocyclization between an upper part, the 3-hydroxypyridine and a lower part, the diethylenetriamine, both functionalized. The two macrocycles are then complexed with manganese ions (figure 1). They are characterized by mass spectrometry, 17O NMR to evaluate the number of coordinated water molecules inside the inner sphere of the complexes1 and the exchange time of these coordinated water molecules, and relaxometry over a wide range of magnetic fields (from 0.01 MHz to 60 MHz) at 37°C in order to evaluate the efficacy of the complexes for MRI.

In a second time, polymersomes composed of poly(e-caprolactone)-block-poly(ethylene oxide) PCL-b-PEO are synthesized by nanoprecipation techniques3 and characterized.

Results/Discussion

Both complexes were synthesized and characterized. One coordinated water molecule was determined by 17O NMR1 and by NMRD profiles at 25°C2. 17O NMR was also used to evaluate the residence time of this coordinated water molecule (τM) and values of 4.7 and 4.95 ns were obtained for MnPy(COO-)2 and MnPy-(COO-)2-OCH2COO- respectively. This reflects a very fast exchange of the coordinated water molecule. The fitting of the NMRD profiles at 37°C allowed to estimate the rotational correlation time (τR) at 31.3 ps for MnPy(COO-)2 and 56.3 ps for MnPy-(COO-)2-OCH2COO-. These results show an increase for the second complex due to the presence of the additional arm on the pyridine. The efficacy at 20 MHz and 37°C of both complexes was also evaluated and relaxivities of 2.26 s-1 mM-1 for MnPy(COO-)2 and 2.98 s-1 mM-1 for MnPy-(COO-)2-OCH2COO- were obtained.

In a second time, the polymersomes were synthesized without the manganese complexes and their size was evaluated by dynamic light scattering.

Conclusions

These manganese-pyclen derivatives are promising contrast agents to replace gadolinium complexes as T1 MRI contrast agents. Moreover, a functionalization of the macrocycle is possible through the additional arm on the pyridine, allowing the coupling of different molecules. An encapsulation of the complex in the lumen or in the membrane of polymersomes is also envisaged in order to improve the rotational correlation time and hence the relaxivity.

Acknowledgment

This work was performed with the financial support of SIRIC BRIO (COMMUNCAN), FNRS, 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).

References
[1] Gale EM, Zhu J and Caravan P, 2013, 'Direct measurement of the Mn(II) hydration state in metal complexes and metalloproteins through 17O NMR line widths',  J Am Chem Soc, 135, 49, 18600-18608
[2] Pujales-Paradela R, Carniato F, Esteban-Gomez D, Botta M and Platas-Iglesias C, 2019, Dalton Trans., 'Controlling water exchange rates in potential Mn2+ - based MRI agents derived from NO2A2-', 48, 3962-3972
[3] Hannecart A, Stanicki D, Vander Elst L, Muller R N, Sandre O, Schatz C, Lecommandoux S, Brûlet A, Laurent S, 2019, 'Embedding of superparamagnetic iron oxide nanoparticles into membranes of well-defined poly(ethylene oxide)-block-poly(ε-caprolactone) nanoscale magnetovesicles as ultrasensitive MRI probes of membrane bio-degradation.', J. Mater. Chem. B, 7, 4692-4705
Structure of the two synthesized complexes
Keywords: Contrast agents, Manganese complexes, MRI, Pyclen derivatives
713

Adapting ADEPT: towards smart MRI contrast agents

Ilse M. Welleman1, 2, Rudi A. J. O. Dierckx1, Ben L. Feringa2, Hendrikus Boersma1, Wiktor Szymanski2, 1

1 University Medical center Groningen (UMCG), Department of Radiology, Groningen, Netherlands
2 University or Groningen(RUG), Strating institute for chemistry, Groningen, Netherlands

Introduction

While MRI is a powerful, safe, whole-body imaging technique, it has a low sensitivity. This hinders the imaging of molecular imaging targets, such as enzyme/receptor (over)expression and physiological processes in the human body.[1]
Contrast agents (CAs) have been developed to improve the sensitivity of MRI, yet a high concentration of CA is still required for contrast. This required concentration is much higher than that of imaging targets. The goal of this project is to use a bio-catalytic approach to locally amplify MRI signal, by adapting the ADEPT prodrug activation strategy.

Methods

Antigen-Directed Enzyme Prodrug Therapy (ADEPT) is a drug delivery modality, which has been developed for cancer treatment with the aim to activate the cytotoxic payload only to the tumour cells to prevent damage to healthy tissue (figure 1.A).[2] Here we aim to apply the ADEPT logic to MR imaging, to activate the MR CA only around the target site. In the ADEPT-MRI method, a MRI pro-contrast agent (e.g. compound 1-Gd, Fig 1.B) is activated at the tumour site by an enzyme (CPG2) which is conjugated to a tumor-directed antibody (D). Upon release of unstable molecule 2, 1,6-elimation leads to the final compound 3. The difference in relaxation between 1 and 3 is expected to be induced by the difference in inner-sphere relaxivity. [1]

Results/Discussion

To provide a proof-of-principle for ADEPT-MRI, we planned to synthesize three molecules (Figure 2): complex 1-Gd as the ‘’off’’ contrast agent, complex 3-Gd as the ‘’active’’ contrast agent, and complex 4-Gd as a model to check if the expected difference in relaxation between 1 and 3 is caused by size, number of water molecules, or water exchange rate.

The synthesis of 1-Gd, consists of 4 steps: synthesis of two building blocks (red) & (blue[3]), their coupling, deprotection, and complexation of gadolinium. We successfully synthesized the two building blocks with 74 and 72 % yield, and are currently optimizing their coupling and deprotection towards compound 1. Simultaneously we successfully prepared (deprotected) ligand 3 and ligand 4 for complexation studies.

Conclusions

We developed an efficient synthesis route for the two building blocks of compound 1, and towards ligands 3 and 4. Short-term future plans include the completion of the synthesis, gadolinium complexation studies and in vitro evaluation of the ADEPT effect using FFC relaxometry. Long-term plans involve the use of specially modified CPG2-antibody conjugates [4] in in vivo studies.

AcknowledgmentThe financial support from the Dutch Organization for Scientific Research (VIDI grant no. 723.014.001)
References
[1] J. Wahsner, E. M. Gale, A. Rodríguez-Rodríguez, and P. Caravan., 2019, Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers,Chem. Rev., 119, 957−1057.
[2] S. K. Sharma and K. D. Bagshawe., 2017,Translating antibody directed enzyme prodrug therapy (ADEPT) and prospects for combination.,Expert Opin. Biol. Ther., 17, 1-13.
[3] A. la Reberdière F. Lachaud F. Chuburu C. Cadiou G. Lemercier, 2012, Synthesis of a new family of protected 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derivatives with thioctic acid pending arms,Tetrahedron Letters., 53 ,6115–6118.
[4] K.D Bagshawe, C.J. Spinger, F. Searle, P Antoniw, S.K. Sharma, R.G Melton and R.F. Sherwoord., 1988,  A cytotoxic Agent can be Generated selecively at cancer sites, Br. J. Cancer., 58, 700-703.
Figure 1.A: Principle of ADEPT 1.B: Mechanism of contrast agent activation.
Figure 2. schematic overview of designed molecules and there synthetic pathways.
Keywords: MRI contrastagent, MRI signal amplification, Enzyme responsive MRI contrast agent, ADEPT, CPG2
715

