15th European Molecular Imaging Meeting
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Multimodal/Multifunctional | Probe Chemistry

Session chair: Fernando Herranz (Madrid, Spain); Olga Koshkina (Nijmegen, Netherlands)
 
Shortcut: PW09
Date: Wednesday, 26 August, 2020, 5:30 p.m. - 7:00 p.m.
Session type: Poster

Contents

Abstract/Video opens by clicking at the talk title.

700

Wazaby platform for the development of bimodal probes and theranostic agents

Robin Lescure1, Malorie Privat1, 2, Jacques Pliquett1, Franck Denat1, Catherine Paul2, Ali Bettaïeb2, Pierre-Simon Bellaye3, Bertrand Collin3, Lucie Sancey4, Christine Goze1, Ewen Bodio1

1 University of Burgundy, ICMUB, Dijon, France
2 University of Burgundy, EPHE, Dijon, France
3 Georges-François LECLERC Cancer Center, Dijon, France
4 Institute for Advanced Biosciences, La Tronche, France

Introduction

Nowadays, more and more sophisticated molecular systems are designed for addressing the needs of biologists: development of selective contrast agents, bimodal probes, theranostics… Such systems often require long and tricky syntheses, especially when in vivo applications are targeted. Thus, a growing interest for new versatile platform molecules has been noted in order to facilitate the access to these. Here, we chose to focus on an aza‑BODIPY dye platform, which already proved to be effective for in vivo molecular imaging (near infrared emission), but which displays poor water-solubility.

Methods

We found a way to water-solubilize the aza-BODIPY via the functionalization of the boron atom.1 Moreover, we were able to develop a four-steps synthesis, which gives access to the platform at the gram-scale in a few days. It displays numerous functionalizable positions, but we focused our attention on grafting the moieties of interest on the two arms borne by the boron atom. Obviously, the introduction of two identical moieties is easy, but, interestingly, we were able to develop an effective one-pot method for introducing two different moieties. Several groups of interest were grafted thanks to these methods: PET/SPECT probe, bio-vector, MRI probe, therapeutically relevant gold complexes.

Results/Discussion

The different vectorized contrast agents, bimodal probes, theranostics were obtained in 1-2 days starting from the water-soluble aza-BODIPY platform at the scale of dozens of milligrams. Two derivatives were obtained with slightly different approaches of this method, presenting respectively a NODAGA and a DOTA moiety. The latest was further studied, being first bioconjugated to a Trastuzumab antibody and tested through in vitro and in vivo experiments. This bioconjugated probe showed promising results, and was also tested in a fluorescence-guided surgery experiment on a mouse. Theranostics were synthesized from the precursor and their toxicity on various cancer cell lines (CT26, MC38, 4T1, SW480) shown IC50 values comparable to those of auranofin.

Conclusions

This communication will focus on the synthesis and the scope of functionalization of the platform. An overview of the different in vitro and in vivo biological results will be given to highlight the great potential of this platform.

Acknowledgment

Support was provided by the Conseil Régional de Bourgogne (PhD JCE grant # 2015-9205AAO033S04139/BG0003203), and the Conseil Régional de Bourgogne Franche-Comté. The Ministère de l’Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), and the French Research National Agency (ANR) via project JCJC “SPID” ANR-16-CE07-0020 and project JCJC “WazaBY - ANR-18-CE18-0012” are gratefully acknowledged. Pr. Anthony Romieu and Iris Biotech are warmly thanked for the TOTA and the Cy5 synthesis, and Dr. Victor Goncalves for the diethylsquarate. The authors thank CheMatech for providing chemicals used in the scope of this work. This work is part of the projects “Pharmacoimagerie et agents theranostiques” et “Chimie durable, environnement et agroalimentaire” supported by the Université de Bourgogne and the Conseil Regional de Bourgogne through the Plan d’Actions Regional pour l’Innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs. FrenchBIC and GDR AIM are acknowledged for fruitful discussion. Oncodesign is acknowledged for fruitful discussion and for their support. The authors thank the "Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne” (http://www.wpcm.fr) for access to analytical instrumentation.

References
[1] Pliquett, J., 2019, ‘A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging’, Bioconjugate Chem., 30, 1061-1066
Overview presentation of the platform approach for the synthesis of bimodal probes and theranostics
Keywords: fluorescent platform, optical imaging, theranostics, bimodality, SPECT
701

In Vivo Validation of Fluorine-18 Labeled Iron Oxide/Aluminum Hydroxide Nanoparticles for Simultaneous PET/MRI Imaging – Potential Applications in Stem Cell Tracking

Sarah Belderbos1, Manuel Antonio González-Gómez2, Yolanda Piñeiro2, Frederik Cleeren3, Jens Wouters4, Christophe M. Deroose5, Willy Gsell1, An Coosemans6, 7, Guy Bormans3, José Rivas2, Uwe Himmelreich1

1 KU Leuven, Biomedical MRI, Department of Imaging and Pathology, Leuven, Belgium
2 Universidade de Santiago de Compostela, NANOMAG Group, Department of Applied Physics, Santiago de Compostela, Spain
3 KU Leuven, Radiopharmaceutical Research, Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium
4 KU Leuven, Molecular Small Animal Imaging Center (MoSAIC), Leuven, Belgium
5 KU Leuven/ UZ Leuven, Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, Leuven, Belgium
6 KU Leuven, Laboratory for Tumor Immunology and Immunotherapy, Department of Oncology, Leuven, Belgium
7 UZ Leuven, Department of Gynaecology and Obstetrics, Leuven, Belgium

Introduction

In a previous study, we have reported on the potential of fluorine 18F-labeled iron oxide nanoparticles embedded in aluminum hydroxide (Fe3O4@Al(OH)3 NPs) as efficient in vitro PET/MRI contrast agents1. Preliminary in vivo studies indicated the potential to visualize both radiolabeled (RL) NPs and RL NP-labeled mouse mesenchymal stem cells (mMSCs) in mice1,2. However, more research is required to understand mMSCs migration processes3. In this study, we evaluated these RL NPs and RL NP-labeled mMSCs as dual contrast agents to assess their in vivo biodistribution, stability and biocompatibility.

