EMIM 2018 ControlCenter

Online Program Overview Session: PW-05

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New Probes | Synthesis and Enabling Technologies I

Session chair: Lorenzo Tei - Alessandria, Italy; Francesca Arena - Torino, Italy
 
Shortcut: PW-05
Date: Thursday, 22 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 051

Lanthanide-based contrast agents for Single Photon Counting CT (#155)

G. Digilio1, 2, F. Capuana2, R. Stefania2, C. Carrera2, S. Si-Mohamed3, L. Boussel3, L. Poggi4, P. Douek3, S. Aime2

1 Università del Piemonte Orientale, Department of Sciences and Technologic Innovation, Alessandria, Italy
2 University of Turin, Department of Molecular Biotechnology and Health Science, Turin, Italy
3 University Claude Bernard Lyon 1, CREATIS, Lyon, France
4 Bracco Imaging s.p.a, Colleretto Giacosa (TO), Italy

Introduction

Spectral Photon Counting Computed Tomography (SPCCT) is a X-ray based imaging technique promising to conjugate a superb anatomic resolution with molecular imaging (MI) capabilities, provided that contrast agents (CAs) with suitable properties are made available. Gadolinium is indicated as one of the most promising element for K-edge imaging.[1] A number of Gadolinium-based contrast agents (GBCA) are routinely employed in clinical MRI, and can find new diagnostic applications in SPCCT imaging without requiring extensive safety assessment.

Methods

The active pharmaceutical ingredient of a clinically approved GBCA was injected into healthy rabbits to assess the capability of new SPCCT technology to obtain K-edge Gd images and to assess the limit of quantitation. Dynamic angiography of the aorta and pulmonary vasculature and perfusion of the lung and myocardium was performed during 45 seconds after injection. A multimeric Gd-chelate targeted to the atherosclerotic plaque was synthetized and initial biodistribution was assessed in mice.

Results/Discussion

Gadolinium K-edge images showing perfusion of the CA in the lungs and myocardium were obtained with a dose of GBCA five times larger than that used in routine clinical examinations. Absolute quantification of gadolinium was possible. To enable molecular imaging, a peptide targeting the atherosclerotic plaque was successfully functionalized with a multimer containing four gadolinium units to increase the density of gadolinium at the biological target.

Conclusions

K-edge imaging of gadolinium is feasible with current prototype SPCCT technology and clinically approved GBCA, but requires a large dose of CA. Molecular imaging of the atherosclerotic plaque might be possible by targeted molecular probes containing multiple Gd centers, such to increase the amount of gadolinium accumulating at the biological target after a single binding event.

References

[1] Schirra et al., Contrast Media Mol. Imaging 2014, 9 62–70

[2] www.spcct.eu

 

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 643694 [2]

# 052

Tuning the photophysical properties of small organic fluorophores for cellular microscopy (#268)

J. A. Fauerbach1, M. Vahldieck1, W. Al Rawashdeh1, C. Dose1

1 Miltenyi Biotec GmbH, R&D reagents, Chemistry, Bergisch Gladbach, North Rhine-Westphalia, Germany

Introduction

Fluorescence is the method of choice for many biological applications thanks to its high sensitivity and ease of implementation.1 Yet, results depend on the photo-physical and chemical properties of the implemented fluorescent reporters (i.e. organic fluorophores), and the ability to modulate them.2 We have focused on tunning the photo-stability of organic fluorophores to obtain either: i) highly photo-stable fluorophores, or ii) highly bright but fast photodegradable ones, aiming tailored cellular fluorescence microscopy applications.

Methods

Small organic fluorophores belonging to different chemical class classfication families (i.e. oxazoles, rhodamines, xanthenes, cyanines) where irradiated with a high power light for fixed periods of time. Absorbance and emission spectra were recorded in between irradiation times. Using these data (i.e. the value of maxima intensity), degradation curves were plotted and fitted with a mono-exponential decal through non-least squares.3 The results obtained from fitting were used to quantify and compared degradation rates. Examples from Miltenyi's own fluorophore portfolio (VioDye series) are compared to Alexa and Cye-dyes. Organic fluorophores were also functionalization with i.e. linear and branched PEGs to modulate their photoresponse appropiately.