An Yb3+- complex as a pH-responsive paraCEST agent for MRI

James Ratnakar1, Sara Chirayil1, Alexander Funk1, Shanrong Zhang1, Joao F. Queiró3, Carlos F. G. C. Geraldes4, Zoltan Kovacs1, Dean Sherry1, 2

1 UTsouthwestern Medical Center, Advanced Imaging Research Center, Dallas, Texas, United States of America
2 University of Texas at Dallas, Department of Chemistry, Plano, United States of America
3 University of Coimbra, Department of Mathematics, Coimbra, Portugal
4 University of Coimbra, Department of Life Sciences and Chemistry Center, Coimbra, Portugal

Introduction

Paramagnetic lanthanide-based DOTA-tetraamide complexes provide contrast in MRI by chemical exchange saturation transfer (paraCEST).1 The presence of the Ln3+- bound inner sphere water reduces the T2 through a T2ex mechanism.2 A pH responsive paraCEST agent lacking the bound water is designed by having three bulky coordinating methylphosphonate pendant arms, and a paraCEST signal via the Ln3+-bound–OH group HP-DO3P (Chart 1) was observed. The three phosphonates also shows pH sensitivity to the chemical shift via protonation of the non-coordinating phosphonate oxygen atoms.

Methods

 Ligand 1 was synthesized by a multistep process. The hydroxypropyl pendant arm is formed in a ring opening reaction between an epoxide and cyclen (1,4,7,10-tetraazacyclododecane). The three phosphonates were introduced by the reaction with triethylphosphite and formaldehyde followed by acid hydrolysis to yield the final ligand. The Yb3+ complex was prepared by reacting the free ligand with YbCl3 at pH 7. Yb(1) was characterized by high resolution 1H and 31P NMR at 400 MHz and 161 MHz, respectively, on a 9.4 T NMR spectrometer. All CEST spectra were recorded in water at 25°C and 9.4 T. Phantom CEST images were obtained on a 9.4 T small animal imaging system.

Results/Discussion

 The 1H NMR spectrum of Yb(1) shows 27 resonances consistent with a ligand structure having C1 symmetry. The broad highly-shifted resonance at 133.6 ppm is assigned to the hydroxypropyl (-OH) proton on the fourth pendant arm. The 31P NMR spectrum of Yb(1) showed three resonances and were simultaneously fitted to a three-step equilibrium model to obtain three log K values (7.0, 5.8 and 4.2). The Yb(1) complex displayed a single highly shifted CEST peak from the exchangeable -OH proton (Figure 1a). The pH-dependent chemical shift of the CEST signal reflects stepwise protonation of the three Yb  coordinated phosphonate groups. CEST images of phantoms at different pH were collected and a false color pH map of each phantom was constructed (Figure 1b). The pH value estimated from the frequency of the CEST peak showed good agreement with the solution pH measured using a glass electrode (Figure 1c).

Conclusions

 The CEST spectrum of the Yb(1) complex reflects a single highly shifted exchanging -OH proton that is also well-outside the chemical shift range of the broad tissue MT signal (±100 ppm). The chemical shift of the Yb3+- OH CEST peak is quite sensitive to pH over a biologically relevant range. These features make Yb(1) a very promising sensor for imaging tissue pH in vivo by chemical shift measurement of the CEST peak.

Acknowledgment

Financial support for this work National Institutes of Health (CA-115531 and EB-02584) and the Robert A. Welch Foundation (AT-584). JFQ FCT-Portugal (Portuguese Foundation for Science and Technology) and FEDER (European Regional Development Fund -PT2020) (UID/MAT /00324/2019). CFGCG UID/QUI/00313/2019 and POCI-01-0145- FEDER-027996) and a Fulbright visiting scholarship.

 

References
[1] S. Viswanathan, Z. Kovacs, K. N. Green, S. J. Ratnakar, A. D. Sherry, Chem. Rev., 2010, 110, 2960 - 3018.
[2] T. C. Soesbe, M. E. Merritt, K. N. Green, F. A. Rojas-Quijano, A. D. Sherry Magn. Reson. Med. 2011, 66, 1697-703.
Figure 1

a) CEST spectra of Yb(1) at different pH values (9.4T, 37°C); b) The false colored CEST images of 25 mM Yb(1) at different pH values; c) Linear plot showing the agreement between the CEST measured pH compared to electrode measurements.

Chart 1
Structure of free ligand designed for this work.
Keywords: ParaCEST, MRI, pH sensor, Lanthanide Complexes, MRI Contrast agents
716

Preparation and initial physicochemical characterization of novel theranostic probes: Gd-loaded polylactide-albumin nanospheres as carriers of the antitumor metal complexes KP1019 and RuCl3(H2O)(Hind)2

Irena Pashkunova-Martic1, 2, Berta C. Losantos2, Daniela Gruber3, Steffanie Ottofuelling4, Thomas Helbich1, Bernhard Keppler2

1 Medical University of Vienna, Department of Biomedical Imaging and Image-guided Therapy, Vienna, Austria
2 University of Vienna, Institute of Inorganic Chemistry, Vienna, Austria
3 University of Vienna, Faculty of Life Sciences, Vienna, Austria
4 University of Vienna, Department for Environmental Geosciences, Vienna, Austria

Introduction

For Magnetic Resonance Imaging (MRI), targeted nanoparticle contrast media (CM) with high relaxivities are required in order to image specific pathological changes in tissues with an adequate signal-to-noise ratio. Herein we report the synthesis of biodegradable drug loaded polylactide-albumin–gadolinium nanoparticles (drug-PLA-HSA-DTPA-Gd) as transport devices for two antitumor metal complexes with pronounced cytotoxic activity. In addition, the novel theranostic probes possess a big potential to monitor applied cancer therapy by MRI.

Methods

The drugs used were (H2ind)[trans-RuIIICl4(Hind)2] (KP1019) (1), a ruthenium complex which has recently finished Phase I clinical trials and its active hydrolysis product mer,trans-[RuIIICl3(H2O)(Hind)2] (2). The nanoparticles have been prepared by single oil in water (o/w) emulsion method. All nanoparticles variations were purified by fast protein liquid chromatography (FPLC) prior to analyses. Initial physical and chemical characterizations were undertaken. The particles´ diameter and size distributions were determined by photon correlation spectroscopy (PCS), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The amount of Ru and Gd in the nanoparticles has been determined by inductively coupled plasma mass spectrometry (ICP-MS).

Results/Discussion

Biodegradable nanoparticles based on PLA, HSA, and DTPA-Gd have been prepared and loaded with the anticancer agents KP1019 and its hydrolysis product. Combination of water solutions of HSA-DTPA-Gd with solutions of each drug-PLA complex in acetone/CH2Cl2 resulted in formation of two phases. After emulsification of the organic and aqueous phase a blue precipitate (pellet) appeared, indicating that both drugs have reacted. These macromolecular constructs are promising candidates for use in MRI and cancer therapy. The sizes of the nanoparticles were 188 nm for KP1019 and 213 nm for mer,trans-[RuIIICl3(H2O)(Hind)2], rendering them suitable for application in cancer treatment as well as an imaging diagnostic tool. 
The ICP-MS analysis of Ru and Gd in the particles showed 9.4 mol Gd atoms per 1 mol Ru in the nanoparticles with entrapped complex mer,trans-RuCl3(H2O)(Hind)2 (2) while that calculated for the nanoparticles loaded with KP1019(1), resulted in 22.4 mol Gd per 1 mol Ru.

