Abstract/Video opens by clicking at the talk title.
Abstract/Video opens by clicking at the talk title.
|3:45 p.m.||PS 10-01||
Introductory Lecture (#669)
1 Radboud University, Nijmegen, Netherlands
|4:03 p.m.||PS 10-02||
Aryl and amine substitution to induce large Stokes shift in BODIPY fluorophores for STED microscopy applications (#503)
Srinivas Banala1, 2, Jean M. Merkes2, Magnus Rueping2, Fabian Kiessling1
1 RWTH Aachen University, Experimental Molecular Imaging, Aachen, Germany
Super-resolution microscopy techniques enable visualization of structure and dynamics of cells in nm-resolution. In particular STimulated Emission Depletion (STED) technique has gained popularity, in which fluorophores-labeled cells are excited first, and by applying an intense donut-shaped depletion laser nm-resolution is achieved. To date widely used fluorophores are based on coumarin, xanthene, cyanine cores for 592 and 760 nm depletion lasers, but are not suitable for newly developed 660 nm laser. Hence novel stable fluorophores for 660 nm STED are needed, which is explored here.
Highly photostable and fluorescent BODIPY was unsymmetrically substituted in a multi-step synthetic sequence in a 2-aryl-3-amino fashion. These substituents were also used as anchor to attach linkers. The obtained dyes were purified, characterized by UV-vis, NMR and ESI-MS. Dyes exhibiting suitable optical properties were studied for XTT cell viability assays, and passive cell uptake in growth medium. The most suitable dye 1 was applied in cellular labeling, first by passive diffusion, later for active targeting and applied in STED imaging with 660 nm depletion. The dye 1-conjugates were synthesized for active targeting. Achieved actin imaging using 100 pmol of 1-phalloidin in formalin fixed HeLa cells. Confocal and STED images were obtained using Leica TCS SP8 STED system.
Unsymmetrical 2-aryl 3-amine substitution on BODIPY core (ArAmBDP) yielded an 80 nm Stokes shift exhibiting (λabs = 510 nm, λem = 590 nm) bright dye 1 with molar extinction coefficient ~50,000 M-1cm-1, and fluorescence quantum yield (Φf ) of 66 % (Fig. 1B). In addition, both the 3-amine or 2-aryl positions were explored to attach linkers, targeting moieties and to prepare bio-conjugates for labelling experiments. The dye 1 exhibited high biocompatibility in XTT assay (A549, PC3 cells) in up to 24 µM. The photostability of 1 was exceptional, as we could visualize the dye in labelled cells stored at ambient temperature, even after 5 months. Furthermore, photostability towards STED depletion pulse was proved, by measuring up to 85 STED-images in a 3-dimensional XZ-scan and multiple images at the same site and compared with commerical dye. The application of 1 in actin imaging was also achieved, using 1-phalloidin conjugate (Fig 1G).
The 2-aryl 3-amine substitution of BODIPY proved to be successful to yield large Stokes shift exhibiting fluorophores, without affecting inherent high fluorescence quantum yields and photostability of BODIPYs. Future work is focussed on live-cell STED imaging, multi-colour nanoscopy applications, and further tuning the optical properties for other STED lasers.
AcknowledgmentFinancial support from Excellence Initiative of the German federal and state governments through the I3TM Seed Fund, I3TM Step2Projects, and Dr. Ulf Schwarz from Leica Microsystems for imaging are acknowledged.
 Sahl, SJ, Hell. SW, Jakobs, S, 2017, 'Fluorescence nanoscopy in cell biology', Nat Rev Mol Cell Biol. 18, 685- 701, Nature Publishers
 Sednev, MV, Belov, VN, Hell SW, 2015, 'Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review', Methods Appl. Fluoresc. 3 042004, IOP Publishing;
 Banala, S, Rueping, M, Kiessling, F, Merkes, JM, 2019, 'Fluorescent dyes' Patent application, Ref. no. DE 10 2018 132 305.0, German Patent Office
A) Core structure of ArAmBDP, B) optical characteristics of 1, C) XTT cell viability assay for 1, D) proof of chemical photostability by confocal imaging over 5 months, E), F) confocal and STED images using (passive diffused) 1 for excitation at 540 nm and depletion with 660 nm, respectively, G) confocal and STED images of actin filaments using 1-phalloidin conjugate.