Results/Discussion

This study comprises a comprehended study on commecially available fluorophores (i.e. VioDyes belonging to Miltenyi's portfolio) in regards to their photo-stability response upon light irradiation and the ability to modulate such response with PEGs. Figure 1 includes a table summarizing the fluorophores included in this study as well as their general fluorescent properties and a neat example comparing photodecay curves for Cy5, Alexa647 and Vio667 (Fig. 1B), where the photo-degradation curve are plotted for each one, showing how Miltenyi's Vio667 fluorophores is aproximately 10-12 times more photo-stable than Alexa647, a usual choice for cellular microscopy. Furthermore, covalent conjugation to PEGs led to accelerated photodegradation (Fig. 2). Here we compare the effects and correlation between covalent conjugation vs non-covalent conjugation as well as linear vs branched PEGs. The effect was found to affect all fluorophores independently of the chemical class classfication.

Conclusions

A comparisson of small organic fluorophores and their photophysical properties is presented. We stress the high performance and superior photostability of Miltenyi's small organic fluorophores in comparisson to other commercial ones. We also show a general approach to modulate the photostability properties via additive molecules, such as PEG, with interest for certain microscopy applications where fast quenching of stained cells may be required. We believe this might have further applications in combination with bleaching cellular confocal microscopy techniques such as FRAP.

References

1. Andreas Ettinger and Torsten Wittmann, 'Fluorescence Live Cell Imaging', Methods Cell Biol. 2014; 123: 77–94.

2. Qinsi Zheng and Luke D Lavis, 'Development of photostable fluorophores for molecular imaging', Current Opinion in Chemical Biology 2017, 39:1–7 

3. Loling Song, E. J. Hennink, Ted Young and Hans J. Tanke, 'Photobleaching Kinetics of Fluorescein in Quantitative Fluorescence Microscopy', Biophysical Journal, 1995, 68: 2588-2600. 

Acknowledgement

 

 

Figure 1. Photophysical properties of organic fluorophores

A. Table with organic fluorophores and their excitation and emission wavelengths, as well as the photodecay fitting values.

B. Photodecay equations used to fit mono-exponential photodegradation curves

C. Comparisson of mono-exponential photodegradation curves for Cy5, Alexa647 and Vio667.

Figure 2. Modulation of photostability via PEG conjugation

A. Modulation of the photodegradation curve for a cyanine fluorophore comparing the effects of a linear and branched PEG as well as covalent and non-covalent conjugation.

B. Examples of PEGylation of organic fluorophores belonging to diferent chemical classifications.

Keywords: photo-bleaching, dyes, fluorescence microscopy, PEG
# 053

Imaging whole-body biodistribution of red blood cells (RBC)-derived extracellular vesicles (EVs) in young adult versus old mice. (#552)

T. Viel1, 2, D. Charue3, B. Tavitian1, 2, 4, O. Blanc-Brude3

1 Paris Descartes University, PARCC - INSERM U970, Paris, France
2 Paris Descartes University, Life Imaging Facility (PIV), Paris, France
3 PARCC-U970, INSERM, Hopital Européen Georges Pompidou, Paris, France
4 APHP, Radiology Department, Hopital Européen Georges Pompidou, Paris, France

Introduction

EVs are cell membrane fragments including exosomes, microvesicles and apoptotic bodies. EVs enable cell to cell communication and fulfill a panel of biological functions such as inflammation, coagulation, extracellular matrix degradation. RBC are major producers of circulating EVs. Imaging the fate of RBC-derived EVs would entail a significant insight into their biological effects. However, labeling EVs without affecting their composition and function remains a challenge. Here, we labeled mouse RBC EVs with FDG and imaged their whole-body biodistribution by PET in young adult versus old mice.

Methods

RBC-derived EVs were produced in vitro from RBC freshly isolated from young adult (4-6 months old) or old (12 months old) C57Bl6 mice, labelled with [18F]FDG. About 1 MBq of [18F]FDG-labelled EVs were injected in young adult or old mice, respectively(n=4-5). Dynamic PET scans were then acquired over 60 min using a nanoScan PET-CT (Mediso, Hungary). A few days later, the same mice received 1 MBq of ‘free’ [18F]FDG for comparison. In vivo PET acquisitions were reconstructed using the Tera-Tomo reconstruction engine (3D-OSEM-based manufactured, customized algorithm) with expectation maximization iterations, scatter and attenuation correction. Images were analyzed using the PMOD software (PMOD Technologies LLC).

Results/Discussion

The biodistribution of FDG-labeled RBC EVs differed significantly from ‘free’ FDG consumption in young adult mice, 60 minutes after injection (p<0,001; paired Anova (1 way) followed by Tukey multiple comparison). RBC EVs produced significantly reduced signals in brains and hearts, but higher signals in livers, lungs, spleens and kidneys, supporting successful encapsulation of FDG in our EVs.