Methods

Synthesis and labeling of Fe3O4@Al(OH)3 NPs with [18F]NaF and NP labeling of mMSCs was performed as described before1. To evaluate their in vivo biodistribution, RL NPs, 105 RL NP-labeled mMSCs or [18F]NaF were injected intravenously in healthy C57Bl/6 mice. A 1h PET scan was simultaneously acquired with dynamic contrast-enhanced (DCE)-MRI, whole-body 3D T2-weighted MRI and parametric T2 maps using a 7T MRI with PET insert (Bruker PCI). Afterwards, the mouse bed was transferred to a Bruker µCT scanner to determine [18F]F- bone uptake. Furthermore, long-term follow-up (until 7 days post injection) of NPs/cells was performed using anatomical MRI, T2 maps, BLI and analysis of white blood cell (WBC) count. After sacrificing the animals, liver, lungs and spleen were analyzed using a γ-counter.

Results/Discussion

After injection of RL NPs or RL NP-labeled mMSCs, NPs could be visualized in liver using both PET and MRI (within 1h and 4h after NP and cell injection, respectively). NP clearance from the liver was detected 7 days after injection. On the other hand, mMSCs lung trapping could not be visualized with MRI, but only with PET (Fig1A). Serial PET/CT acquisition revealed [18F]F- uptake in the mice’s skeleton, indicating in vivo radiolabel dissociation from NPs. However, this did not hamper NP/cell visualization (Fig1B). BLI confirmed mMSCs lung uptake and showed rapid cell death within 24h of injection (Fig1C-D). Furthermore, both groups presented with spleen radiotracer uptake, indicating NPs clearance via the reticuloendothelial system. All in vivo results were confirmed by ex vivo γ-counter measurements. Leukocytosis analysis showed no differences in the number of WBC or its subtypes over 7 days or between the different groups.

Conclusions

The novel 18F-labeled Fe3O4@Al(OH)3 NPs are promising in vivo PET/MRI contrast agents as they can be tracked with both imaging modalities and do not induce any effect on WBC count. However, fluorine coupling stability needs to be improved. Future research will focus on mMSCs therapeutic potential in different tumor types and spinal cord injuries, and mMSCs migration towards site of injury, which will be studied using these dual contrast agents.

Acknowledgment

This work was funded by the European Horizon 2020 ‘PANA’ project (grant agreement 686009), the Flemish Agency for Innovation by Science and Technology (IWT agreement 140061, SBO ‘NanoCoMIT’) and the KU Leuven program financing ‘In Vivo Molecular Imaging Research’ (IMIR, PF10/017).

The authors would like to thank Prof. Greetje Vande Velde (Biomedical MRI), Ms. Roxanne Wouters and Ms. Ann Vankerckhoven (Laboratory for Tumor Immunology and Immunotherapy) for technical assistance, advice and discussions.

References
[1] González-Gómez, MA, Belderbos, S, Yañez-Vilar, S, Piñeiro, Y, Cleeren, F, Bormans, G, Deroose, CM, Gsell, W, Himmelreich, U, Rivas, J. 2019, 'Development of Superparamagnetic Nanoparticles Coated with Polyacrylic Acid and Aluminum Hydroxide as an Efficient Contrast Agent for Multimodal Imaging', Nanomaterials, 9(11), 1626
[2] Belderbos, S, González-Gómez, MA, Piñeiro, Y, Cleeren, F, Wouters, J, Manshian, BB, Soenen, SJ, Deroose, CM, Gsell, W, Bormans, G, Rivas, J, Himmelreich, U. 2019, 'Radiolabeled Iron Oxide/Aluminum Hydroxide Nanostructures as New Dual Contrast Agents for Simultaneous PET/MRI', 14th European Molecular Imaging Meeting, Glasgow (UK)
[3] Fu, X, Liu G, Halim, A, Ju, Y, Luo, Q, Song, G. 2019, 'Mesenchymal Stem Cell Migration and Tissue Repair', Cells, 8(8), 784
Figure 1: Biodistribution of mesenchymal stem cells labeled with radiolabeled nanoparticles.
A-B) Representative 1h static PET scan overlaid with A) the 3D T2-weighted MRI scan (axial orientation), simultaneously acquired, and B) the bone-masked CT scan (coronal orientation), serially acquired, indicating the trapping of the cells in the lungs, and the presences of free [18F]F- in the bones (indicated by the white arrows). C-D) Bioluminescence imaging demonstrates the presence of viable cells in the lungs 1h post injection and rapid cell death from 24h after injection on. All images were acquired within 1 hour of cell injection in a healthy mouse.
Keywords: cell tracking, nanoparticles, preclinical imaging, radiolabeling, simultaneous PET/MRI
702

Intracellular Uptake, Localization, and Excretion Kinetics of PCFE Nanoparticles used for Cardiac Stem Cell Labeling