Keywords: Synthesis, BODIPY, large Stokes shift, super resolution
|4:15 p.m.||PS 10-03||
Effect of OATP-mediated transport on the biodistribution of fluorescently-labeled molecules (#365)
Stefanie Rosenhain1, Tim M. Wiechmann1, Fabian Kiessling1, 2, Felix Gremse1
1 University Hospital RWTH Aachen, Institute for Experimental Molecular Imaging, Aachen, Germany
Fluorescence-mediated computed tomography (FLT-CT) using fluorescently labeled molecules plays an important role in preclinical cancer research. Organic anion transporting proteins (OATP) recently gained attention, because they are involved in the uptake of these molecules. It is proven that not only hepatocytes but also most cancer cells overexpress different OATPs. Since this can cause false-positive results, especially in tumor and liver tissue, it is important to understand the OATP-mediated transport of fluorescence dyes and their influence on the biodistribution.
OATP-mediated uptake of AlexaFluor750 (AF750), Cy7, DY-730, DY-736, DY-750, ICG, IR-783, IRDye 750, and 800 CW was investigated by FLT-CT phantom and absorption measurements. OATP-mediated uptake of the dyes in A431, HepG2, and HUVECs were investigated at 4°C and 37°C using fluorescence microscopy (20 µM; 2h). Inhibition of the OATPs were performed using Rifampicin (20µM). qPCR was performed to assess OATP expression (SLC01A2, SLC01B1, SLC01B3, and SLC02B1). Effects on biodistribution and tumor accumulation of free AF750, Cy7, DY750, and IR-783 were analyzed in five A431 tumor-bearing athymic nude mice. After injection of the dyes (2 nmol/animal), mice were measured by FLT-CT at 0h, 3h, 6h, 24h, 48h, 72h, and 96h. After 96h, excised organs were imaged in the 2D fluorescence mode.
qPCR demonstrated a higher expression of OATP1A2 and OATP1B1 in both A431 and HepG2. Microscopic analysis revealed no uptake of AF750 and Cy7 into A431, in contrast to the uptake of DY-750 and IR-783. These finding were also confirmed in vivo. After injection of AF750, no tumor signal but a strong bladder signal along with a fast clearance from the whole body (6h) can be seen. Fluorescence intensities of Cy7 were strong both in liver and gut at early points in time. Most of the dye is cleared not later than 48h post injection via the hepatobiliary elimination route, although some signal remained in the kidneys. IR-783 was showing a strong tumor accumulation even at 96h. These findings were supported by the ex vivo results. The preliminary results indicate a possible impact of NIRF dyes on biodistribution assessment of fluorescence-tagged molecules. Especially IR-783 shows a strong tumor signal, possibly influencing tumor accumulation of molecules labeled with IR-783.
A comprehensive knowledge of OATP-mediated transport processes and their effect on the biodistribution and tumor accumulation will increase proper fluorescence labeling strategies without bias the pharmacokinetic properties and optimize the specificity of favorable targeting. Further studies are needed to investigate the biodistribution of fluorescence-tagged molecules and the role of OATP mediated transport in vitro and in vivo.
 Vasquez, KO, Casavant, C, Peterson, JD, 2011, 'Quantitative Whole Body Biodistribution of Fluorescent-Labeled Agents by Non-Invasive Tomographic Imaging', PLoS ONE 6(6): e20594. doi:10.1371/journal.pone.0020594
 Patik, I, Szekely, V, et al. 2018, 'Identification of novel cellimpermeant
fluorescent substrates for testing the function and drug interaction of Organic Anion-
Transporting Polypeptides, OATP1B1/1B3 and 2B1', Scientific Reports, 2018) 8:2630 | DOI:10.1038/s41598-018-20815-1
 Roth, M, Obaidat, A, Hagenbuch, B, 2011, 'OATPs, OATs and OCTs: the
organic anion and cation transporters of the SLCO and SLC22A gene superfamilies', British Journal of Pharmacology, 165 1260–1287
In vitro characterization of OATP-mediated transport of AlexaFluor750 (AF750), Cy7, DY750, and IR-78
(A) Absorption spectrum of AF750, Cy7, DY750, and IR-783. (B) Increase of fluorescence intensities dependent on fluorescence dye amount measured by phantom FLT-CT. (C) Fluorescence microscopy of dye uptake at 37°C, 4°C, and after incubation with the Rifampicin (unspecific inhibition of OATPs). (D) Gene expression analysis of different OATPS using real time PCR.