Moreover, the uptake of FDG-labeled RBC EVs was increased in the brains (1.57 vs 0.78 %ID; p<0.001) and livers of old versus young adult mice (7.50 vs 5.19 %ID; p<0.001). Conversely, FDG-labeled RBC EV accumulation was lower in spleens (2.84 vs 4.17 %ID; p<0.001) and livers of old versus young adult mice (1.54 versus 2.90 %ID; p<0.001). Low accumulation of FDG-labeled RBC EVs was also registered in the hearts of old and young adult mice (2.91 vs 2.45 %ID at 60 min; ns).

Conclusions

We developed a novel protocol to successfully produce natural and non modified FDG-labeled EVs derived from RBC and image their biodistribution over 1h in whole, live mice by PET. Our initial results revealed that RBC derived EV biodistribution was significantly altered by age in mouse brain, liver, spleen and lungs.

This novel method will now be leveraged to study the differential distribution of RBC-derived EVs in hematologic disorders, and adapted to image the fate of EVs of different cellular origins in various models of cardiovascular disease and inflammation.

Figure 1
Differential biodistribution of RBC-derived [18F]FDG-labelled RBC EVs versus ‘free’ [18F]FDG in a young adult C57Bl6 mouse, 60 min post-injection.
# 054

Carbon-11 labelling with [11C]carbon disulfide: recent advances and applications  (#322)

T. Haywood1, S. Kealey1, C. Plisson2, S. Cesarec1, P. W. Miller1

1 Imperial College London, Chemistry, London, United Kingdom
2 Imanova Ltd, London, United Kingdom

Introduction

Methylation using [11C]methyl iodide is the most common route to produce carbon-11 radiolabelled PET tracers. Despite its widespread use [11C]CH3I has limitations and cannot be used to prepare all desired carbon-11 tracers. Consequently a range of other small reactive molecules such as: 11CO, H11CN, 11CH2O and 11COCl2 have been developed. Recently, our group reported the synthesis of [11C]carbon disulfide (11CS2) for the generation of novel carbon-11 thiocarbonyl compounds.[1,2] Herein, we report the facile production [11C]ammonium thiocyanate and its applications towards C-11 radiolabelled.

Methods

The high temperature (500 oC) gas phase reaction of [11C]CH3I with elemental sulfur at 500 oC generates 11CS2 quantitatively.  The 11CS2  can be easily trapped in organic solvents such as DMSO, acetonitrile and THF at room temperature.  Reaction of 11CS2 solution with methanolic ammonia (2M) at mixture  95 oC generates the  [11C]ammonium thiocyanate (NH4SCN) which can be used for further reaction. Reaction of [11C]NH4SCN with alpha-bromo-ketone precursors (5 min at 95 oC) generated a range of novel 11C-labelled phenacyl-thiocyanates (scheme 1).   Addition of acid to 11C-labelled phenacyl-thiocyanates acid resulted in the formation of a series of new 11C-labelled thiazole structures. 

Results/Discussion

Established literate routes for the preparation of [11C]KSCN or [11C]NaSCN are complex multistep syntheses and require the initial preparation of [11C]HCN followed by changing reaction with sulfur.  Our method for the synthesis of [11C]NH4SCN is achieved in high radiochemical yield via the reaction of easily prepared 11CS2 with ammonia at lower temperatures and reasonably short, and as of yet unoptimized, reaction times. The [11C]thiocyanate ion (SCN-) is a potent nucleophilic species, that has the potential for a wide range of organic reactions and transformations.  A range of [11C] phenacyl-thiocyanates were produced in high radiochemical yields ( >95%) via reaction alpha-bromo-ketone precursors.  Interestingly, these structures could be cyclized to generate heterocycle thiazole structures in good radiochemical yields (70-95%).  Further reaction and applications of [11C]NH4SCN will be reported in due course.  

Conclusions

[11C]NH4SCN can now be efficiently formed via the reaction of 11CS2 with ammonia.  This presents a new and facile route to access the [11C]SCN- nucleophile. We have demonstrated that [11C]NH4SCN can be used to prepare a range of interesting 11C-labelled phenacylthiocyanates and thiazoles that would be very difficult to access via established methods.  We are investigating further reactions of [11C]NH4SCN, including routes to more complex heterocylic derivatives that may have application for PET imaging.   

