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Introductory Talk by Kai Licha - Berlin, Germany
This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.
Lanthanide(III)-Based Molecules, Macromolecules and Nanomaterials: New Generation of the Near-Infrared Emitting Probes for Optical Imaging (#34)
I. Martinic1, S. V. Eliseeva1, T. Nguyen3, D. Gosset1, S. Routier2, F. Suzenet2, V. L. Pecoraro3, S. Petoud1
1 CNRS, Center for Molecular Biophysics, Orleans, France
Fluorescence optical imaging is a highly sensitive technique. In particular, imaging in the near-infrared (NIR) region attracts significant attention due to reduced autofluorescence and light scattering. New generation of optical imaging agents, lanthanide(III) (Ln3+)-based probes (LnPs), possess unique optical properties e.g. sharp emission bands not affected by the microenvironment and high resistance toward photobleaching. Low absorbance of free Ln3+ requires the use of appropriate chromophores for sensitization of their luminescence.1 Herein, we present several families of LnPs.
Yb3+[Zn(II)MCpyzHA] was synthesized by reaction of pyrazinehydroxamic acid with Zn2+ and Yb3+ triflates. The L1Nd3+ complex was obtained by interaction between the ligand L1 and Nd3+ nitrate; AQ1,nOH-Yb@PS/PEG (n=4,8) nanoparticles by encapsulation of 1,4- or 1,8- dihydroxyanthraquinones and Yb3+ triflates in polystyrene beads and their functionalization with polyethylene glycol. (Ln8-)G3P-(aza-BODIPY)n (n=16,32) were synthesized by coupling of aza-BODIPY with generation-3 polyamidoamine (G3P) dendrimer and interaction with Yb3+ or Nd3+ nitrates. Absorption/excitation/emission spectra, luminescence lifetimes, quantum yields and photostabilities were measured. Uptake of LnPs by HeLa cells was analyzed by confocal and epifluorescence microscopies and by flow cytometry in the visible/NIR.
Novel LnPs have been designed and successfully used for visible/NIR in vitro imaging and their advantages over the current fluorescent probes have been demonstrated. In particular, i) non-permeable and photostable polymetallic metallacrown (MC) complexes, Yb3+[Zn(II)MCpyzHA], demonstrated their applicability for labelling of cell necrosis2; ii) monometallic L1Nd3+, the first LnP with excitation and emission wavelengths within the ideal diagnostic window, demonstrated a deeper penetration through tissues of different origin and a more sensitive detection; iii) polymetallic AQ1,nOH-Yb@PS/PEG nanoparticles represent a major breakthrough in the design of LnPs with improved properties by simplifying screening of efficient chromophores; and iv) polymetallic G3P dendrimers, (Ln8-)G3P-(aza-BODIPY)n, demonstrated possibility for tuning of photophysical properties, photostability and cellular uptake, by controlling the number of chromophores at their periphery and by presence and nature of Ln3+.
Presented monometallic and polymetallic Ln3+-based molecules, macromolecules and nanomaterials possess a superior and complementary optical properties over the currently available commercial fluorescent probes. Their unique potential makes them attractive candidates for advanced applications in optical imaging and therefore represents significant breakthroughs toward the creation of a new generation of the NIR optical imaging probes for in vitro and in vivo applications with possibilities for replacement of current probes.
1. I. Martinić, S.V. Eliseeva, S. Petoud, Journal of Luminescence, 189 (2017) 19-43.
2. I. Martinić, S.V. Eliseeva, T.N. Nguyen, V.L. Pecoraro, S. Petoud, JACS (2017), 139, 8388-8391.
This research has received funding from the European Community's Marie Curie Seventh Framework Programme (FP7/2007-2013: n° 316906 (ITN Luminet) and n° 611488 (IRSES Metallacrowns)), La Ligue Contre le Cancer, La Region Centre and ANR Lumiphage.
Figure 2. Results of epifluorescence and confocal microscopies of HeLa cells incubated with LnPs.