Christakis Constantinides1, Louisa Potamiti2, Petros Patsali2, Carolyn Carr3, Mangala Srinivas4, Andreas Hadjisavvas2, Kyriacos Kyriacou2

1 Chi Biomedical Ltd, Limassol, Cyprus
2 The Cyprus Institute of Neurology and Genetics and The Cyprus School of Molecular Medicine, Dept: of Electron Microscopy /Molecular Pathology, Nicosia, Cyprus
3 University of Oxford, Department of Physiology, Anatomy & Genetics, Oxford, United Kingdom
4 Radboud University Medical Center, Radboud Institute for Molecular Life Sciences (RIMLS), Nijmegen, Netherlands

Introduction

While fluorinated nanoparticles (NPs) have been successfully used to label/track stem cells in the brain [1, 2], cardiac applications have languished. Recently, we successfully labelled/imaged/tracked cardiac progenitor stem cells (CPCs) in mice in vivo [3], and showed that the signal persisted for ~7 days. To understand the etiology, this study investigates the temporal accumulation, localization, and excretion of perfluoro-crown-ether nanoparticle labels (PFCE-NPs) in CPCs in vitro using transmission electron microscopy (TEM) and fluorescence imaging.

Methods

CPCs were isolated from adult, C57BL/6 mouse atria, expanded in culture, and incubated with PFCE-NPs (with Atto647) for ~24 h and with FuGENE (25 μl in ~106 cells). Live/fixed cells were used to study fluorescence/morphology. TEM: Fixed cells were dehydrated at increasing ethanol concentrations and were embedded in an epon araldite/resin mixture. After polymerization, ultrathin sections (80–100 nm) were cut, stained, and imaged with a JEOL TEM at 80 kV. Fluorescence Imaging: Live labeled (LB)/FuGENE-labeled (F-LB) cells (n=3) were cultured/imaged (D1-D3) to assess survival/fluorescence responses with an Olympus IX73 microscope (exposure time=1 s, λexcitationreception overlapped Atto647 wavelengths). Fresh media were added (D2/D3) before imaging and supernatant solutions were re-imaged.

Results/Discussion

TEM images show endocytic morphologies (Fig. 1, labeling (LB), FuGENE-labeling [F-LB]). Fig. 2 shows increased endosomes (E)/lysosomes (L) after F-LB (G-I), compared with control (A-C) and LB (D-F), LB). NP sizes were quantified in control water emulsions of NP/premixed NP/FuGENE (NP-F) based on TEM (J, K). Noted is a) morphological regularity and b) size/distribution differences with FuGENE (diameters: NP 141±36 nm vs. NP-F 236±128 nm, mean±sd). NPs are likely repackaged in FuGENE vesicles before being endocytosed. Early E sizes match NP sizes, while L sizes match the L sizes of stem cell-derived macrophages [4]. By D2, there is a shift from early E/E-L hybrids to late Es and Ls. The intense cellular responses of D1 (LB/F-LB) subside on D2/D3 [Fig. 2(E-F, H-I)]. Contrary to LB, few early Es and numerous Ls and E/L hybrids are observed on D1 (F-LB) possibly owing to the rapid uptake kinetics. Fluorescence decreased to undetectable levels by D2 for LB and decreased by D3 for F-LB.

Conclusions

1. NPs/NP-Fs were taken up by phagocytosis/receptor-mediated endocytosis, with NP-F taken up in packaged form
2. Cell responses were immediate in LB and faster/more prominent in F-LB cells
3. NPs/NP-Fs were located close to the membrane (D1), in early Es/Ls, and E/L hybrids (D1, D2). By D3, NP/FuGENE vesicles were processed and the byproducts exocytosed. Increased apoptosis was noted at D2/D3

References
[1] Gaudet JM et al, PloS One 10(3):e0118544, 2015.
[2] Boehm-Stum P, et al., PloS One 6(12):e29040, 2011.
[3] Constantinides C et al., Nanomedicine 18:391-401, 2019.
[4] Naphade S et al., Stem Cells 33(1):301-309, 2015.
Figure 1:
TEM images attesting to the proposed uptake mechanisms (D1) and localization of uptaken PFCE-NPs in early/late endosomes and lysosome/endosome hybrids (D1, D2) in CPCs following labeling (LB), and example of apoptotic cell following FuGENE-labeling (F-LB) at D3.
Figure 2:
Temporally dependent cellular responses from control, labeled (LB), and FuGENE-labeled (F-LB) cells in TEM images. (A-C) Typical TEM images from control CPCs harvested on D1 after plating. Sequence of TEM images (D1-D3) following (D-F) labeling (LB) and (G-I) F-LB. (J, K) Histograms of sizes of water emulsions of control NPs/FuGENE-NPs based on TEM images.
Keywords: FuGENE, Stem cells, Cardiac, Transverse Electron Microscopy, Nanoparticle labeling
703

Gd3+ and Eu3+ Loaded Iron Oxide@Silica Core-Shell Nanocomposites as Trimodal Contrast Agents for MRI and Optical Imaging

Carlos F. G. C. Geraldes1, 2, 3, Sonia L. C. Pinho4, 5, José Sereno3, Antero J. Abrunhosa3, Marie-Hélène Delville6, João Rocha5, Luis D. Carlos5

1 University of Coimbra, Life Sciences, Coimbra, Portugal
2 University of Coimbra, Coimbra Chemistry Center, Coimbra, Portugal
3 University of Coimbra, CIBIT/ICNAS-Instituto de Ciências Nucleares Aplicadas à Saúde, Coimbra, Portugal
4 University of Coimbra, Center for Neurosciences and Cell Biology, Coimbra, Portugal
5 University of Aveiro, Departments of Chemistry and Physics, CICECO-Aveiro Institute of Materials, Aveiro, Portugal
6 University of Bordeaux, CNRS, Bordeaux INP, ICMCB, UMR 5026, Bordeaux, France