Conclusions

The use of polymers in nanoparticles’(NPs) manufacturing has shown to be a very efficient method for increasing the NPs size and prolonging the dwell time of the entrapped active compounds in blood. Advantages can be made from such a macromolecular thearanostic agent for selective uptake and reliable diagnostics of many pathological processes such as cancer and inflammation as well as to following up and monitor the effectiveness of chemotherapy.

Acknowledgment

We thank Dr. Steffanie Ottofuelling for the photon correlation spectroscopy (PCS) experiments and D.I. Norbert Kandler for the ICP-MS measurements.

References
[1] Maysinger, D, 2007, Org. Biomol. Chem. , 5, 2335-2342
[2] Muller, R.N, 1996, Encyclopaedia of NMR. John Wiley, 1438-1444: New York
[3] Caravan, P, Ellison, JJ, McMurry, TJ, Lauffer, RB,1999, Chem Rev, 99 , 2293-2352
[4] Montisci, MJ, Giovannuci, G, Duchene, D, Ponchel, G, 2001. Int J Pharm, 215, 153-161
[5] Stollenwerk, MM,  Pashkunova-Martic, I, Kremser, C, Talasz, H, Thurner, GC, Abdelmoez, AA, Wallnoefer, EA, Helbok, A, Neuhauser, E, Klammsteiner, N, Klimaschewski, L, Von Guggenberg, E, Froehlich, E, Keppler, B, Jaschke, W, Debbage, P,  2010, Histochem Cell Biol , 133, 375–404
Scanning electron micrograph (SEM) of theranostic nanoparticles (KP1019-PLA-HSA-DTPA-Gd), pellet
The nanoparticles in this pellet population, possess a mean particle diameter between  840-997 nm, with a polydispersity index (PI) of 0.4-0.9 and spherical shape seen under SEM. Interestingly, substructures could be seen on the NPs surface indicating the binding of the HSA-DTPA conjugates on it.
Model structure of a theranostic PLA-HSA-Gd based nanoparticle loaded with antitumor metal complexes
Keywords: novel theranostic probes, PLA-nanoparticles, antitumor metal complexes, MRI
717

Development of a Polymer CEST MRI Contrast Agentthat Measures Extracellular pH in the Tumor Microenvironment

Chathuri Kombala1, 2, Aikaterini Kotrotsou2, William Schuler2, Jorge de la Cerda2, Shu Zhang2, Mark D. Pagel2

1 University of Arizona, Chemistry and Biochemistry, Tucson, United States of America
2 MD Anderson Cancer Center, Cancer Systems Imaging, Houston, United States of America

Introduction

AcidoCEST MRI can measure the extracellular pH (pHe) of the tumor microenvironment in mouse models of human cancers and in patients who have cancer.  However, Chemical Exchange Saturation Transfer (CEST) is an insensitive MRI contrast mechanism, requiring a high concentration of small molecule agent, iopamidol, to be delivered to the tumor for the pHe measurement.  Herein, we developed a nanoscale CEST agent that can measure pH using  acidoCEST MRI, which may decrease the requirement for high delivery concentrations of agent.  We also developed a monomer agent for comparison to the polymer.

Methods

4-acrylamidosalicylic acid was polymerized using 4,4’-azobis(4-cyanovaleric acid) as initiator, and was purified by dialysis. The monomer agent, 4-acrylamidosalicylic acid, was synthesized by acylating 4-aminosalicylic acid, which was purified using HPLC.  CEST MRI saturation conditions were optimized to detect the polymer and monomer agents, and a ratio of the two CEST signals at 5.0 and 9.2 ppm was used to correlate CEST with pH.  This correlation was tested over a temperature range of 25-49C, and a concentration range of 0.02-0.3 mM for the polymer, and 2.5-40 mM for the monomer.  Cytotoxicity was evaluated for both agents.  In vivo acidoCEST MRI was performed with the polymer and monomer agents, as well as the standard agent iopamidol, to measure extracellular pH in a tumor model.

Results/Discussion

The polymer and monomer agent showed CEST signals at 5.0 and 9.2 ppm, as expected.  A ratio of these signals was linearly correlated with pH.  After optimizing CEST experimental conditions, the polymer agent and monomer agent could be used during in vivo acidoCEST MRI studies at 0.016 mmol/kg and 2.0 mmol/kg, respectively, while the standard agent used for acidoCEST MRI, iopamidol, is used at 7.8 mmol/kg.  The pH measurement with the polymer and monomer agent had negligible dependence on temperature, but was dependent on concentration of the agent.  In vivo acidoCEST MRI using the polymer and monomer agents, as well as iopamidol for comparison, were performed to study a xenograft MDA-MB-231 model of mammary carcinoma.  The tumor pHe measurements were 6.85 ± 0.15 units with the polymer, 6.70 ± 0.15 units with the monomer, and 6.33 ± 0.02 units with iopamidol.  These results indicated that the polymer and monomer agents overestimated in vivo tumor pH relative to the standard agent.

Conclusions

Our polymer agent can reduce the agent concentration for CEST detection by >100-fold, and can measure pH with via an acidoCEST MRI method.  However, these pH measurements were dependent on agent concentration, which was attributed to changes in hydrogen bonding and/or steric hindrance.  This concentration dependence caused the in vivo tumor pH measurements with acidoCEST MRI to be overestimated relative to measurements with a standard agent.

Acknowledgment

We thank the Mass Spectrometry Facility at the University of Arizona and the High Resolution Electron Microscopy Facility (NIH P30CA016672) at MDACC for their support.  CJK was supported by NIH T32 GM008804. Support was provided through NIH R21 EB027197, R01 CA231513 and P30 CA016672.

acidoCEST MRI calibrations
in vivo acidoCEST MRI
Keywords: magnetic resonance imaging, CEST, pH imaging, polymer contrast agent, tumor acidosis
718

Mn(II)-based probes for molecular imaging applications

Gyula Tircsó1, Richárd Botár1, Balázs Váradi1, Tibor Csupász1, Zoltán Garda1, Réka Anna Gogolák1, Éva Jakab Tóth4, Sandra Même4, William Même4, György Trencsényi2, Imre Tóth1, Ferenc Krisztián Kálmán1, 3

1 University of Debrecen, Department of Physical Chemistry, Faculty of Science and Technology, Debrecen, Hungary
2 University of Debrecen, Division of Nuclear Medicine, Department of Medical Imaging, Faculty of Medicine, Debrecen, Hungary
3 Le Studium, Loire Valley Institute for Advanced Studies, Orléans, France
4 CNRS-UPR 4301, Université d’Orléans, Centre de Biophysique Moléculaire, Orléans, France

Introduction

Complexes of Mn2+ have attracted considerable attention recently because they considered as safer alternatives to Gd3+-based MRI CAs. Our approach to find acceptable alternative to Gd3+ takes the advantage offered by the PCTA chelator forming thermodynamically stable Mn2+ complex of high inertness.[1] By “truncating” the PCTA ligand, we have designed, synthesized, and studied several PC2A derivatives including a bifunctional ligand and monoaquated (q = 1) Mn2+ complexes applicable for angiographic and pH MRI (Fig. 1).[2]

Methods

Ligands were prepared using standard chemical synthetic techniques. The thermodynamic stability of theMn2+ complexeswas determined by the combination of pH-potentiometry and 1H-relaxometry, whereas solvent exchange kinetics was studied via variable temperature 17O-NMR method. Dissociation kinetics of the complexes was studied by studying metal exchange reactions with essential (Cu2+ and Zn2+) metal ions, and their serum stability was also evaluated by using commercially available human blood serum. T1-weighted images of phantoms were acquired at 25°C, by using preclinical (Mediso NanoScan PET/MRI 1 T) and clinical (Siemens Magnetom Essenza 1.5 T and Philips Achivea 3 T) MRI scanners.