References

[1] P. W. Miller, and D. Bender, Chem. Eur. J., 2012, 18, 433.

[2] T. Haywood, S. Kealey, S. Sanchez-Cabazez, J. Hall, L. Allot, G. Smith, C. Plisson and P. W. Miller, Chem. Eur, J., 2015, 21, 9034.

Acknowledgement

The EPSRC is gratefully acknowledged for funding (grant no. EP/L025140/1)

Scheme 1
Formation of [11C]NH4SCN and reaction with alpha-bromoketones to generate carbon-11 phenacylthiocyanates and thiazoles. 
Keywords: carbon-11, radiolabelling methods, thiocyanate, thiazole, labelled heterocycles
# 055

Standardization of magnetic IONPs' surface coating as key factor to achieve a potential ‘companion diagnostic’ agent for cancer stratification based on optical/MRI molecular imaging. (#257)

M. Filice1, 2, E. Yazdanparast1, I. Martin Padura1, P. Morales3, M. A. Del Pozo1, J. Ruiz-Cabello4, 2, M. Marciello3

1 National Research Centre for Cardiovascular Disease (CNIC), Madrid, Spain
2 Biomedical Research Networking Center for Respiratory Diseases (CIBERES), Madrid, Spain
3 Materials Science Institute of Madrid (ICMM)/CSIC, Madrid, Spain
4 Universidad Complutense de Madrid, Madrid, Spain

Introduction

Since the interactions of NPs with biological environments are mediated by their surface coating, the quality and reproducibility of the IONPs' surface is a crucial parameter for their successful biomedical application and clinical potential translatability. The oleic acid (OA)/dimercaptosuccinic acid (DMSA) ligand exchange is one of the most widely used coating reaction that, unfortunately, has not been fully characterized and optimized yet. Thus, the potential imaging activity of DMSA-coated IONPs modified with bioactive compounds will be highly unpredictable.

Methods

We produced DMSA-coated IONPs of 10 nm core diameter and characterized those by TGA, TEM, DLS, FTIR, VSM, CLSM, Relaxometry and thiol titration assay. By means of chemical strategies, we elucidated the mechanistic aspects hampering the reproducibility of OA/DMSA ligand exchange reaction. Hence, we optimized a novel protocol accessing monodisperse IONPs with controlled and reproducible DMSA-surrounding shell (n=4) in comparison with untreated DMSA-IONPs. To assess their biomedical potential, after PEGylation and fluorophore labelling, the treated DMSA-IONPs were assessed as dual contrast agent promoting optical and magnetic resonance imaging (OI/MRI) in the diagnosis of an orthotopic xenograft mice model of human breast cancer. The imaging results were confirmed by immunohistochemistry.

Results/Discussion

Firstly, we elucidated the mechanisms involved in the uncontrolled OA/DMSA ligand exchange reaction. Thus, we optimized a strategy overcoming such a drawbacks and permitting to achieve 4 different lots of IONPs with reproducible DMSA coating. Due to the high density of carboxyl and thiol groups newly generated on NPs’ surface, the pegylation and fluorophore covalent linking have been successfully promoted by orthogonal chemistry reactions. All the physic-chemical characterizations confirmed our hypothesis. After assessing the imaging potential in situ with phantoms of OI and MRI, the optimized IONPs have been used for in vivo diagnostic of human breast cancer mice models by EPR passive targeting. Within all the studied models (n=5), the cancer mass has been successfully tracked and analysed in 4 by OI and MRI (DT2=-24%) respectively. In one case, despite the clear presence of tumour, no molecular imaging was promoted at all. All these results were confirmed by immunohistochemistry.

Conclusions

Here, we elucidate the mechanistic aspects of OA/DMSA ligand exchange on IONPs and present a standardized protocol that enables controlled and reproducible DMSA shell grafting. These optimized IONPs successfully accumulate into the tumour’s interstitium by EPR effect enabling cancer diagnosis by OI and MRI. Thus, these optimized NPs showed a potential ability as ‘companion diagnostic’1 enhancing the stratification of tumour mass based on its sensitivity to nanocarrier permeability. This prediction to benefit from nanomedicine could improve the success rate of new therapeutic NP formulations.

References

1 Miller MA, Arlauckas S, Weissleder R. Prediction of Anti-cancer Nanotherapy Efficacy by Imaging. Nanotheranostics2017, 1(3):296-312.

Acknowledgement

M.F. acknowledges the financial support of MINECO and FEDER for research grant no. SAF2014-59118-JIN.