Introduction

While conventional MRI contrast agents respond in a single imaging mode, nanoparticles with multimodal capabilities can provide complementary diagnostic information, optimally obtained using hybrid imaging systems.1 Here we report the synthesis, characterization and labeled cell imaging studies of superparamagnetic maghemite core-porous silica shell nanoparticles, γ-Fe2O3@SiO2 impregnated with paramagnetic complexes b-Ln ([Ln(btfa)3(H2O)2]) (btfa =  4,4,4-trifluoro-l-phenyl-1,3-butanedione, Ln = Gd, Eu, and Gd/Eu), performing as promising trimodal T1-T2 MRI and optical imaging contrast agents.

Methods

Aqueous maghemite NPs suspensions (FS) were used to obtain core-shell γ-Fe2O3@SiO2 NPs using Stöber’s process, surface-activated by citric acid, allowing incorporation of b-Ln complexes in the NPs porous shell,2 and characterized by SEM, TEM, EDS and DRIFT. Hydrodynamic radii and zeta potentials in aqueous suspensions were obtained by DLS, optical and magnetic properties by spectrofluorimetry and magnetometry. r1 and r2 relaxivities and MRI phantom images were obtained at 9.4 T on a Bruker small animal scanner, from T1 Map-RARE images using a SE sequence and a T2-Map from a 2D MSME sequence. Leaching of Gd3+ was evaluated by the xylenol orange test. HeLa cells internalization of the NPs was studied using confocal microscopy and ICP-MS. The resulting MRI contrast effect was assessed by MRI.

Results/Discussion

The obtained core-shell γ-Fe2O3@SiO2 NPs (50 ± 6 nm size, 10 ± 1 nm core size) exhibit very negative NPs zeta potential (- 40 mV) in highly stable water dispersions at neutral pH and less than 0.39% Gd leaching. Embedding the b-Eu complex in the silica pores endowed the NPs with strong luminescence in the visible region (eg. strong 5D0 → 7F2 signal at 612 nm), with lifetime τ= 0.345 ms. The b-Eu complex kept its water coordination number q= 2 upon NP embedding. The structure and superparamagnetic properties of the maghemite core nanocrystals were preserved upon imbedding, with a saturation magnetization MS = 55.2 emu. g-1 Fe. Hela cells efficiently and swiftly internalized the NPs into the cytosol, with no observable cytotoxicity below 62.5 mg mL-1 concentration. The final NPs performed better than the free b-Gd complex as T1 (positive) contrast agents in cellular pellets, while their performance as T2 (negative) contrast agents was similar to the FS (Figure 1).

Conclusions

The impregnation of b-Gd and b-Eu complexes in a 1:1 ratio in the porous shell of the core-shell γ-Fe2O3@SiO2 NPs afforded a trimodal nanoplatform performing as a high quantum efficiency luminescent probe and a double T1-T2 MRI contrast agent even more efficient than b-Gd used on its own, as observed in vitro in cell labeled confocal microscopy experiments and T1w and T2w MR imaging of labeled cell pellets.

Acknowledgment

We thank support from FCT and FEDER (Portugal), CNRS and Région Nouvelle Aquitaine (France) and COST Action D38.

References
[1] Gao, J, Gu, H, Xu, B., 2009, Acc. Chem. Res. 42, 1097-1107 

[2] Pinho, SLC, et.al., Inorg. Chem., in press.
Figure 1

T1- and T2-weighted nuclear magnetic resonance images of cellular pellet phantoms (i) cells + b-Gd, (ii) cells + FSb-Gd, (iii) cells + FSb-EuGd (iv) cells + FS NP and (v) control: cells (T1-weighted images: repetition time = 450 ms, echo time 12 ms; T2-weighted images: repetition time = 4000 ms, echo time 80 ms.

Keywords: trimodal nanosystems, superparamagnetic nanoparticles, fluorescence microscopy, MRI contrast agents, Ln complexes
704

Comparing in vivoMRI/PETimaging with SPIONs: The no effect of iron concentration.

Ana B. Miguel-Coello1, Susana Carregal-Romero1, 2, Jordi Llop1, 2, Jesús Ruiz-Cabello1, 2

1 CIC BiomaGune, Donostia-San Sebastián, Spain
2 Ciberes, Madrid, Spain

Introduction

SPIONs are used in biomedica technologies as contrast agent in MRI. Synthetic protocols allow tailor the size, shape, surface chemistry and magnetism.1 SPIONs can be doped with radioisotopes to obtain MRI/PET dual probes.2 In vivo studies could require toxic amounts of iron for the animal. We have studied with molecular imaging in a murine model the influence of NP concentration and demonstrated that MRI/PET contrast was not affected by the NP concentration. We have used a peptide as angiogenesis marker, to functionalize the NP surface obtaining a specific dual probe to target tumors.

Methods

Synthesis of 68Ga-SPION radiotracer was microwave assisted, using a 68Ge/68Ga generator as radionuclide source. Conjugation of peptide marker was carried out using EDC/sulfo-NHS reaction. NPs were characterized by TEM, DLS, Z-potential and XPS. Relaxometry studies were carried out both in Bruker minispec magnet and MRI instrument. Iron concentration was determined by ICP-MS. For the in vivo studies, female mice were inoculated with melanoma cells to grow a subcutaneous tumor. Then, mice were injected with two different iron concentration doses: the concentrated one was four times the diluted one concentration, and PET/CT scans were acquired. After decay of radioactivity, MRI images were obtained.