Epifluorescence microscopy of: necrotic cells incubated with Yb3+[Zn(II)MCpyzHA] (λex: 447nm/60nm, λem> 805nm, τexp: 10s) (A) and viable cells incubated with L1Nd3+ (λex: 655nm/40nm; λem> 805nm, τexp: 20 s) (B). Confocal microscopy of viable cells incubated with: AQ1,4OH-Yb@PS/PEG (λex: 488nm; λem: 580–750nm) (C) and Yb8-G3P-(aza-BODIPY)16 (λex: 633nm; λem: 650-800nm) (D).
Keywords: Optical Imaging, Luminescence, Near-Infrared, Lanthanide, Coordination, Polyamidoamine Dendrimer, Nanomaterial, Metallacrown, Anthraquinone, Polyaminocarboxylate
Ex Vivo Tracking of Endogenous CO with Ruthenium(II) Complexes (#285)
J. A. Robson1, A. Toscani1, C. Torre2, C. Marín-Hernández2, M. Terencio2, M. Alcaraz2, R. Martínez-Máñez2, F. Sancenón2, J. Wilton-Ely1
1 Imperial College London, London, United Kingdom
In the body, endogenous CO acts as a gaseous signalling molecule serving many functions, including anti-inflammatory, antiapoptotic and anticoagulative roles.1 The potential of CO as a therapeutic agent has received significant attention, particularly since CO-releasing molecules (CORMs) are capable of liberating controlled amounts of CO in biological systems.2 One of the major obstacles limiting progress in understanding the biological role of CO and its therapeutic application is the lack of real-time methods to selectively track CO in biological systems.
Our novel molecular probes provide one- or two-photon fluorogenic detection of CO in biological environments using the IDA paradigm pioneered by Anslyn.3 In vitro studies use confocal and multi-photon microscopy to visualise the release of CO by carbon monoxide releasing molecules CORM-3 and hemin in live cells. Ex vivo studies on mice with a subcutaneous air pouch use Lipopolysaccharides (LPS) to induce an inflammatory reaction characterized by plasmatic exudation and migration of leukocytes to the cavity. LPS also induces HO-1 protein expression in leukocytes migrating to the air pouch exudates, causing them to exhibit higher levels of CO than would be expected in the absence of LPS. Exudates in the pouch are collected and visualized using two-photon microscopy.
Equipped with fluorophores suited for imaging in biological environments, new water-soluble ruthenium vinyl probes have been designed based on the system we have used for detection of CO in air.4 In the presence of CO in vitro, these probes exhibit a rapid and selective fluorogenic response, the probes are selective for CO even in high reactive oxygen species (ROS) environments. In addition, they avoid the drawbacks of current palladium-based systems such as slow response times, pH limitations and the toxicity of unligated Pd salts. Coupled with their pH stability and low toxicity, these attributes allow fluorescence imaging of range cellular environments and permit the visualisation of very low levels of CO in cells. For the first time, these two-photon fluorescent probe detected endogenous carbon monoxide ex vivo using mice with a subcutaneous air pouch.5
The ruthenium probes are nontoxic to cells and can be used in low doses for the in vitro two-photon fluorescence imaging of CO in cells in the presence of CORM-3 or hemin. Even more significantly, the probes can be used in ex vivo two-photon fluorescence, detecting CO in cells collected from the exudates of an air pouch inflammation mouse model. The combination of selectivity, sensitivity and inexpensive, straight-forward synthesis make the system described here a very attractive and efficient probe for the facile fluorogenic detection of this gas in realistic biological environments.
I thank the EPSRC for PhD studentship
Near-infrared fluorescent probe for detecting NF-kB activation in a mouse model of type 1 diabetes (#311)
T. Taghian1, V. G. Metelev1, 2, S. Zhang1, A. A. Bogdanov1
1 University of Massachusetts Medical School, Radiology, Worcester, Massachusetts, United States of America
Detecting inflammation markers in the endocrine pancreas during the silent phase of type 1 diabetes (T1D) will aid in diagnosis and enable early therapeutic interventions . Transcription factor NF-kB plays a pivotal role in regulating beta cell function and is involved in development of T1D. We designed and synthesized near-infrared fluorescent oligodeoxyribonucleotide (NIR-ODN) probes for imaging NF-kB-DNA interactions , Fig.1. The goal of our research was to perform NIR imaging of pancreatic cells and whole pancreatic islets using NIR-ODN probe by using models of experimental diabetes.