Abstract
# 056

Towards dual optical-PET imaging agents based on transition metals (#383)

Q. Li1, S. Cesarec1, J. Wilton-Ely1, P. W. Miller1

1 Imperial College London, Chemistry, London, United Kingdom

Introduction

Dual modality imaging agents integrate the features of two imaging agents in one discrete molecular entity.  The aim of multimodality imagining is to overcome the physical limitations of each imaging method.  Optical imaging has high spatial resolution but low depth penetration, whereas PET has lower spatial resolution but good depth penetration.  Herein, we aim to develop dual optical-PET imaging agents based on transition metal complexes.  Our transition metal probes are based on a Ru-pyrenyl fluorophore system that is capable of rapidly binding to C-11 labelled dithiocarbmates (figure 1).  

Methods

Dithiocarbamate ligands are well-known to coordinate to a wide range of transition metals, rapidly and at room temperature. In order to prepare carbon-11 labelled dithiocarbamate ligands, 11CH3I was first reacted with suflur at high temperature to generate11CS2, subsequent reaction secondary amines (HNR2, R = alkyl group) was found to generate carbon-11 dithiocarbamate (DTC) ligands in high yield.[1]  Preliminary reactions of these carbon-11 DTCs with Pd, Pt and Au precursors were found to rapidly generate their equivalent carbon-11 DTC labelled complexes. Reactions of DTCs with a range Ru-pyrenyl fluorophores are currently under investigation, where the fluorescence of these transition metal complexes is turned on when coordinated to a DTC molecule. 

Results/Discussion

Carbon-11 labelled diethyl dithiocarbamate (CS2NEt2-) was prepared in high radiochemical yields (>95% based on analytic HPLC) via the reaction 11CS2 and diethyl amine. Reaction of [11C]CS2NEt2aqueous solution of KAuCl4 solution, PtCl2(cyclooctadiene) and PdCl2(cyclooctadiene) for 5 min at room temperature resulted in the formation of their equivalent carbon-11 DTC labelled complexes in good radiochemical yields (50-95%) (Scheme 1).  We are interested in developing this method further for radiolabelling 6 coordinate Ru(II) complexes that contain a fluorophore moiety.  Ru-pyrenyl fluorophores were prepared via the reaction of [RuHCl(CO)(PPh3)3] with benzothiadiazole (BTD) and 1-ethynylpyrene (Pyr) [Ru(CH═CHPyr)Cl(CO)(BTD)(PPh3)2] (complex 1). [1] We are currently investigating the synthesis of derivatives of complex 1 and undertaking reactions labelling with [11C]CS2NEt2- to develop a dual optical-PET probe.  

Conclusions

Carbon-11 labelled DTCs can be efficiently prepared in short reaction times using our 11CS2 method.   Labelled DTCs have been reacted with a range of transition metal precursors to generate C-11 labelled complexes.  Fluorescent Ru precursors can be easily prepared and have been found to readily react with unlabelled DTC ligands. Cell uptake and toxicity studies of these compounds are under investigation.  Application of C-11 labelled DTCs for the preparation of dual PET-optical fluoresent Ru complexes is currently underway and results will be reported in due course. 

References

[1] Haywood T, Kealey S, Sanchez-Cabezas S, Hall JJ, Allott L, Smith G, Plisson C, Miller PW, Chem. Eur. J., 2015, 21, 9034-9038

[2] Moragues ME, Toscani A, Sancenon F, Martinez-Manez R, White AJP, Wilton-Ely JDET, J. Am. Chem. Soc., 2014,136, 11930-11933

Acknowledgement

We are grateful to the EPSRC (grant no. EP/L025140/1)

figure 1
Dual imaging optical-PET probe 
Scheme 1
preparation of carbon-11 DTC and labelling of transition metal complexes
Keywords: dual imaging, optical-PET, carbon-11, transition metal complex, multimodal
# 057

Entrapment of nanoparticles in a thermoresponsive polymer during temperature-induced precipitation (#384)

V. Herynek1, K. Kolouchová2, O. Sedláček3, M. Hrubý2, O. Kaman4, A. Gálisová1, D. Jirák1, M. Hájek1

1 Institute for Clinical and Experimental Medicine, Radiodiagnostic and Interventional Radiology Department, Prague, Czech Republic
2 Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic
3 University of Ghent, Ghent, Belgium
4 Institute of Physics, Czech Academy of Sciences, Prague, Czech Republic

Introduction

An interest in thermoresponsive polymers reflects their potential in medical applications as MR traceable drug carriers. Change of their structure with temperature substantially influences not only mechanical properties, but also release of the carried molecules or particles. We studied entrapment of small hydrophilic molecules and bigger nanoparticles by a thermoresponsive polymer during temperature-dependent structure changes by using dynamic MR relaxometry.