Results/Discussion

Successful synthesis of targeting radiotracer for angiogenesis in tumor was demonstrated by comparison of DLS, Z-potential and XPS measurements between 68Ga-SPION and 68Ga-SPION-peptide data. Tumor was localized by MRI and the resulting T​​​​​​2 maps were compared. Images point out that T​​​​​​2 transverserelaxation time is similar in the tumor location for both doses and there are no significant differences between the mouse injected with the diluted and with the concentrated dose (Figure 1). Analysis and quantification of PET images shows that the injected dose percentage in general biodistribution and in tumor uptake is similar for diluted and concentrated doses. That demonstrates that lower concentrations of this SPION radiotracer are enough to evaluate the in vivo uptake.

Conclusions

We demonstrated that reducing the SPION dose and therefore their potential toxicity, these probes were efficient contrast agents to perform both MRI and PET, without losing information and imaging quality. This indicates that often the effect of nanoprobe concentration is overrated and potential toxic effects of nanoparticles can be reduced by optimizing the experimental conditions of molecular imaging measurements.

AcknowledgmentThis work was supported by grants from the Ministerio de Economía, Industria y Competitividad (MEIC) (SAF2017-84494-C2-R), and from the Gobierno Vasco, Dpto. Industria, Innovación, Comercio y Turismo under the ELKARTEK Program (Grant No. KK-2019/bmG19). JRC thanks the funding obtained from the BBVA Foundation (Ayudas a Equipos de investigación científica Biomedicina 2018) and the Fundación contra la Hipertensión Pulmonar (2018). CIC biomaGUNE thank the support of the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720.
References
[1] Magnetic Nanoparticles and Biosciences, 2002, 133, 6, 737–759.
[2] Scientific reports, 2017, 7, 13242
Figure 1
MRI image of subcutaneous tumor after injection of nanotracer. Diluted dose (left) 
and concentrated dose (right). T​​​​​​2 values are similar in both images.
Keywords: MRI/PET dual probes, SPION, contrast agent
705

Fine tailored multimodal PET/MRI probes based on manganese ferrite

Susana Carregal-Romero1, 2, Ana B. Miguel-Coello1, Lydia Martínez-Parra1, Jesus Ruiz-Cabello1, 2

1 CIC biomaGUNE, Molecular and Functional Biomarkers, San Sebastian, Spain
2 Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain

Introduction

All clinical imaging techniques have intrinsic limitations in terms of spatial and temporal resolution or limited sensitivity. In addition, molecular imaging often requires contrast agents that are potentially toxic and could irrevocably accumulate within the patient organism. Some of the key intrinsic properties to improve molecular imaging with contrast agents are: i) multimodality, ii) targeting and iii) toxicity. Our work is focused in developing probes based on nanoparticles with fast protocols which allow simultaneous multimodality and surface labelling for targeting.

Methods

A microwave assisted method was used to synthesize chelator-free trimetallic nanoparticles based on Mn, Fe and 68-Ga following the method developed by Herranz et al.[1] Electron microscopy, superconducting quantum interference device (squid), inductively coupled plasma mass spectrometry, ultra-high performance liquid chromatography, x-ray photoelectron spectroscopy, dynamic light scattering, relaxometry, magnetic resonance imaging (MRI) and positron emission tomography (PET) were used for the characterization of the multimodal nanoparticles. The murine model C57BL/6NCrl was used to determine the blood circulation time by MRI and PET was used for biodistribution studies.

Results/Discussion

We were able to produce multimodal Mn/Fe nanoparticles which allow for radiolabelling with 68-Ga and functionalizing their surface with targeting moieties using a fast protocol lasting less than one hour. By modification of the synthetic conditions we finely tailored the ratio of Fe:Mn from 20 to 0.3 within the nanoparticles which triggered a different T1/T2 contrast in MRI. Moreover, we demonstrate their efficient doping with 68-Ga which allow to study their biodistribution. Future studies will include toxicity in vitro and in vivo and labelling with targeting molecules for tumour accumulation.

Conclusions

We demonstrated that it is possible to finely control the chemical composition of multimodal PET/MRI nanoprobes and this chemical modifications induce different MRI contrast and magnetic properties between others while maintaining similar physichochemical properties such as surface chemistry, surface charge, size and shape.

AcknowledgmentThis work was supported by grants from the Ministerio de Economía, Industria y Competitividad (MEIC) (SAF2017-84494-C2-R), and from the Gobierno Vasco, Dpto. Industria, Innovación, Comercio y Turismo under the ELKARTEK Program (Grant No. KK-2019/bmG19). JRC thank the funding obtained from the BBVA Foundation (Ayudas a Equipos de investigación científica Biomedicina 2018) and the Fundación contra la Hipertensión Pulmonar (2018). CIC biomaGUNE thank the support of the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720.
References
[1] Pellico J, Ruíz-Cabello J, Saiz-Alía M, del Rosario G, Caja S, Montoya M, Fernández de Manuel L, Puerto Morales M, Gutiérrez L, Galiana B, Enríquez J A, Herranz F 2016, ‘Fast synthesis and bioconjugation of (68) Ga core-doped extremely small iron oxide nanoparticles for PET/MR imaging’ Constrast Media Mol. Imaging, 11, 203, Wiley
Figure 1
(A) MRI contrast effects of ultrasmall MnxFe3-xO4 nanoparticles upon changes in the Mn doping level. (A) r1 relaxivities and r2/r1 ratios of ultrasmall MnxFe3-xO4 nanoparticles. (B) and (C) Transmission electron microscopy (TEM) and high resolution TEM of manganese ferrites with different Mn doping.
Keywords: manganese ferrite, ultrasmall nanoprobes, PET/MRI
706