Multiple low dose streptozotocin (MLD-STZ) T1D model was developed by using SKH1 mice which received 50 mg/kg/d STZ for 5 d. Pancreata were harvested on day 8 and snap frozen for sectioning. Islets and exocrine tissue fragments were separated using collagenase P with subsequent density centrifugation. Frozen non-fixed tissue sections were incubated with 40-250 nM Cy5.5-labeled NIR-ODN probe in the presence of Mg2+, DTT and tRNA for 4 h followed by staining for insulin. Additionally, tissue sections were examined after treating with anti-NF-kB p65phospho-(S536). Fluorescence images were acquired using 14-bit 20 MHz CCD and quantified using ImageJ. Electrophoretic shift (EMSA) results were obtained by using cell lysates and NIR-ODN probe, visualized and quantified using a NIR imaging system.
Following MLD-STZ treatment of mice, insulin-positive fraction of the STZ-treated pancreas on tissue sections was significantly decreased in observed area compared to control. However, blood glucose levels remained in the normal range throughout the study. Treated mice exhibited significantly higher level of NF-kB expression in the nuclei and cytoplasm of the islet cells vs. control as assessed by fluorescence images using anti-NF-kB p65phospho-(S536) and EMSA. In agreement with these results, Cy5.5 labeled NIR-ODN exhibited highly significant quantitative differences in binding to nuclei and cytoplasmic sites of STZ treated islet cells vs. control cells as demonstrated in frozen sections (Fig.2). Therefore, we demonstrated that the sensitivity of NIR-ODN duplex probe specific for mature NF-kB heterodimer was sufficient for detecting NF-kB expression and activation in MLD-STZ treated normoglycemic non-fixed pancreas in a mouse model of T1D, prior to the onset of diabetes.
Results of biochemical (EMSA) and fluorescence microscopy studies showed that NIR-ODN imaging probe was specific for NF-kB both in cell cytoplasm and nuclei of pancreatic cells. The binding to nuclei and cytoplasmic fraction of pancreatic islet cells correlated with the increased NF-kB activity/expression after MLD-STZ treatment damage to the pancreas. The tested NIR probe is not species-specific and can be used in studies of various animal models of diabetes as well as other inflammatory disease.
1. Mathis, D. et al. Nature, 2001; 414(6865):792-8. 2. Zhang, S. et al. Proc Natl Acad Sci, 2008; 105(11):4156-61.
NIH grants R01 DK095728, R01 EB000858 (AAB).
NIR-ODN NF-kB binding to nuclei and cytoplasmic fractions of NFkB. A- MLD-STZ treated mouse islet cells in non-fixed tissue treated with NIR-ODN probe. B- control islet section treated with NIR-ODN. red - Cy5.5-NIR-ODN, green - anti-insulin, blue- DAPI. C- results of fluorescence intensity measurements performed using sections of MLD-STZ treated and control nuclei and cytoplasm (n=100-150/group).
Keywords: diabetes, NFkB, fluorescence, near-infrared
In vivo Cerenkov imaging of cellular energetics in breast cancer cells with different metastatic potential (#313)
A. D. Arroyo1, B. Moon1, A. V. Popov1, E. J. Delikatny1
1 University of Pennsylvania, Radiology, Philadelphia, Pennsylvania, United States of America
The objective of this project is to develop functional fluorinated bioactivatable molecules to utilize the emission of Cerenkov radiation to distinguish tumors based on metabolic markers. The metastatic potential of tumors has been correlated with several metabolic markers including acidic pH in tumor microenvironment. Deregulated cellular energetics were recently added to the Hallmarks of Cancer. 
Resazurin (RA), or Alamar Blue, is a commonly used viability dye and redox sensor. Reduction by the mitochondria converts RA into resorufin, a highly fluorescent molecule. Cold- and radio-labeled monofluorinated resazurin (MFRA) and difluorinated resazurin (DFRA) were synthesized by electrophilic fluorination. Tumor probe reduction was imaged by fluorescence and Cerenkov imaging in breast cancer cell lines and solid tumors with different metastatic potentials: MDA-MB-231 (triple-negative breast cancer) and 4175-Luc+ (MDA-MB-231-variant isolated from murine lung metastases). This was correlated with in vitro glycolytic activity as determined by glucose consumption, lactate production, and LDHA levels. In vivo pharmacokinetics were determined using PET imaging of the 18F derivatives.