Methods

A thermoresponsive polymer poly[(2,2-difluorethylacrylamide)-co-(N-imidazopropylacrylamide)], was prepared and diluted (1:9) in a phosphate buffer (pH=7.4).

The process of polymer crosslinking and particle entrapment was monitored dynamically during temperature increase by continuous measurement of T2 relaxation times. A pure polymer solution, a polymer solution with gadolinium chelates (Gd-DO3A-butrol, concentration c = 1 mM), and a polymer solution with suspended nanoparticles (30 nm silica-coated ferrites (1), c = 0.02 mM) were measured together with the same solutions without the polymer. T2 was measured at 0.5 T. Relaxation rates of the pure polymer solution and buffer were calculated as R2 = 1/T2, relaxivities of samples with Gd chelates or nanoparticles were expressed as r2 = 1/T2/c.

Results/Discussion

The dissolved polymer at low temperature had a little effect on water relaxation rate (Fig. 1A). R2 decreased with increasing temperature similarly to that of water. Polymer crosslinking started around 18°C, which limited water diffusion leading to temporary R2 increase. Further temperature increase caused further crosslinking and precipitation, which squeezed water molecules out of the polymer hydrophobic inside. A precipitate had negligible influence on water R2.

Similar results were found for the polymer solution with Gd chelates (Fig. 1B). Precipitation did not change the dependence substantially. Gd chelate molecules were eliminated from the polymer together with water during precipitation.

Ferrite nanoparticles significantly influenced relaxivity of the polymer solution (Fig. 1C). We observed steep decrease of relaxivity below the values of the pure nanoparticle suspension at temperatures above 20°C. We presume that the nanoparticles remained entrapped in the precipitate.

Conclusions

Small and diluted molecules were easily released from the thermoresponsive polymer together with water during precipitation. Bigger nanoparticles remained entrapped in the precipitate or bound to hydrophilic groups on the surface during polymer crosslinking, which effectively eliminated particles from the rest of the buffer.

We demonstrated that binding or release of particles by the thermoresponsive polymer depends on their size and surface, and might be monitored by dynamic MR relaxometry.

The study thus contributes to examination of controlled drug release from the polymer.

References

1. Kaman O et al. JMMM 2017; 427:251–257

Acknowledgement

The study was supported by the Czech Science Foundation, projects No. 16-04340S and P205–16-03156S

Temperature dependent relaxivity of solutions containing the polymer
Temperature dependence of the relaxation rate of the pure polymer and buffer (A), temperature dependence of the relaxivity of the polymer solution with dissolved Gd chelates (B), and  temperature dependence of the relaxivity of the polymer solution with suspended ferrite nanoparticles (C).
Keywords: thermoresponsive polymer, nanoparticles, MR relaxometry, drug carriers
# 058

Multimodal nanoparticles for structural and functional tracking of stem cell therapy on muscle regeneration. (#260)

G. Loudos1, 2

1 National Center for Scientific Research (NCSR) “Demokritos”, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, Athens, Greece
2 Technological Educational Institute of Athens (TEIA), Department of Biomedical Engineering, Athens, Greece

Introduction

Stem cell therapy keeps gaining ground as a promising approach for a broad range of diseases, with currently no alternative effective therapies. However, tools providing real-time tracking of transplanted cells on their early biodistribution and viability, are missing from the current therapeutic approaches. Thus, the need to develop methods which could evaluate and predict the safety and success of cell-based treatments is crucial.

Methods

nTRACK is a new H2020 project, envisaging to overcome the current barriers of cell therapeutics, by the development of safe and functional nano-based multimodal imaging agents. The nTRACK agent will compromise a magnetic core and a gold shell, to allow for simultaneous use of different imaging techniques, such as CT, MRI and PET for tracking stem cells used in muscle injury. This will also enable non-invasive whole-body monitoring of living stem cells in large animal models, to resemble human complexity. It will furthermore provide tailored software for the interpretation and optimization of the imaging acquisition, including embedded mathematical models, to support the data analysis and ultimate distribution of transplanted cells, viability and prediction of the therapeutic success.