Au@Fe2O3nanoparticles as multi-modal imaging agents targeting cancer

Jennifer Lamb1, Marcus Yaffee1, Jason P. Holland1

1 University of Zurich, Department of Chemistry, Zurich, Switzerland

Introduction

Iron oxide nanoparticles (NP) are well-known Tcontrast agents used in magnetic resonance imaging.1,2 We have recently synthesised iron oxide nanoparticles which are encased in a gold coating, enabling biocompatibility and functionalisation viathe formation of gold-thiol bonds. By synthesising disulphide derivatives of the prostate specific membrane antigen (PSMA) binding motif (Glu-NH-C(O)-NH-Lys) and a 68Ga chelator (DOTA-GA), multi-functionalised the NPs were produced that showed selective accumulation in cancer cells with potential applications in PET/MRI.

Methods

The NHS-ester of lipoic acid (LA) was used to modify Glu-NH-C(O)-NH-Lys and DOTA-GA-(PEG)4. Radiolabelling reactions to prepare [68Ga]LA-(PEG)4-DOTA-GA-Ga were accomplished by addition of a 68Ga stock solution to an aqueous solution of LA-(PEG)4-DOTA-GA(0.2 M NaOAc, pH4.4, 70 oC, 10 min). Radiolabelling was monitored by using radio-iTLC and the product characterised by analytical HPLC using the natGa complex as a reference.

Functionalisation of the NPs was accomplished by incubation of the Au@Fe2O3 NP with [68Ga]LA-(PEG)4-DOTA-GA-Ga (pH7) and Glu-NH-C(O)-NH-Lys-LA. Radiochemical stability, hydrodynamic size, zeta (ζ) potential and Trelaxation times of the modified NPs were determined. Targeting effects were studied by in vitro experiments using LNCaP (PSMA +ve) and PC-3 (PSMA -ve) cells.

Results/Discussion

[68Ga]LA-(PEG)4-DOTA-GA-Gawas synthesised with a RCC >99% (n= 5). NP-1was then synthesised via the incubation of Au@Fe2O3NP with [68Ga]LA-(PEG)4-DOTA-GA-Gagiving a RCC >97% (radio-iTLC,n = 3) and a RCP of 94% (SEC-PD10, n = 1). Monitoring the reaction indicated completion within 90 s. NP-2 was synthesised in a similar manor with the simultaneous addition of Glu-NH-C(O)-NH-Lys-LA giving a RCC>94% (radio-iTLC, n = 3) and a RCP of 95% (SEC-PD10, n=1). Stability tests in saline, PBS, EDTA and cysteine showed minimal loss of activity from the NP-1 and NP-2 constructs, and T2relaxation times were measured as 23.70±0.01 and 28.77±0.02 ms, respectively. Hydrodynamic diameters of modified NPs remained similar (±2 nm) to the bare nanoparticles (48.6±25.0 nm) and no significant decrease in ζ-potential was observed. Cell binding assays (Fig. 1b) showed NP-2 has an increased uptake (1.5-fold increase in associated activity) in LNCaP cells compared to the non-targeted NP-1.

Conclusions

Experiments demonstrated that Au@Fe2Onanoparticles can be easily modified with disulphide derivatives producing constructs which are both radiolabelled with 68Ga and functionalised with biologically active targeted vectors. Cellular work indicated increased uptake in the LNCaP cell line suggesting that functionalised Au@Fe2Onanoparticles have potential to act as targeted PET/MRI imaging agents.

AcknowledgmentJ.P.H thanks the Swiss Cancer League (Krebsliga Schweiz; KLS-4257-08-2017), the Swiss National Science Foundation (SNSF Professorship PP00P2_163683), the European Research Council (ERC-StG-2015, NanoSCAN – 676904), and the University of Zurich for financial support. 
References
[1] Rosen et al. Nanomedicine: Nanotechnology, Biology, and Medicine, 2012, 8, 275–290
[2] Lamb et al. J Nucl Med, 2018, 59, 382–389
Figure. 1

(A) Schematic of NP-1 ([68Ga]Au@Fe2O3-LA-(PEG)4-DOTA-GA-Ga) and NP-2([68Ga]Glu-NH-C(O)-NH-Lys-LA-Au@Fe2O3-LA-(PEG)4-DOTA-GA-Ga) (B) Cellular binding assay of NP-1and NP-2with the LNCaP (PSMA +ve) and PC-3 (PSMA -ve) cell lines. Data given as the percentage of activity bound normalised per 1 mg mL-1of total protein. Blocking involved the pre-treatment with free Glu-NH-C(O)-NH-Lys ligand (20 mM) before addition of radiotracers. Note: Student's t-test analysis: ns=not significant, *=P-value < 0.05, **=P-value < 0.01.