Reduced and oxidized MFRA and DFRA were distinguished optically by differences in their Cerenkov emission with a ratio of 3.5 and 1.3 respectively at 640 nm. 4175-Luc+ cells showed more rapid reduction of RA, MFRA and DFRA than MDA-MB-231 cells (Fig 1). Modulation of intracellular glutathione levels had no effect on probe reduction. The 4175-Luc+ cells displayed higher consumption of glucose and production of lactate than MDA-MB-231s. PET imaging of 18F-DFRA in mice with 4175-Luc+ tumor xenografts showed 0.25-0.9% of the injected dose accumulating in the tumor. Cerenkov time course studies showed probe accumulation in the periphery of the tumor. Increased emission at 640 nm relative to 580 nm indicated increased emission of the reduced fluorescent probe via Cerenkov Radiation Energy Transfer (CRET). DFRA-treated 4175-Luc+ tumor slices ex vivo showed immediate probe reduction. IT injections into 4175-Luc+ tumors confirmed fast probe reduction in vivo (Fig 2).
The highly metastatic 4175-Luc+ cell line displayed both increased glycolytic and Krebs cycle activity relative to the MDA-MB-231 parent line. NADH dehydrogenases such as Complex 1 in the electron transport chain reduce resazurins,. Cerenkov radiation and CRET detected probe reduction in vitro and in vivo. Resazurins could be used to determine mitochondrial energy imbalance and assess metastatic potential of tumors. The use of 18F-labeled isotopomers allows for dual modality PET and Cerenkov imaging to measure probe concentration and metastatic potential simultaneously.
We would like to acknowledge the Small Animal Imaging Facility and the Cyclotron Facility at the University of Pennsylvania and our funding sources, NIH grants R01 EB018645, F31 CA206453
Figure 1: Reduction of resazurin and fluorinated resazurins in 4175-Luc+ and MDA-MB-231.
Graphs show activation of resazurin probes in 2 cell lines: MDA-MB-231 and its metastatic variant 4175-Luc+, as measured by the time-dependent increase in fluorescence. Activation of resazurin and monofluororesazurin is more rapid and significantly higher in 4175-Luc+ cells. In the case of difluororesazurin, the difference is not as great, possibly due to its weak fluorescence.
Figure 2: Cerenkov imaging time course of 18F-DFRA in athymic nude mice
An in vivo tumor injected with 400 mCi 18F-DFRA and 18F-FDG and an ex vivo tumor injected with 400 mCi of 18F-FDG were imaged at the same time to approximate the absorption of Cerenkov radiation by tumor tissue. Using the data from the ex vivo tumor injection of FDG, we were able to calculate how much signal we could theoretically see at 640 nm, taking into account tissue scattering.
Development framework for GMP translation of optical tracers cetuximab-800CW and trastuzumab-800CW (#360)
M. D. Linssen1, 2, E. J. ter Weele1, D. P. Allersma1, M. N. Lub-de Hooge1, 4, G. M. van Dam3, 4, A. Jorritsma-Smit1, W. B. Nagengast2
1 University of Groningen, University Medical Center Groningen, Department of Clinical Pharmacy and Pharmacology, Groningen, Netherlands
Optical molecular imaging can be of significant added value to guide surgical or endoscopic procedures using fluorescently labeled tracers. Monoclonal antibodies have favorable properties for dye delivery. However, development of tracers for clinical trials is a complex process, and therefore implementation of new tracers in the clinic is slow. We present a framework for development and translation into a Good Manufacturing Process (GMP) compliant manufacturing process of monoclonal antibody tracers, illustrated by the results of cetuximab-800CW and trastuzumab-800CW.
Cetuximab and trastuzumab were conjugated under GMP conditions to IRDye 800CW according to standardized operating procedures. Optimal label ratio and formulation buffer were investigated according to our standardized framework. Best performing formulations were advanced to full-scale stability study. Tracers were analyzed for stability by SE-HPLC, pH-measurement, osmolality, visual inspection and sterility, as required by the European Pharmacopeia and GMP guidelines.