Results/Discussion

The results show that the PC2A ligand forms a stable (log K[Mn(PC2A)] = 17.09, pMn = 8.64) complex with the Mn2+ ion, yet its dissociation remains relatively fast. Attachment of electron withdrawing groups (ie, para-nitrobenyl, 4-phenylbenzyl, etc.) to the nitrogen atom being trans- to the pyridine nitrogen allowed us to improve the inertness of the corresponding Mn2+ complexes, and these modifications have also improved the interaction of the complexes with HSA. The [Mn(PC2A-BP)] complex possess good thermodynamic (log K[Mn(PC2A-BP)]=14.86, pMn=8.35), excellent dissociation kinetic (half-life about 72.5–95.0 hours at 37°C to minimize the rate of decomplexation in vivo), and relaxation parameters (r1p = 3.8 vs r1pbound = 23.5 mM−1s−1). Attachment of ethylamine moiety to the given nitrogen atom resulted in a stable (log K[Mn(PC2A-EA)] = 19.01, pMn = 9.27) pH-responsive Mn2+ chelate capable of responding to the changes in pH (r1p = 3.6 vs 2.1 mM−1s−1) above pH >5.8.

Conclusions

We have demonstrated that the PC2A platform can be used to build smart/responsible MRI CAs. A potential bifunctional ligand (pNO2BzPC2A), angiographic
(PC2A-BP), and pH-responsive (PC2A-SA and PC2A-EA) MRI CA candidates possessing high stability, inertness, as well as excellent relaxation properties
(all of these are being crucial for a chelate being considered for the in vivo applications) were designed, synthesized, and characterized.

Acknowledgment

This research was funded by the GINOP-2.3.2-15-2016-00008 project supported by the EU and co-supported by the European Regional Development Fund, by the Hungarian National Research, Development and Innovation Office (NKFIH K-120224 project), the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (Gy. T. and F. K. K.) and COST Action CA15209 European Network on NMR Relaxometry.

References
[1] Z. Garda, E. Molnár, F. K. Kálmán, B. Richárd, V. Nagy, Zs. Baranyai, E. Brücher, Z. Kovács, I. Tóth, Gy. Tircsó, Front. Chem. 2018. 6, 232.
[2] R. Botár, E. Molnár, Gy. Trencsényi, J. Kiss, F. K. Kálmán, Gy. Tircsó, J. Am. Chem. Soc., 2020, 142, 4, 1662.
Fig. 1. Formulae of the ligands studied.
Keywords: MRI CAs, Mn2+ alternatives to Gd3+, stability, interness, smart probes
719

Imaging glucose-stimulated zinc secretion from the prostate and pancreas using a Mn(II)-based sensor

Andre F. Martins1, 2, 3, Sara Chirayil1, Veronica Clavijo-Jordan1, 4, Namini Paranawithana2, James Ratnakar1, Dean Sherry1, 2

1 UT Southwestern Medical Center, Advanced Imaging Research Center, Dallas, United States of America
2 University of Texas at Dallas, Chemistry and Biochemistry, Richardson, United States of America
3 University Hospital of Tuebingen, Werner Siemens Imaging Center, Tuebingen, Germany
4 Harvard Medical School, Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, United States of America

Introduction

Imaging glucose-stimulated zinc secretion (GSZS) from secretory tissues has proven useful at assessing organ function and health; current probes to detect zinc secretion by MRI have so far been limited to gadolinium-based sensors. In this work we introduce a manganese-based zinc sensor and show that pancreatic and prostatic zinc detection is not compromised when using Mn instead of Gd for imaging GSZS in vivo. Given the current concerns associated with Gd after repeated usage of some Gd-based MRI contrast agents, we introduce an effective manganese alternative for the MRI detection of GSZS.

Methods

The precursor for the ligand was synthesized following previously published protocols1. Bis(pyridine-2-ylmethyl)ethane-1,2-diamine was coupled to the precursor using PyAOP  and DIPEA. Male C57Bl6 mice were imaged at 4.7 T and 9.4 T using Varian/Agilent scanners. Two ge3d T1-weighted scans were obtained as a baseline (TE/TR = 1.69/3.35 ms, Average = 4, θ=20°). Mice then received 0.07 mmol/kg MnL i.v. plus 2.2 mmol/kg D-Glucose i.p. and sequential 3d T1-weighted scans were obtained for 90 minutes until clearance of the agent was evident. Images were analyzed using ImageJ. Biodistribution of MnL was measured by ICP-AES in tissues 15 and 90 min after injection of 0.07 mmol/kg MnL (or saline) with or without 2.2 mmol/kg D-glucose into male C57Bl6 mice.

Results/Discussion

The newly synthesized MnL zinc sensor shows strong zinc binding capabilities (KD(Zn)  = 90 nM; Figure 1). MnL appears to be excreted as an intact complex after renal filtration (as observed by LC-MS of urine), but degraded during hepatobiliary clearance (as observed by LC-MS of bile). Control experiments showed marginal enhancement in the prostate and pancreas when D-glucose was not co-injected suggesting that MnL is uniquely sensitive to zinc (Figure 2). Tissue measurements of Mn content by ICP-AES together with imaging show the clearance show that after 15 minutes post-injection, MnL is found primarily in the kidneys, liver, pancreas, and prostate. After 90 minutes, the Mn content was greatly diminished in kidney and liver tissues but accumulated in the spleen and in the prostate.  The later observation may reflect Mn in the prostatic urethra during excretion of the agent. MnL distribution shows no accumulation in the heart indicating that MnL remains intact during circulation.

Conclusions

In summary, a zinc-responsive Mn-based MRI contrast agent has been used to image GSZS in mice, similar to that reported previously using Gd-based agents2,3. The tissue Mn content showed that the MnL chelate is excreted intact through renal filtration, and appears to be degraded in the liver and bile as free Mn2+ ions. These data suggest that a Mn-based zinc sensor provides an alternative to the use of Gd agents for detection of GSZS by MRI.

References
[1] Gale, E. M., Atanasova, I. P., Blasi, F., Ay, I. & Caravan, P. A, 2015, Manganese Alternative to Gadolinium for MRI Contrast. J Am Chem Soc 137, 15548-15557.
[2] Yu, J. et al. 2015, Amplifying the sensitivity of zinc(II) responsive MRI contrast agents by altering water exchange rates. J Am Chem Soc 137, 14173-14179.
[3] Esqueda, A. C. et al. 2009, A new gadolinium-based MRI zinc sensor. J Am Chem Soc 131, 11387-11391.
[4] Martins, A.F. et al. 2018, Imaging Insulin Secretion from Mouse Pancreas by MRI Is Improved by Use of a Zinc-Responsive MRI Sensor with Lower Affinity for Zn2+ Ions. J Am Chem Soc 140, 17456-17464.
Zinc detection mechanism with Mn-based contrast agent.