Keywords: PET/MRI, nanoparticles, targeted, PSMA
707

Radiolabelling of gold nanoparticles for accessing their ability to multimodal imaging: an investigation of their potentials to track stem cells in muscle regeneration models and their preliminary in vivo evaluation with SPECT/CT imaging

Sophia Sarpaki1, Eirini Fragogeorgi2, Maritina Rouchota1, Irinaios Pilatis1, Racheli Ofir3, Rachela Popovtzer4, Marc Masa5, Panagiotis Papadimitroulas1, George Loudos1, 2

1 Bioemission Technology Solutions, Research & Development, Athens, Greece
2 National Center for Scientific Research (NCSR) “Demokritos”, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, Athens, Greece
3 Pluristem Therapeutics Inc, Research & Intellectual Property, Haifa, Israel
4 Bar Ilan University, Faculty of Engineering and the Institutes of Nanotechnology and Advanced Materials, Tel Aviv, Israel
5 Leitat Technological Center, Biomed Division, Barcelona, Spain

Introduction

Biocompatible and functional nano-based multimodal imaging agents are developed, within the nTRACK (H2020) project, as a tool to overcome the barriers of current cell therapeutics on tracking non-invasively the transplanted cells and monitor their viability. In our approach, modified and labelled with the long-lived radio-isotope indium-111, gold coated-magnetic core NPs (Au@IONPs) were developed aiming in real-time non-invasive whole-body monitoring of living stem cells in small animal models through the simultaneous use of different imaging techniques.

Methods

The surface of Au@IONPs was initially modified to allow the chelation of the long-lived radioisotope [111In]InCl3. Quality control of [111In]In-Au@IONPs resuspended pellet was carried out with paper chromatography and kinetic stability assays were performed from t=0 up to 24h post-preparation, over a range of temperatures (5⁰C, 25⁰C, 37⁰C) and under different incubation conditions in both aqueous solution (saline 0.9% v/v and glucose (5%)) and mouse serum at 37⁰C. Additionally, the potentials of the modified Au@IONPs to be labelled in living stem cells was investigated. Finally, imaging studies were performed on a SPECT and on a CT imaging system by Molecubes (γ-CUBE and x-CUBE), providing a spatial resolution of 0.6 mm and 0.05 mm, respectively.

Results/Discussion

The radiochemical yield for [111In]In-Au@IONPs was > 95 % providing a single radioactive species. In vitro stability up to 24 h post-labelling was high (>98 %) at all different sets of temperature and while diluted ten times in aqueous conditions [saline and glucose 5 %: >93 %]. Radioactivity was mainly found at the point of the injection and was then excreted via the kidneys into the urinary bladder. The clearance of the [111In]In-Au@IONPs is shown to be much faster than the one found at control experiment using [111In]InCl3 in saline solution.

Conclusions

The first results on radiolabelling Au@IONPs, examining their in vivo biodistribution and their potentials as multimodal imaging agents on living stem cells, presented herein. The encouraging results suggested that the successfully labelled contrast agents follow the desired clearance from the body that will allow for effective monitoring of the cells’ fate in the point of targeted cell therapy

Acknowledgment

This study is part of a project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 761031. Part of the study was funded from the program of Industrial Scholarships of Stavros Niarchos Foundation.

References
[1] Betzer, O., et al., 2015, 'In-vitro Optimization of Nanoparticle-Cell Labeling Protocols for In-vivo Cell Tracking Applications.' Scientific Reports, 5, 15400.
[2] Meir, R., et al., 2015, 'Nanomedicine for Cancer Immunotherapy: Tracking Cancer-Specific T-Cells in Vivo with Gold Nanoparticles and CT Imaging.' ACS Nano, 9(6),  6363-6372.
[3] Motiei, M., et al., 2019 'Trimodal Nanoparticle Contrast Agent for CT, MRI and SPECT imaging: Synthesis and Characterization of Radiolabeled Core/Shell Iron Oxide@Gold Nanoparticles.' Chem. Lett, 48, 291-294
Figure 1:
Schematic illustration of in vivo imaging experimental workflow.
Keywords: Indium-111, Stem cells, cell therapy, gold-shell NPs, multimodal imaging
708

Functionalized Silica Nanoplatform as Bimodal Contrast Agent for MRI and Optical Imaging (OI)

Sarah Garifo1, Indiana Ternad1, Dimitri Stanicki1, Sébastien Boutry1, 2, Lionel Larbanoix1, 2, Robert N. Muller1, 2, Sophie Laurent1, 2

1 University of Mons (UMONS), Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, Mons, Belgium
2 Center for Microscopy and Molecular Imaging (CMMI), Gosselies, Belgium

Introduction

Among the numerous imaging techniques, magnetic resonance imaging has imposed a powerful diagnosis tool for owing to its high spatial resolution, unlimited tissue penetration and non-ionizing nature. Nevertheless, the inherent lack of sensitivity constitutes a major drawback, especially in the field of molecular imaging. Its combination with optical imaging offers the high spatial resolution of the former and the high sensitivity of the latter. In this context, we focused on the improvement of clinically approved Gd chelates by their confinement in silica nanoparticles, SiO2 NP[1,2,3].

Methods

Thanks to their exceptional properties (i.e. biocompatibility, chemical stability, low toxicity) SiO2 NPs have been chosen as a matrix[2,3]. The systems were obtained by reverse micro-emulsion procedure in the presence of a hydrosoluble paramagnetic contrast agent (Gd-HP-DO3A). Then, the particle surface was modified by silanol-PEG chains to ensure aqueous stability. Functional groups were introduced by mean of a photochemical treatment4 in the presence of a diazirine system. Bimodality was reached by introducing ZW800-2, a near-infrared emitting molecule, on the carboxylic functions of the linkers on the top of the coating.