In vivo and ex vivo biodistribution analysis of intravenously injected free dyes: AF750, Cy7, DY750,
After injection of IR-783, there is a strong tumor accumulation that last until 96h p.i. AF750 exhibit a fast clearance from the whole body which is evidenced by the strong bladder signals. Cy7 depicted strong fluorescence intensities both in liver and gut at early points in time. Although some signal remains in the kidneys, most of Cy7 is hepatobiliary cleared not later than 48h p.i. DY750 shows indistinct signals in the liver, but is completely cleared after 24h with no remaining signal in the organs 96h post injection. Further fluorescence intensity quantification needs to be performed.
Keywords: OATP, fluorescence, labeling, preclinical imaging
|4:27 p.m.||PS 10-04||
Perylene Diimide-Based Nanoparticles as Photoacoustic Contrast Agents for in vivoCell Tracking (#270)
Claudia Fryer1, 2, Haifei Zhang1, Patricia Murray2, Yonghong Yang1, 2
1 University of Liverpool, Department of Chemistry, Liverpool, United Kingdom
The ability to non-invasively track the fate and biodistribution of transplanted stem cells is imperative for the successful design of regenerative medicine therapies (RMTs).1 Multispectral optoacoustic tomography (MSOT) is a preclinical bioimaging modality with excellent spatial resolution and high sensitivity, without the need for ionising radiation. Near-infrared absorbing nanoparticles, based on perylene diimide (PDI) derivatives, have been developed as contrast agents for MSOT with excellent uptake into mesenchymal stromal cells (MSC), strong NIR attenuation and high biocompatibility.
N,N’-Bis(cyclohexyl)-1,7-di(pyrrolidinyl)perylene-3,4,9,10-tetracarboxy bisimide was prepared using a well-established method (Fig. 1).2,3 PDI nanosuspensions were prepared via nanoprecipitation with hyperbranched block copolymer (DEAEMA50-c-DEGDMA2)-b-(OEGMA80), developed by our group, as a stabiliser.
An NIR-absorbing PDI was successfully prepared with peak absorption at 710 nm, where each step of the synthesis was confirmed with ‘H NMR and UV-vis spectroscopy. This PDI derivative has good solubility in common solvents such as acetone. NIR PDI was formulated into nanoparticles by first dissolving in acetone and adding dropwise to water with our novel stabiliser. The nanosuspension shows excellent stability with a particle size of 100 nm and surface zeta potential of 0 mV.
Novel NIR-absorbing probes have been prepared, based on perylene diimide, for the tracking of MSCs. These nanoparticles have peak absorption of 710 nm, excellent stability in aqueous medium and a particle size of 100 nm. PDI nanoparticles showed limited cytotoxicity, good uptake and intense MSOT signal in MSCs. Therefore, these probes show high potential for in vivo tracking of cells with MSOT, in order to aid the design of RMTs.
AcknowledgmentYonghong Yang, Haifei Zhang, Patricia Murray, Jack Sharkey, Aiden Thomas, Ulrike Wais, Alexander W. Jackson, EPSRC
 Scarfe, L, Brilliant, N, Kumar, JD, Ali, N, Alrumayh, A, Amali, M, Barbellion, S, Jones, V, Niemeijer, M, Potdevin, S, Roussignol, G, Vaganov, A, Barbaric, I, Barrow, M, Burton, NC, Connell, J, Dazzi, F, Edsbagge, J, French, NS, Holder, J, Hutchinson, C, Jones, DR, Kalber, T, Lovatt, C, Lythgoe, MF, Patel, S, Patrick SJ, Piner, J, Reinhardt, J, Ricci, E, Sidaway, J, Stacey, GN, Starkey Lewis, PJ, Sullivan, G, Taylor, A, Wilm, B, Poptani, H, Murray, P, Goldring, CEP, Park, BP 2017. 'Preclinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies', NPJ Regenerative Medicine, 2:28, 1-13
 Sengupta, S, Dubey, RK, Hoek RWM, van Eeden, SPP, Gunbas, DD, Grozema FC, Sudholter, EJR, Jager, WF 2014, 'Synthesis of Regioisomerically Pure 1,7-Dibromoperylene-3,4,9,10-tetracarboxylic Acid Derivatives', The Journal of Organic Chemistry, 79, 6655-6662
 Sukul, PK, Datta, A, Malik, S 2014. 'Light Harvesting and Amplification of Emission of Donor Perylene–Acceptor Perylene Aggregates in Aqueous Medium', Chemistry A European Journal, 20, 3019-3022
Figure 2: Confocal microscope image of PDI nanoparticles in MSCs
MSCs were seeded and left to grow for 24hr before labelling with PDI nanoparticles (25 µg/ml) for a further 24hr. MSCs were then fixed and stained with DAPI (nuclei, blue) and AlexaFluor 488® (cytoskeleton, green), where PDI nanoparticles can be seen in red (after 24 hr incubation).