For cetuximab-800CW, 7 out of 7 formulations showed potential for long-term stability, whereas for trastuzumab, this was the case for 6 out of 10 tested formulations. Based on these results, 2 formulations for each antibody were investigated in a full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product.
We designed a framework to standardize the development, formulation and GMP translation of molecular fluorescent tracers. Using our standardized approach, we developed two stable antibody-based tracers for clinical use. When developing tracers, the proposed framework can be used to efficiently develop a GMP compliant formulation and improve translation of newly developed optical tracers to first in human use.
Structured tracer development framework
The proposed framework for development and translation of a cGMP compliant manufacturing process. The framework assists the researcher by giving a bird’s eye view of the steps to be taken leading up to the first clinical-grade production, and facilitating the cooperation between the different disciplines involved.
Development of near infrared probe allowing non invasive in vivo imaging of fibrosis in mouse (#392)
C. Robin-Jagerschmidt1, E. De Lemos2, D. Merciris3, E. Berrocal1, M. Pizzonero2, P. Clément-Lacroix1, R. Gosmini2
1 Galapagos, In vivo pharmacology, Romainville, France
Currently, evaluation of fibrosis extent in preclinical rodent fibrosis models is achieved post-mortem, histologically or biochemically. Starting from a green fluorescent based collagen and elastin binding probe, replacement of the fluorescent dye and modulation of the linker moiety studies generated a new probe with near infrared (NIR) emitting properties allowing in vivo monitoring of fibrosis disease or predicting therapeutic responses in mice. The probe was evaluated in two preclinical models of CCl4-induced liver injury and BLM-induced lung injury.
Liver fibrosis was induced by repeated treatment of male Balb/C J mice with CCl4. Lung fibrosis was induced by a single instillation of Bleomycin to C57BL/6 N male mice. After 4 weeks of disease induction, the probe was injected in the retro-orbital sinus of anesthetized mice (100µL, 40µM). To determine the optimal timing for signal to background ratio, images were captured 15 min, 30min, 1h, 2h, 3h, 4h, 5h and 24h after probe injection using Bruker In-Vivo Xtreme imaging system Ex730 Em790. Confirmation of increase fibrosis extent was performed by measurement of collagen production in tissue lysate using a hydroxyproline assay (QuickZyme Bioscience), or in FFPE livers using Sirius red staining or type I collagen immunohistochemistry.
CCl4-treated mice displayed increased binding of the probe in the liver with 3 hours as the optimum timing for highest signal to background ratio. Similarly, BLM increased binding of the probe in the lungs with 2 hours as the optimum timing for highest signal to background ratio. For both models, binding was demonstrated both in vivo and ex vivo. In addition, increase collagen extent was also shown with classical histology techniques (collagen detection with immunohistochemistry or Sirius red labelling).
We describe the first near-infrared collagen-binding probe allowing non-invasive, specific and rapid imaging of fibrosis extend, in several preclinical models of lung and liver fibrosis. In addition to predict therapeutic responses in mice, it makes possible randomization of mice in homogeneous groups prior dosing, thus decreasing intra-group variability, leading to lower number of mice to be included in the study.
Biela E et al., 2013 Cytometry A, 83 : 533-9
Starkel P., 2011 Best practice & Research Clinical Gastroenterology 25: 319-333
Peng et al. 2013, PLoS One, 8: e59348.
Keywords: fibrosis, near infrared probe
In vivo calcium imaging with photoacoustics (#484)
S. Roberts1, 2, 3, M. Seeger5, 2, Y. Jiang2, 3, A. Mishra2, 3, F. Sigmund2, 3, A. Stelzl2, 3, A. Lauri2, 3, P. Symvoulidis1, 2, 3, H. Rolbieski1, 2, 3, M. Preller6, L. Deán-Ben2, D. Razansky2, T. Orschmann3, S. Desbordes3, P. Vetschera2, T. Bach4, V. Ntziachristos5, G. G. Westmeyer1, 2, 3
1 Technical University Munich, Nuclear Medicine, Munich, Germany
Photoacoustic imaging has the key advantages that its resolution is insensitive to photon scattering and that it can provide volumetric data with high frame rates wihtout the need for scanning procedures. To exploit these powerful features for molecular imaging, we have synthesized the first cell-permeable photoacoustic sensor that reversibly changes its photoacoustic signal in response to specific binding of the the key second messenger calcium (Ca2+).