Results/Discussion

Despite its early stage, nTRACK's preliminary results strongly support the concept and feasibility of the proposed project's scheme. More specifically, it has already been demonstrated, both in vitro and in vivo, that glucose coating of gold NPs (GNP) facilitates their cellular uptake, and generates ideal imaging agents for real-time, quantitative and prolonged monitoring of stem cells, using both MRI and CT[1-3]. A complete quantification of the gold concentration required to induce x-ray image contrast at different energies has also been performed, to allow an optimized gold shell design of the NPs and also optimize the imaging protocols that will be followed.

Conclusions

n-TRACK's preliminary results show its great potential in the area of predictive and precise medicine, surpassing the current limitations of cell therapy. The design and implementation of the nTrack imaging agent will allow for real-time tracking of transplanted cells and thus for an early prediction on the stem cell therapy success, by also exploiting the advantages of the most prominent clinically-used multimodal imaging systems.

References

1.Betzer, O., et al., In-vitro Optimization of Nanoparticle-Cell Labeling Protocols for In-vivo Cell Tracking Applications. Scientific Reports, 2015. 5: p. 15400.

2. Meir, R., et al., Nanomedicine for Cancer Immunotherapy: Tracking Cancer-Specific T-Cells in Vivo with Gold Nanoparticles and CT Imaging. ACS Nano, 2015. 9(6): p. 6363-6372.

3. Motiei, M., et al., Differentiating Between Cancer and Inflammation: A Metabolic-Based Method for Functional Computed Tomography Imaging. ACS Nano, 2016. 10(3): p. 3469-3477.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 761031. George Loudos is presenting this work on behalf of the n-TRACK consortium.

Figure 1:
Schematic illustration of the n-TRACK’s project plan.
Figure 2
Schematic illustration of the nTRACK NPs, to be used as a multimodal imaging agent.
# 050

An automated synthesis method for the production of Ga-68 labelled ubiquicidine. (#338)

J. S. Le Roux1, S. Rubow1, T. Ebenhan2, C. Wagener3

1 Stellenbosch University, Medical Imaging and Clinical Oncology, Tygerberg, South Africa
2 Pretoria University, Department of Nuclear Medicine, Pretoria, South Africa
3 The South African Nuclear Energy Corporation, Radiochemistry, Brits, South Africa

Introduction

Radiosynthesis of radiopharmaceuticals and more specifically radiopharmaceuticals for positron emission tomography (PET), often involves the use of automated synthesis modules for the production process (Decristoforo, Pickett and Verbruggen, 2012). Ubiquicidine (UBI) 29-41 is an antimicrobial peptide and synthetic forms of this peptide have been suggested as possible agents for imaging infections (Kahrom et al., 2014). Methods for radiolabelling of this tracer with Ga-68 only describe manual processes (Thomas Ebenhan et al., 2014; Vilche et al., 2016).

Methods

Ga-68 for radiolabelling was freshly eluted from an iThemba Labs Ge-68/Ga-68 generator, using the fractional elution method with 0.6 N HCl. NOTA-UBI was provided by BL Biochem (Shanghai, China). The approach to developing an automated method was to first duplicate the manual method developed by Ebenhan et al. (Thomas Ebenhan et al., 2014),  using the generator, eluant and consumables available at our PET Centre. Next, the manual method was compared to a currently well-established automated methods using the Scintomics GRP synthesis unit. The manual method was then adapted to suit the Scintomics procedures, e.g. adapting volumes for the synthesis unit. The radiolabelling yield and radiochemical purity were determined after each labelling experiment.

Results/Discussion

Initial work required three successful manual labelling procedures before the method can be transferred to an automated synthesis method. It was decided to use a 1.5 M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer to adjust the eluate to an acceptable pH. Eluate volumes ranging from 1.0 – 2.0 ml were used to which 1.2 – 1.6 ml of HEPES buffer was added and the pH measured in order to find the most suitable combination of eluate volume and volume of HEPES buffer to render a labelling mixture with a pH of between 3.5 – 4.0. An automated synthesis protocol for the Scintomics GRP module was created and tested. Results indicated that an eluate volume of 2.0 ml and a HEPES buffer volume of 1.5 ml proved to be suitable for use in the development of an automated synthesis method. Four  successful automated labellings were performed using the HEPES buffer with an average decay-corrected radio-yield of 88.97% and a radiochemical purity of 99.49%.

Conclusions

An automated synthesis protocol using a Scintomics GRP Module has been successfully developed and tested. This protocol can be utilised for the routine synthesis of Ga-68 UBI under GMP conditions.