Keywords: perylene diimide, photoacoustic imaging, cell tracking, stem cells, near-infrared probe
|4:39 p.m.||PS 10-05||
Theranostic ICG-Mesoporous Silica Nanoparticles (MSNs) as photoacoustic contrast agents: development of a ratiometric approach for assessing drug release (#154)
Giuseppe Ferrauto1, Fabio Carniato2, Enza Di Gregorio1, Silvio Aime1, Mauro Botta2, Lorenzo Tei2
1 University of Torino (It), Dept of molecular biotechnologies and health sciences, Torino, Italy
ICG is widely employed in optical and photoacoustic preclinical studies. However, in vivo application is hampered by i) the sequestration from serum albumin and ii) the reduction of optical/PAI efficiency due to interaction with micro-environment (e.g. O2). The loading inside mesoporous silica nanoparticles (MSNs) may prevent these problems.1 Herein, two formulations of ICG-MSNs are compared, i.e.: a) ICG filled inside MSNs’ pores1 and b) ICG covalently bound to MSNs loaded with the drug mitoxantrone (MTX)2. Furthermore, an innovative ratiometric approach to assess MTX release is proposed.
MSNs were prepared through a sol-gel procedure and functionalized on the surface with aminopropyl groups and PEG molecules. In the first formulation, ICG was loaded into MSNs’ pores; in the second formulation, ICG-NHS active ester was covalently attached to NH2 groups of MSNs and MTX confined into the pores by wet-impregnation. Both formulations were fully characterized by UV-Visible and photoluminescence spectroscopy. PAI analysis was carried out by using a VisualSonics Vevo 2100 LAZR Instrument. For in vitro PAI, ICG-MTX-MSNs suspensions were loaded onto plastic capillaries surrounded by agarose gel. For in vivo PAI, Balb/c mice bearing intramuscolar TS/A breast cancer cells were injected with ICG-MSNs and PA images of tumour region were acquired.
Both ICG-MSNs formulations display PA effect, higher when ICG is loaded inside pores than when bound to the surface. Serum albumin sequestering is fully avoided, when ICG is covalently bound to MSNs, making the system suitable for in vivo applications. In this formulation, pores are available to be loaded with a drug; MTX is convenient for this purpose because it displays a proper PA signal (at l=700nm), different from the PA signal of ICG (l=810nm)(Fig.1A). These two PA signals can be used to develop a ratiometric method to quantify MTX release. In fact, upon MTX release there is a reduction of PA signal at 700nm; signal at 810 nm reports on MSNs concentration. The proof of concept of MTX release in tumor region was obtained: in pre-contrast PAI, only haemoglobin signal is present (Fig.2) whereas in post contrast PA image, the presence of ICG-MTX-MSNs is detectable in the tumour; both PA signals of MTX and of ICG are present. At t=30min, only MTX signal is strongly reduced (Fig.2).
ICG-MSNs can be a suitable PAI contrast agent. Optical and optoacoustic properties, as well as chemical stability, are influenced by the ICG-MSNs formulation. MSNs in which ICG is covalently bound to the surface and MTX drug is loaded on MSNs are efficient systems because: i) ICG sequestering by serum albumin is prevented, ii) a good PAI effect is present, iii) the release of the drug MTX can be monitored by an innovative ratiometric PAI method.
 Ferrauto G. et al. Nanoscale 2017 Jan 7;9(1):99-103.
 Ferrauto G. et al. Nanoscale 2019 Oct 10;11(39):18031-18036.