Figure 1. MnL in its “off” configuration has a longitudinal relaxivity of r11.5T = 3.5mM-1s-1 and once in its “on” configuration bound to Zn(II) and HSA relaxivity increases to r11.5T = 14.1mM-1s-1.  

In vivo MR studies with MnL

Figure 2. A) In vivo evaluation of MnL as a zinc sensor by 3D T1W-MRI before and after injection of MnL 0.07 mmol/kg i.v. and  D-glucose 2.2 mmol/kg i.p. B0 = 9.4 T. B) MRI signal intensity change measured from ROIs placed on each organ, normalized to a muscle ROI and calculated as a percentage change compared to pre-injection scans. B0 = 4.7 T,  N = 4. 

Keywords: Zinc sensors, Manganese, GSZS, Responsive agents, Prostae & Pancreas
720

Metal-doped iron oxide nanoparticles for positive contrast in MRI

Irene Fernández-Barahona1, 2, Juan Pellico3, Ignacio Rodríguez2, Jesús Ruiz-Cabello4, Fernando Herranz1, 2

1 Consejo Superior de Investigaciones Científicas, Instituto de Química Médica (IQM-CSIC), Madrid, Spain
2 Facultad de Farmacia. Universidad Complutense de Madrid, Departamento de Química en Ciencias Farmacéuticas, Madrid, Spain
3 Kings College London, School of Biomedical Engeneering and Imaging Sciences, London, Spain
4 CIC biomaGUNE, Ikerbasque, Basque Foundation for Science, Donostia-San Sebastián, Spain

Introduction

Iron oxide nanoparticles have been traditionally studied as a T2 contrast agents for MRI because of their superparamagnetic behaviour. Nevertheless, T1-based positive contrast, being much more advantageous for clinical applications, is still limited to gadolinium- or manganese-based imaging tools. Recently, we have shown how the microwave-driven synthesis of citrate-coated nanoparticles renders maghemite nanoparticles with large r1 values.1 In this work we have studied how different dopant metals (Mn, Co, Ni, Cu and Zn) affect relaxometric properties of these nanoparticles.

Methods

IONP@X-cit (X being dopant metal) synthesis was performed following a microwave-driven protocol. A mixture of FeCl3, citric acid trisodium salt and the dopant metal (Mn, Co, Ni, Cu or Zn) was dissolved in water. Subsequently, hydrazine monohydrate was added and the mixture was introduced into the microwave. Samples were heated for 10 minutes at 120 °C. Once this step was completed, nanoparticles were purified through a gel filtration column and stored for further characterisation.
After thorough sample characterisation, MR contrast agent capability of samples with improved relaxometric properties were tested in vivo in tumour-bearing mice. Specific tumour accumulation was achieved by conjugating the nanoparticles to RGD peptide, that specifically binds avb3 integrin.

Results/Discussion

Hydrodynamic size distributions measured by dynamic light scattering show a narrow size distribution for all samples. Zeta potential values are around -30 mV as expected for citrate coated nanoparticles. Core size of the nanoparticles, measured by STEM, is around 4 nm for all samples. Magnetisation curves for all samples show a superparamagnetic behaviour. Relaxometric properties of the samples were studied to assess how they were affected by metal doping. The best longitudinal relaxivity obtained was for the sample doped with Cu, while transversal relaxivity value was maintained low for all samples, yielding r2/r1 ratios suitable for T1-weighted contrast for all samples. In vivo MR angiography experiments were performed to test samples’ capacity as blood pool contrast agents. Best sample was posteriorly used for targeted-tumour detection; proving its superior T1 contrast agent capability and its potential use in vivo for diverse imaging applications.

Conclusions

We produced metal-doped IONPs with improved T1 contrast agent capability and relaxometric properties that can be used in vivo for different biomedical applications.

References
[1] Pellico, J. et al. One-Step Fast Synthesis of Nanoparticles for MRI: Coating Chemistry as the Key Variable Determining Positive or Negative Contrast. Langmuir 33, 10239–10247 (2017).
[2] Fernández-Barahona, et al. Cu-Doped Extremely Small Iron Oxide Nanoparticles with Large Longitudinal Relaxivity: One-Pot Synthesis and in Vivo Targeted Molecular Imaging. ACS Omega. 2019, 4 (2), 2719–2727.
Sample characterisation
In vivo MRI with IONP@Cu-cycloRGD
Keywords: iron oxide nanoparticles, metal doping, T1 contrast
721

Responsive phosphorus–containing contrast agent for 1H/31P MR

Natalia Ziółkovská1, 2, Martin Hrubý3, Martin Vít4, 2, Zdislava Pechrová3, 5, 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 and Clinical Experimental Spectroscopy, Prague, Czech Republic
3 Czech Academy of Sciences, Institute of Macromolecular Chemistry, Supramolecular polymer systems department, Prague, Czech Republic
4 Technical University of Liberec, Technical cybernetics, Liberec, Czech Republic
5 Charles university, Faculty of Sciences, Department of Physical and Macromolecular Chemistry, Prague, Czech Republic

Introduction

The aim of this project is to develop and test a responsive phosphorus–containing contrast agent for 1H/31P MR. Presented nanoparticles are based on phytate crosslinked with Ca2+ ions (CaIP6) and doped with different concentrations of Fe3+ ions, which can be detached from the probe and can act as a response to external physiological changes (lower pH in cancer tissue1, bacteria producing iron chelating siderophores2). Here we present in vitro study focusing on the effect of different iron concentrations on 31P MR signal and its rechelation using iron-binding deferoxamine (DFOA, siderophore).

Methods

Probes were doped with different concentrations of Fe3+ ions (cFe3+= 0, 0.68, 2.04, 2.73, 5.43, 13.6 mM). In vitro simulation of Fe3+ release was performed with DFOA (2.73 mM Fe3++DFOA; 1:1)3, a compound possessing high affinity to Fe3+ ions. Contrast was tested at 4.7 T MR using self-made 1H/31P RF coil. Single pulse sequence was used for obtaining 31P T1 relaxation times (TR=160–3000ms, 3000 acquisitions) and for spectra comparison between probes (TR=500ms, ST=16h 40m). For 31P MRI chemical shift imaging was optimized (TR=500ms, ST=1h, resolution 2.5x2.5x5.8mm3). For visualization of Fe3+ influence on MR signal 1H MRI of phantoms was measured (T2-weighted imaging, TR/TE=2000/24ms). SNR was calculated from 1H, 31P MRI and 31P MRS. Contrast cytotoxicity was assessed using alamarBlue.

Results/Discussion

1H MRI of phantoms confirmed iron influence on T2-weighted images (13.6 mM Fe3+: SNR=1.2; 0 mM Fe3+: SNR=86.2). Effect of iron chelation of DFOA was also observed in 1H MRI (2.73 mM Fe3+: SNR=25.4; 2.73 mM Fe3++DFOA: SNR=34.7) (Fig. 1) and is visible in 31P MRS data (2.73 mM Fe3++DFOA: SNR=72.2; 2.73 mM Fe3+: SNR=13; no iron doping: SNR=45.9) (Fig.2). We confirmed linked iron ions impact on 31P MRS signal widening with increasing Fe3+ concentrations with no signal obtained from concentrations higher than 2.73 mM Fe3+. In 31P MRI signal was observed for low Fe3+ concentrations, with the highest SNR=57.1 for 0.68 mM Fe3+ (Fig. 1). 31P T1 relaxation times are decreasing with iron doping increase (0.68 mM Fe3+: T1=487ms; 2.04 mM Fe3+: T1=305ms). Cytotoxicity testing stated that our probe is not significantly influencing cells viability.