Results/Discussion

The as-synthetized nanoplatforms were characterized by dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, relaxometry measurements, UV-Vis and IR spectroscopies and transmission electron microscopy (TEM). The encapsulation of Gadolinium ion complexes inside the silica matrix have been optimized during the study and in vitro MRI images have been performed on stable paramagnetic particles. The amount of Gd3+ reached 20 µmol per milligrams of particles. Photochemistry process was used to allow post-functionalization of the PEGylated NPs by the introduction of the fluorophore via an amide bound. The final experimental step consisted in the incorporation of a peptide, the R832, which, according to the literature, targets VCAM-15.

Conclusions

A stable fluorescent paramagnetic nanoplatform was successfully prepared and characterized. Narrow size distribution (Dh: 80 nm) were obtained and relaxometric measurements have proven its efficiency to decrease water proton relaxation times. A biological vector (R832 peptide) has been grafted on the surface of our functionalized bimodal platform and its efficiency is under investigation.

Acknowledgment

This work was performed with the financial support of the FNRS, the ARC, the Walloon Region, NanoCardio, 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] S. Laurent et al., Springer, MRI Contrast Agents: From Molecules to Particles, 2017.
[2] E. Lipani et al., Langmuir 29, no 10, 3419‑27, 2013.
[3] N. Wartenberg et al., Chemistry - A European Journal 19, no 22, 6980‑83, 2013.
[4] V. Pourcelle et al., Bioconjugate Chemistry 26, no. 5, 822–29, 2015.
[5] C. Burtea et al., J. Med. Chem., 52, 15, 4725-4742, 2009.

Figure 1

Figure 1: Schematic silica nanoplatform

Keywords: Silica nanoparticles, bimodality, targeting
709

Novel anticancer drugs-lipiodol emulsions: preparation and initial characterisations as potential theranostic agents

Irena Pashkunova-Martic1, 2, Berta C. Losantos2, Norbert Kandler2, 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

Introduction

Transcatheter arterial chemoembolization (TACE) with Lipiodol (Lp, an oily lymphographic agent) is widely used to treat patients with unresectable hepatocellular carcinoma. After infusion, most of its volume accumulates in the tumor rather than in the liver tissue. Therefore, Lp can be used for selective delivery of drugs. Mixing anticancer agents with Lp may enhance the anticancer effect by increasing the drug local concentration. In this pilot study, we used Lp as a potential carrier of three promising antitumor metal complexes KP46, KP1019 and the active species of the latter compound.

Methods

Lipiodol emulsions of the anticancer complexes KP46 (tris(8-quinolato)gallium(III)), KP1019 (tetrachlorobis(indazole)ruthenate(III)) and the hydrolysis product of KP1019 (mer,trans-[RuIIICl3(Hind)2(H2O)]) were prepared by directly solution of these drugs in lipidol and application of ultrasonicator until total dissolution. Their stability in lipiodol was examined for three consecutive days by means of high pressure liquid chromatography (HPLC). The release profile into the aqueous phase has been investigated by incubating the emulsions in 0.9% NaCl solution at 37°C for the duration of one month by HPLC, atom absorption spectroscopy (AAS) and electrospray ionization mass spectrometry (ESI-MS). KP1019 and KP46 showed good solubility in lipiodol, whereas the active species did not dissolve.

Results/Discussion

Both KP1019 and KP46 showed good compatibility in forming an emulsion with Lp. In contrast, evaluation of Lp-emulsion with the hydrolysis product, mer,trans-[RuIIICl3(Hind)2(H2O)], was not possible due to its insolubility in Lp. The stability of KP46 and KP1019 in the Lp-emulsion was analyzed by HPLC–UV/vis detection. Both complexes showed good separation suitable for their quantification when using phosphate buffer/acetonitrile (ratio 4:6) as mobile phase.

The complexes were stable and remained in the Lp-emulsion (about 90 % of KP1019 and 95 % of KP46) over 3 days. With respect to the release profile from the lipiodol emulsion, KP1019 released rapidly into the aqueous phase in the first week and after 1 month, it was not possible to detect the complex in the emulsion. KP46 showed a gradual release with the time and about 6.4 % of the complex was released into the aqueous phase after 1 month of incubation.

Conclusions

These first results show that Lp can be successfully applied as drug carrier and showed a potential for simultaneous embolization and sustained release of anticancer Ru and Ga metal complexes. Furthermore, advantages can be taken by overcoming the poor water solubility of the metal complexes and opening new perspectives for use of Lp-emulsions for selective molecular imaging and cancer therapy as novel theranostic agents.

Acknowledgment

We gratefully acknowledge the support of the University of Vienna within the exploratory focus „Functionalized Materials and Nanostructures“.

References
[1] Nakakuma, K, Tashiro, S, Hiraoka, T, Ogata, K, Ootsuka, K, 1985, Radiology, 154: 15-7
[2] Bhattacharya, S, Dhillon, AP, Winslet, MC, Davidson, BR, Shukla, N, Gupta, SD, Al-Mufti, RA, Hobbs, KE, 1996, British Journal of Cancer, 73: 877-81
[3] Al-Mufti, RA, Pedley, RB, Marshall, D, Begent, RH, Hilson, A, Winslet, MC, Hobbs, KE, 1999, British Journal of Cancer, 79: 1665-71
[4] Lambert, B, Van de Wiele, C, 2005, European Journal Nuclear Medicine and Molecular Imaging, 32: 980-989
[5] Ikoma, A, Kawai, N, Sato, M, Minamiguchi, H, Nakai, M, Nakata, K, Tanaka, T, Sonomura, T, 2012, Hepatology Research, 42: 1227-1235
Keywords: drugs-lipiodol emulsions, antitumor metal complexes, selective targeting, theranostic agents