References

Decristoforo, C., Pickett, R. D. and Verbruggen, A. (2012) ‘Feasibility and availability of 68Ga-labelled peptides’, European Journal of Nuclear Medicine and Molecular Imaging, 39(SUPPL.1). doi: 10.1007/s00259-011-1988-5.

Ebenhan, T. et al. (2014) ‘Peptide synthesis, characterization and68Ga-radiolabeling of NOTA-conjugated ubiquicidin fragments for prospective infection imaging with PET/CT’, Nuclear Medicine and Biology. Elsevier Inc., 41(5), pp. 390–400. doi: 10.1016/j.nucmedbio.2014.02.001.

Kahrom, M. et al. (2014) ‘Poor sensitivity of 99mTc-labeled ubiquicidin scintigraphy in diagnosis of acute appendicitis’, European Surgery - Acta Chirurgica Austriaca, pp. 173–176. doi: 10.1007/s10353-014-0278-4.

Vilche, M. et al. (2016) ‘68Ga-NOTA-UBI-29-41 as a PET Tracer for Detection of Bacterial Infection’, Journal of Nuclear Medicine, 57(4), pp. 622–627. doi: 10.2967/jnumed.115.161265.

 

Keywords: automated synthesis, gallium-68 ubiquicidine, fractional elution
# 059

Preparation of [11C]Acetate using an Arduino-based synthesizer, and biodistribution analysis in healthy mice (#289)

A. Maurer1, C. Parl1, M. Krüger1, B. J. Pichler1

1 Eberhard Karls Universität Tübingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Baden-Württemberg, Germany

Introduction

Various [11C]acetate radiosyntheses have been published, but most of them require a dedicated hot cell or use adaptations in a complex synthesizer, blocking the equipment for the duration of synthesis and subsequent cleaning. Homemade solutions published rely on commercial software and expensive equipment. We established a radiosynthesis of [11C]acetate based on an inexpensive open-source platform with small footprint, enabling back-to-back production in a hot cell already occupied by other modules. To establish a PET protocol we analyzed dynamic tracer uptake in healthy mice.

Methods

[11C]CO2 was trapped and released using a Tracerlab FX MeI module and then captured in a loop coated with MeMgCl. [11C]acetate was passed through H+ and Ag+ cartridges, trapped on a anion exchange cartridge and eluted with saline solution [1]. Following elution, the product was buffered with citrate buffer and purged with pressurized air for 2 min. The whole process was fully automated using a ATmega328P microcontroller (Arduino Uno), with tube liquid detectors for sensing completion of individual steps.

Animal experiments were approved by the local authorities. Healthy C57BL/6 mice were injected with 354+/-21 µCi of [11C]acetate and dynamic activity distribution was measured for 60 minutes on an Inveon D-PET scanner, followed by anatomical MRI scans and ex vivo biodistribution analysis.

Results/Discussion

A module consisting of six "recycled" solenoid valves and one optical liquid detector was successfully automated using Arduino Uno. Components were assembled using 3D-printed and lasercut parts, with material costs of < 100 € (excluding valves).

The synthesis of Soloviev et al. [1] served as starting point but occasionally yielded contamination with [11C]CO2. Thus we optimized the protocol by adding a bubbling step, now achieving radiochemical yields of 40+/-21% and radiochemical purities of 94+/-7% (n=9) in a synthesis time of 15 min.

In line with available literature, biodistribution analysis showed a low background in healthy animals, with the highest activity in liver (2.4+/-0.5%ID/g) and kidneys (1.3+/-0.3%ID/g) and only 0.4+/-0.1%ID/g in the blood. Currently we are evaluating the tracer performance in different tumor models.

Conclusions

This setup allows us stable and flexible supply with [11C]acetate and paves the way for further measurements in our lab. The setup can easily be replicated at other sites using inexpensive equipment.

Products based on the Arduino platform have become valuable and versatile tools in our lab, automating processes where full radiochemistry synthesizers are too bulky and uneconomical. Due to its inexpensive and simple character, this approach has the potential to increase tracer production capabilities in many radiopharmacies, thereby improving preclinical research.

References

[1]          Soloviev, D. and C. Tamburella (2006). "Captive solvent [11C]acetate synthesis in GMP conditions." Applied Radiation and Isotopes 64(9): 995-1000.

Acknowledgement

The research leading to these results has received funding from the Clinical Research Unit FOR2314 of the DFG.