Conclusions

1H and 31P MR imaging modalities offer complementary information about physiological conditions in living tissue and can be easily combined at the same experiment. Presented phosphorus probe can act as a response for sensing early tissue pathology and biochemical processes in living organism. Our results confirms nontoxicity of probes and possible use as a 1H/31P MR responsive probe influenced by iron-chelating siderophores (DFOA).

Acknowledgment

The study was supported by the Charles University, GA UK No 358119; Institute for Clinical and Experimental Medicine IKEM, IN00023001; Charles University, First Faculty of Medicine; Ministry of Education of the Czech Republic through the SGS project no. 21332/115 of the Technical University of Liberec.

References
[1] Kato, Y, et al, 2013, Acidic extracellular microenvironment and cancer. Cancer Cell Int., 13, 89
[2] Miethke, M, Marahiel, M.A, 2007, Siderophore-Based Iron Acquisition and Pathogen Control, Microbiol. Mol. Biol. Rev, 71(3), 413–45
[3] Porter, J, Viprakasit V, 2014, Iron overload and chelation, In:Cappellini MD, Cohen, A, Porter, J, et al. Guidelines for the Management of Transfusion Dependent Thalassaemia (TDT) [Internet]. 3rd edition. Nicosia (CY): Thalassaemia International
Fig. 1

MR images of CaIP6 containing 0, 0.68, 2.04, 2.73, 5.43, 13.6 mM Fe3+ and 2.73 mM Fe3+ probe with DFOA (1:1). UPPER: 1H MRI (T2-weighted imaging, TR/TE=2000/24ms). MIDDLE: 31P MRI (CSI, TR=500ms, ST=1h, resolution 2.5x2.5x5.8mm3). BOTTOM: SNR calculated from both 1H and 31P MR images.

Fig. 2

MR signals comparison of CaIP6 with no iron linked (A), 2.73 mM Fe3++DFOA (B), 2.73 mM Fe3+ (C). UPPER: 31P MRS (single pulse sequence, TR=500ms, ST=16h 40m). MIDDLE: 31P MRI (CSI, TR=500ms, ST=1h, resolution 2.5x2.5x5.8mm3). BOTTOM: SNR calculated from both 31P MRS and 31P MRI.

Keywords: 31P magnetic resonance imaging, 31P magnetic resonance spectroscopy, responsive contrast agent
722

Novel probe based on fluorinated 2-oxazoline polymer for 19F magnetic resonance imaging

Martin Vít1, 2, Leonid Kaberov4, Daniel Jirák1, 2, 3

1 IKEM, Institute for Clinical and Experimental Medicine, Praha 4, Czech Republic
2 TUL, Technical university of Liberec, Liberec, Czech Republic
3 UK, Charles university, Praha 1, Czech Republic
4 IMC, Institut of macromolecular chemistry, Praha 6, Czech Republic

Introduction

Fluorine 19F magnetic resonance imaging (MRI) has emerged especially in experimental preclinical and clinical studies. The limited commercial supply of probes leads to further research on new substances including smart contrast agents (thermo-responsive, pH-responsive and more (1)). Comparable sensitivity to proton and no background in the mammal physiological environment enabling absolute quantification of 19F MR signal class fluorine based probes as excellent ones. Here we present new MRI fluorine probe based on utilization of 2-oxazolines.

Methods

Probe is based on poly-2-oxazolines with 2-(3,3,3-trifluoroprophyl) 2-(3,3,3-trifluoropropyl)-2-oxazoline as fluorine containing part.(2). For use in biological, hydrophilic behavior was done by 2-methyl-2-oxazoline (MeOx) and 2-ethyl-2-oxazoline (EtOx). 2-n-octyl-2-oxazoline is hydrophobic modification and 2-n-propyl-2-oxazoline is thermoresponsive modification (Fig.1a). Probe was tested in vitro in tubes with 0.2ml of substance.
Experiment was performed at 4.7T system (Bruker Biospec 47/20, Ettlingen, Germany)
We analyzed 8 samples HF1, HTF7, HTF3, HTF4 HF3, HF4, HF5 and HLF4 (Fig.1). 19F imaging procedure is combination of 1H navigator and 19F imaging sequence; results are on figure 2.
Fluorine T1 times were measured by spectroscopic method. Fluorine T2 times were measured as CPMG

Results/Discussion

T1 relaxation times were calculated in the range 40-251 ms. T2 assessment  was successful only for HF5 due to low signal intensity; T2 relaxation for HF5 is 122 ms. Results of HF5 are summarized on figure 1b.
Images with high signal-to-noise ratio (SNR) were obtain for HF1 (12.8), HF3 (28.4), HF4 (53.6) and HF5 (63.1). These 4 polymers are clearly imaged by MRI. Thermoresponsivity of HF5 probe was measured with results on fig. 1b. SNR changes, or relaxation time changes are negligible.
Low signal intensity for other polymers could be caused by extremely short relaxation times. In our case echo time of imaging sequence is 13.7 ms. Shorter relaxation times are not clinical relevant. It suggests using sequences allowing shorter TE. Another reason of low signal intensity could be intended by low hydrophilicity of substance.

Conclusions

We tested different fluorine contrast probes intended as thermoresponsive agents. The best imaging properties for our experimental set up was achieved for HF5, the probes HF4, HF3 and HF1 are still visible. HLF4, HTF3, HTF4 and HTF7 were not visualized. Next step will be imaging with shorter echo times and biological in vitro experiments to determine biocompatibility of the probes.

Acknowledgment

The work was supported by SGS 21332 TUL (CZ) and by the Ministry of Health of the Czech Republic (CZ-DRO, Project IN 00023001), Ministry of Education, Youth and Sports of the Czech Republic (grant #  LTC19032 and National Sustainability program I, grant # POLYMAT LO1507), Czech Science Foundation GA CR (grant 17-00973S).

References
[1] Jirak D. (2019), MAGMA, Fluorine polymer probes for magnetic resonance imaging: quo vadis? 32(1):173-185
[2] Kaberov, L. I.,2018,ACS Macro Lett. , 7 (1), 7–10.

Figure 1
a)Chemical structure and properties of the substances, b) Thermal dependence of MRI parameters, c) Vizualization of T1 realxation curve KL62 for 26 and 43°C
Figure 2
Imaging of fluorine probes. On each image are two substances with different concentration 40, 20, 10 and 5 mg/mL. On left side are 1H navigators (grey), on the right side are fluorine images (red).
Keywords: 19F MRI, Probe chemistry, High field MR, Smart contrast agent
723

Fluorescein and FMN - comparison of two photosensitizers for 19F nuclear spin hyperpolarization using photo-CIDNP

Frederike Euchner1, Christian Bruns1, Johannes Bernarding1, Markus Plaumann1

1 Otto-von-Guericke University / Medical Faculty, Institute of Biometrics and Medical Informatics, Magdeburg, Germany

Introduction

As demonstrated in earlier works, enhancements of 19F MR signals are possible using hyperpolarization techniques such as PHIP, DNP, and photo-CIDNP.1 To date, only photo-chemically induced dynamic nuclear polarization has enabled hyperpolarization of 19F in aqueous solution.2 The number of fluorinated molecules in medical applications has risen again in recent years and therefore it is still important to investigate those hyperpolarization methods. We show a comparison of two photosensitizers (fluorescein and FMN) with respect to the 19F nuclear spin hyperpolarization of 3-fluorotyrosine.