CaSPA_550 was synthesized via a condensation reaction between 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indol-3-ium iodide, containing an activated methyl group and the appropriate aldehyde of 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetate skeleton resulting in deep purple crystals with 85% yield. Subsequent demethylation was carried out at room temperature and the resulting product was dried under high vacuum to obtain the corresponding free acid before further esterification was carried out. CASPA_550 was characterized photophysically and biochemically in response to various metals. Combined fluorescence and photoacoustic microscopy was conducted in cell and tissue culture as well as in zebrafish larvae using custom-built microscopic or mesoscopic photoacoustic instruments.
We synthesized a selective metallochromic sensor (CaSPA_550) with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca2+ lead to a robust blueshift of the absorbance and photoacoustic spectra (Figure 1 a,b) . Sensitivity for calcium was in the micromolar range and selectivity towards magnesium and other metals was high due to the BAPTA chelation group. Esterification of CaSPA_550 enabled efficient cellular uptake into HEK293 and CHO cells and detection of pharmacologically induced Ca2+ influx by both fluorescence and optoacoustic microscopy (Figure 1 c,d). Calcium transients could be measured in beating heart organoids (Figure 2 a) as well as in zebrafish larval brains via simultaneous fluorescence and optoacoustic imaging (Figure 2 b) .
The efficient non-toxic delivery, high sensitivity and specificity of the Ca2+ sensor for photoacoustics (CaSPA) enabled us to for the first time perform molecular photoacoustic imaging of Ca2+ fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain. The semi-cyanine scaffold of CaSPA_550 may serve as a versatile platform to generate red-shifted variants  for imaging deeper in tissue as well as sensors selective for other metals or small analytes to enable molecular photoacoustic imaging with photoscattering-independent resolution in living tissue.
 Roberts, S. et al. Calcium Sensor for Photoacoustic Imaging. J Am Chem Soc jacs.7b03064 (2017). doi:10.1021/jacs.7b03064
 Mishra, A., Jiang, Y., Roberts, S., Ntziachristos, V. & Westmeyer, G. G. Near-Infrared Photoacoustic Imaging Probe Responsive to Calcium. Anal. Chem. 88, 10785–10789 (2016).
We thank Dr. Robert Pal (Durham University) for relative QY measurements, Dr. Andreas Bauer (Technical University of Munich) for absolute QY measurements. We are grateful for support from the Helmholtz Alliance ICEMED (AM, GGW), the European Research Council under grant agreements ERC-StG: 311552 (GGW), the Priority program SP1665 of the German Research Foundation (DFG), as well as the Laura Bassi Award of TUM (SR), grant agreements CRC 1123 (Z1), and Reinhard Koselleck project (NT 3/9-1).
Figure 1: Calcium Sensor for Photoaoustics (CaSPA) in vitro and in cell culture
(a) Schematic of cellular delivery of esterified CaSPA and intracellular 'unmasking' of the BAPTA chelation group (b) Absorbance and photoacoustic spectral changes in response to increasing concentrations of micromolar Ca2+ (c) Fluorescence and (d) photoacoustic microscopy of CHO cells incubated with CaSPA_550 (2 µM, 30 min.) in response to Ca2+ influx triggered by addition of a Ca2+ ionophore.
Figure 2: Photoacoustic calcium imaging in heart organoids and zebrafish larval brain
(a) Heart organoids loaded with CaSPA_550 ester and visualized as overlay of photoacoustic image (magenta) and two-photon fluorescence volume (gray). right: Median signal difference map and photoacoustic signal time course (b) Combined fluorescence imaging (top) and multispectral photoacoustic tomography (bottom) of CaSPA-injected larval zebrafish brain and time courses after stimulation (right).
Keywords: photoacoustics, optoacoustics, MSOT, multispectral optoacoustic tomography, calcium imaging, in vivo, reversible sensor