Keywords: indocyanine green, mitoxantrone, photoacoustic, drug release, mesoporous silica nanoparticles
|4:51 p.m.||PS 10-06||
Phase-change ultrasound contrast agents as a novel strategy for proton range verification (#177)
Bram Carlier1, 2, Sophie V. Heymans3, Sjoerd Nooijens4, Yosra Toumia5, Marcus Ingram4, Gaio Paradossi5, Emiliano d'Agostino6, Jan D'Hooge4, Koen Van Den Abeele3, Edmond Sterpin1, 2, Uwe Himmelreich2, 7
1 KU Leuven, Department of Oncology, Leuven, Belgium
The favorable depth-dose distribution of proton beams (Bragg profile) theoretically allows to reduce healthy tissue exposure and spare organs-at-risk. However, range uncertainties prevent the full potential of proton therapy by imposing large margins on the treatment plan (1). To reduce these margins, several in vivo range verification tools are under investigation, but clinical translation has proven difficult (2, 3). Here, we examine the radiation-induced phase-change of superheated nanodroplets and evaluate the possibility to correlate the generated ultrasound contrast to the proton range.
The nanodroplets (⌀ = 842 ± 12 nm) comprised of a perfluorobutane (C4F10, boiling point = -2°C) core encapsulated by a UV-polymerized 10,12-pentacosadiynoic acid monolayer. Nanodroplets were fixed in gelatin phantoms and irradiated twice (in forward and reverse position, figure 1b) at 25°C. Differences in microbubble generation in isometric regions of interest (ROI) irradiated with 0, 10 and 20 Gy protons (figure 1d) and control phantoms (kept at 25°C outside of the proton beam) were evaluated using Student’s t-tests (n=3, figure 1e). Extracted bubble count profiles were compared to absolute range measurements for 46.8 MeV and 62 MeV irradiations. Finally, the obtained results were interpreted via the theory of radiation-induced nucleation of superheated emulsions (4).
Figure 1b, 1c and 1e show a statistically significant increase in microbubble generation in the zones irradiated with 10 Gy and 20 Gy with respect to the zone distal to both proton ranges. The bubble counts of the latter did not differ from the control phantoms, confirming radiation-induced vaporization. However, the extracted bubble count profiles did not follow the Bragg curve. This was explained by the insufficient linear energy transfer (LET) of protons, reaching up to 90 keV/µm at the distal end of the Bragg peak. Instead, the radiation-induced nucleation theory described an LET threshold for nucleation of 370 keV/µm. Hence, we hypothesized that vaporization was triggered by nuclear recoils. This also explained the abrupt signal drop before the end of the proton range (figure 1c and 1f). The observed signal shift (2.60 ± 0.26 mm (n=3) for 62 MeV and 3 mm (n=2) for 46.8 MeV irradiations) was related to the proton range with sub-millimeter repeatability.
The radiation-induced phase-change of superheated nanodroplets was confirmed by the strong increase in microbubble generation in proton-irradiated zones. However, the calculated LET threshold in combination with the observed results identified nuclear recoils instead of primary protons as the vaporization stimuli. The generated ultrasound contrast could be related to the proton range with sub-millimeter reproducibility.
This project has received funding from the European Union’s Horizon 2020 research and innovation Programme under grant agreement 766456 (AMPHORA). BC received a PhD fellowship fundamental research from the Research Foundation Flanders (n°11A9520N).
 Paganetti, H 2012, 'Range uncertainties in proton therapy and the role of Monte Carlo simulations', Physics in Medicine & Biology, 57, R99
 Knopf, AC, Lomax, A 2013, 'In vivo proton range verification: A review', Physics in Medicine & Biology, 58, 131
 Jones, KJ, Nie, W, Chu, JCH, Turian, JV, Kassaee, A, Sehgal, CM, Avery, S 2018, 'Acoustic-based proton range verification in heterogeneous tissue: simulation studies', Physics in Medicine & Biology, 63, 025018
 d'Errico, F 2001, 'Radiation dosimetry and spectrometry with superheated emulsions', Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms, 184, 229
Figure 1. Radiation response of superheated nanodroplets
Ultrasound images before (a) and after (b) 62 MeV proton irradiation with 10 Gy in forward and 20 Gy in reverse position. The corresponding bubble count profiles are displayed in (c). Statistical differences in bubble quantification (d) after subtraction of the pre irradiation counts are shown in (e). * p<0.05, ** p<0.01 and *** p<0.001 for n=3. Blue lines from one-tailed Student t-tests, black lines from two-tailed t-tests. (f) depicts the relation between the ultrasound contrast (blue) and the absolute dose measurements (red) for 46.8 MeV and 62 MeV irradiations.
Keywords: range verification, contrast-enhanced ultrasound imaging, nanodroplets, radiation-induced nucleation theory, proton therapy