Methods

The investigated samples contained 3-fluoro-DL-tyrosine (2 mM) and 0.21 mM flavin mononucleotide (FMN) sodium salt hydrate or fluorescein (see Figure1), respectively. All compounds were dissolved in physiologic salt solution or D2O. The NMR-spectroscopic measurements were performed in a 5 mm NMR tube on a 7T MR system (Bruker WB-300 Ultrashield). An optical fiber was connected to a Cree XP E high power LED (455 nm)2-4 and was centrally positioned inside the solution. For comparison, irradiation times between 0 s and 30 s were chosen and a 90° pulse was used for the detection of 19F (P1 = 32.5 µs, PL1 = 17 W) NMR spectra. The temperature was varied in the range of 300-318K to compare possible temperature effects.

Results/Discussion

Signal enhancements are detectable with both photosensitizers when LED light was irradiated into the sample. The illumination took place direclty inside the magnet (7T), so that no transport between place of polarization and detection field had to be covered. Figure 2 shows for example the 19F MR signal of the hyperpolarized 3-fluoropyridine in aqueous solution. The comparison of the two photosensitizers shows slight advantages in terms of signal amplification for the FMN. The corresponding 19F spectra for FMN are shown in reference 2. However, warming up the sample or long time irradiation shows a disadvantage of FMN. The stability of the molecule structure seems to be lower. The low cost LED set-up allows irradiation without warming the sample (opposite to class IV lasers) as well as an irradiation of a spectrum of wavelengths. This allows more flexibility in choosing a photosensitizer.

Conclusions

Enhanced 19F MR signals of 3-fluorotyrosine can be observed when LED light with wavelengths around 455 nm illuminated a sample that contains photosensitizers such as FMN or fluorescein. Fluorescein shows a absorption maximum in a UV/Vis spectrum at around 496nm, while the oxidized form of the FMN has a maximum at 450 nm. This is an explanation for the different observed enhancements and can be easily optimized by replacing the LED in the set-up.

References
[1] Flögel, U, Ahrens, E, Fluorine Magnetic Resonance Imaging, 1. Ed., 2017, Singapore: Pan Stanford Publishing.
[2] Bernarding, J, Euchner, F, Bruns, C, Ringleb, R, Müller, D, Trantzschel, T, Bargon, J, Bommerich, U, Plaumann, M, Low-cost LED-based photo-CIDNP enables biocompatible hyperpolarization of 19F for NMR and MRI at 7 T and 4.7 T, ChemPhysChem, 2018, 19(19), 2453-2456.
[3] Feldmeier, C, Bartling, H, Riedle, E, Gschwind, RM, LED based NMR illumination device for mechanistic studies on photochemical reactions--versatile and simple, yet surprisingly powerful, J. Magn. Reson., 2013, 232, 39-44.
[4] Feldmeier, C, Bartling, H, Magerl, K, Gschwind, RM, LED-illuminated NMR studies of flavin-catalyzed photooxidations reveal solvent control of the electron-transfer mechanism, Angew. Chem., 2015, 127, 1363-1367.
Figure 1

Molecule structures of fluorescein (1), flavin mononucleotide (2) and 3-fluoro-DL-tyrosine (3).

Figure 2
The four 19F NMR spectra show the fluorine signal of 3-fluoropyridine in dependence of the time of light irradiation. Fluorescein was added as photosensitizer. The solvent of this sample was pure D2O.
Keywords: 19F, Hyperpolarization, photo-CIDNP, Fluorescein, FMN
724

Targeting of cancer integrins by using RGD/Gd-Giant Unilamellar vesicles by exploiting Magnetization Transfer Contrast MRI

Giuseppe Ferrauto1, Martina Tripepi1, Enza Di Gregorio1, Silvio Aime1, Daniela Delli Castelli1, Valeria Bitonto1

1 University of Torino (It), Dept of molecular biotechnologies and health sciences, Torino, Italy

Introduction

An important topic in biomedicine is the targeting of cancer. MRI can be the election technique but its application is strongly hampered by the low sensitivity.For overcoming this drawback, herein we investigated the use of RGD-containing-Giant Unilamellar vesicles (GUVs liposomes) as carriers of a high amount of Gd-MRI contrast agents.Furthermore, Magnetization Transfer Contrast (MTC) MRI has been employed. In fact, it has been previously demonstrated that MTC depends on the T1 of the tissue, so it can report about the presence of a Gd-MRI contrast agents.1

Methods

GUVs were prepared by using the gentle swelling method2. Cyclic RGD targeting moiety was added to the external membrane. The inner aqueous cavity was filled with Gd-HPDO3A (Prohance, Bracco Imaging srl) as MRI contrast agents and Rhodamine, as fluorescent probe.  As control, GUVs without cyclic RGD targeting moiety were used. U-87-MG human glioblatoma cells were used for cell targeting experiments. As animal model, nu/nu mice were subcutaneously injected in the legs with 106 U-87-MG cells and imaged by MRI, after 15 days. MRI was carried at 7T, by using a Bruker spectrometer provided with a micro-imaging coil. T2w, T1w and Z-spectra of all the samples (in vitro and in vivo) were acquired.Confocal microscopy images and ICP-MS were used for validating results.

Results/Discussion

In vitro results showed a specific binding of RGD-GUVs to U-87-MG cells. Microscopy revealed that i) RGD-GUVs are anchored to cells, ii) a low number of GUVs per cell is present with a clusterization into specific region, iii) GUVs are attached to the external surface of cells, with no internalization, iv) neither macrophages uptake nor cells toxicity is reported. (Fig.1).MRI of cells’ pellets after incubation with RGD-GUVs or untargeted ctrl-GUVs has been acquired. No difference in T1 signal is present, whereas a 15% MT contrast is present in RGD-GUVs- treated cells in comparison to ctrl-GUVs-treated cells.Then, MR images of tumour region have been acquired pre and post (t=0, 4h and 24h) the administration of RGD-GUVs or ctrl-GUVs. Only at 4h post administration of RGD-GUVs, there is a MTC variation. The effect is ca. 15% (Fig.2).  Immunofluorescence analysis and ICP-MS analysis (for Gd-detection) of explanted tumours confirm the specific accumulation of RGD-GUVs in the tumor regions.

Conclusions

Giant liposomes can overcome some limits of small unilamellar liposomes preserving their main advantages. In fact, they are neither sequestered by macrophages nor internalized inside cells.MTC has been proven to be suitable for the detection of small amounts of Gd-agents.The simultaneous application of the highly sensitive RGD-GUVs and the powerful MTC-MRI technique can allow reaching a success in the in vivo targeting of tumors integrins.

References
[1] Delli Castelli D, Ferrauto G, Di Gregorio E, Terreno E, Aime S. NMR Biomed. 2015 Dec;28(12):1663-70;
[2] J.P. Reeves, R.M. Dowben, J. Cell. Physiol, 1969, 73, 49-60.
Figure 1
Confocal microscopy image of RGD-GUVs liposomes (Red=Rhodamine) anchored to the external surface of U-87-MG cells (green=phalloydin)
Figure 2

MTC enhancement after administration of RGD-GUVs or ctrl-GUVs.

Keywords: liposomes, targeting, giant, MRI, magnetization transfer contrast
726

Gadolinium-labeled Discoidal Polymeric Nanoconstructs as Rationally Designed Magnetic Resonance Imaging Agents

Miguel F. M. M. Ferreira1, Annalisa Palange1, Paolo Decuzzi1

1 Istituto Italiano di Tecnologia, Laboratory of Nanotechnology for Precision Medicine, genova, Italy

Introduction

As compared to other clinically relevant modalities, Magnetic Resonance Imaging (MRI) offers excellent soft-tissue contrast; diffusion-weighted and functional imaging as well as MR spectroscopy capabilities. Nanoparticles can boost contrast enhancement in MR imaging and simultaneously deliver other imaging and therapeutic agents, enabling multimodal imaging and theranostics. In this work, polymeric nanoconstructs, labeled with Gd(DOTA), and exhibiting different geometrical properties are employed to sample the vascular permeability of the diseased brain microcirculation via MR imaging.

Methods

Three different nanoconstruct configurations were considered: i. micelles realized with Gd(DOTA)-DSPE and Gd(DOTA)-PEG-DSPE (Figure 1a); ii. spherical polymeric nanoconstructs (SPNs) coated with Gd(DOTA)-PEG-DSPE or Gd(DOTA)-DSPE; and iii. discoidal polymeric nanoconstructs (DPNs) loaded with Gd(DOTA)-DSPE. SPNs, realized via a bottom-up synthesis strategy, consist of a poly (lactic-co-glycolic acid) (PLGA) core stabilized by an external monolayer of mixed phospholipid (DPPC, DSPE-PEG-COOH, and the Gd monomers). Differently, a top-down fabrication approach was used for the fabrication of DPNs. Specifically, soft-DPNs were realized by mixing 30 mg of PLGA with 6 mg of 1 kDa chains of PEG. MR imaging was performed to assess the particle properties in terms of blood pool MRI agents.

Results/Discussion

SPNs were monodisperse nanoparticles with a diameter of ~ 180 nm, whereas DPNs presented a discoidal shape with a diameter of ~ 1,000 nm and a height of ~ 400 nm (Figure 1b). A progressive enhancement in longitudinal relaxivity r1 was observed with the particle size, rising from ~ 4 mM-1s-1 for the small Gd(DOTA) molecules up to ~ 70 mM-1s-1 for the larger nanoconstructs. Gd(DOTA)-DSPE SPNs presented a maximum of 30 mM-1s-1 at 30 MHz; Gd(DOTA)-PEG-DSPE SPNs exhibited a maximum of 68 mM-1s-1 at 50 MHz, and DPNs showed a maximum of 43 mM-1s-1 at 30 MHz (Figure 1c). Note that these r1 values are 10 to 20 times higher than those measured for clinically available MRI agents. Following the SBM theory, this r1 amplification could be ascribed to the lower tumbling time and more favorable hydration conditions of the larger nanoconstructs over the clinically used molecular Gd(DOTA).

Conclusions

Gd-based theranostic nanoconstructs are proposed with high MRI relaxivity (> 10-fold increase), at clinically relevant magnetic fields. This boost in relaxivity demonstrates that the confinement of Gd-complexes within porous matrices amplifies the longitudinal relaxivity and returns highly efficient MRI contrast agents. These nanoconstructs could be used as vascular pool agents and characterize the permeability of the malignant vasculature.

AcknowledgmentThis project was partially supported by the European Research Council, under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 616695, by the Italian Association for Cancer Research (AIRC) under the individual investigator grant no. 17664, and by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 754490.
Figure 1:
Structure and properties of Gd-based Nanoconstructs.  NMRD profiles for the Gd micelles, and two Gd SPN and two Gd DPN configurations, compared with the clinical agent Gd(DOTA).
Keywords: Gd(DOTA) Nanoconstructs, MRI, vascular permeability
727

The issue of Gadolinium retention in tissues upon the administration of Gd-based MRI Contrast Agents

Chiara Furlan1, Enza Di Gregorio1, Silvio Aime1, Eliana Gianolio1

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

Introduction

Gadolinium based contrast agents (GBCAs) are commonly used at clinical settings to add relevant physiological information to MR images. Recently, a renewed interest on the possibility that GBCAs may cause adverse effects, came out: a number of studies reported the occurrence of Gd retention in the brain of patients that have been previously administered with multiple doses of GBCAs, also in the absence of renal impairment.1 Herein, different preclinical investigations aimed at understanding how and why Gd is retained in the brain and other tissues of mice, are reported. 2-4

Methods

Mice were administered with clinically approved GBCAs, both linear and macrocyclic, following several administration protocols differing in the number and doses of iv injections and in the sacrifice time after the last administration. After sacrifice, tissues were collected, mineralized and the amount of Gadolinium quantified through ICP-MS. UPLC-MS analysis was carried out to identify the chemical form of retained Gd. The same analytical techniques were used in order to investigate the distribution of Gd in the blood components of mice administered with gadodiamide and gadoteridol.

Results/Discussion

Principal results concerning the addressed issues are the following: 1) In all the investigated organs/tissues the linear neutral gadodiamide complex is retained more than the charged linear gadopentetic acid and the macrocyclic gadoteridol; areas of long term deposition appear to be spleen and bones. 2) Significant amounts of Gd has been found in RBCs and WBCs, thus GBCAs can cross blood cells membranes. 3) Both in brain and in blood cells, the less stable Gadodiamide is dechelated while the more robust gadoteridol is not. 4) Whereas GBCAs endowed with different stabilities cross the barriers to the same extent, the successive “journey” is affected by their relative stabilities.

Conclusions

These studies bring new knowledge that goes beyond the field of the potential toxicity of the used Gd-complexes as they allow to get more insight into the complex domain of the thermodynamics and kinetics involving metal containing species in living systems. Moreover, these works allow to face, in a timely manner, the Gd retention issue in order to foresee and, possibly, avoid potential long-term correlated side effects.

References
[1] McDonald, R.J, Levine, 2018 Radiology, 289, pages 517-534.
[2] Gianolio E, Di Gregorio E, and Aime S, 2019, European Journal of Inorganic Chemistry, 137-151.
[3] Di Gregorio E, Furlan C, Atlante S, Stefania R, Gianolio E, Aime S. 2019 Invest. Radiol. 
[4] Di Gregorio E, Ferrauto G, Furlan C, Lanzardo S, Nuzzi R, Gianolio E, and Aime S, 2018 Invest. Radiol. 53, 167-172.
Amount of Gd in RBCs
Amount of Gd found RBCs 24, 48, 96, and 240 hours after the administration of a single dose of Gadodiamide and Gadoteridol (1.2 mmol/kg).
Amount of Gd in WBCs
Amount of Gd found in WBCs 24, 48, 96, and 240 hours after the administration of a single dose of Gadodiamide and Gadoteridol (1.2 mmol/kg).
Keywords: Gadolinium, Retention, In vivo