15th European Molecular Imaging Meeting
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Optical, Ultrasound, Photoacoustic | Probe Chemistry

Session chair: Clemens Bos (Utrecht, Netherlands); Paolo Decuzzi (Genova, Italy)
 
Shortcut: PW27
Date: Friday, 28 August, 2020, 12:00 p.m. - 1:30 p.m.
Session type: Poster

Contents

Abstract/Video opens by clicking at the talk title.

750

Monte Carlo simulations of Cerenkov luminescence imaging using FLUKA

Giulia Aricò1, Federico Boschi2, Antonello E. Spinelli1

1 Experimenal Imaging Centre, San Raffaele Scientific Institute, Milan, Italy
2 Department of Computer Science, University of Verona, Verona, Italy

Introduction

The interest in the detection of Cerenkov radiation (CR) is growing very fast in the preclinical imaging field. CR is produced by charged particles travelling in a medium with energy above a given threshold. CR can be used for example to image the biodistribution of beta+/- radioisotopes both in vivo and ex vivo [1], or as a dosimetry tool for external beam radiotherapy [2].
We investigated, by means of FLUKA, the CR production from isotopes used in nuclear imaging and radionuclide therapy, in materials of interest for Cerenkov and radio luminescence imaging for preclinical applications.

Methods

We considered radioactive isotopes like 177Lu, 64Cu, 18F, 11C, 13N, 32P, 15O, 68Ga and 90Y, commonly used in nuclear medicine. These isotopes mainly
decay by means of beta+/- decays, with endpoint energies between 0.45 and 2.3 MeV and, thus above the Cerenkov threshold in water (0.26 MeV).
Point-like sources surrounded by water were modeled in the FLUKA Monte Carlo code, version 2011.2x.8 [3,4]. The values of refraction index, absorption and scattering coefficient for water were taken from literature. For each source 104 decays were considered, and the results were averaged over 5 independent FLUKA runs. The amount of produced optical photons per decay in the wavelength range between 400 and 800 nm was calculated. The FLUKA results were compared with literature data obtained with Geant4 [5].

Results/Discussion

Figure 1 shows the total number of optical photons per decay, for different radioisotopes in water, as a function of the source’s end point energy. As mentioned before, the results obtained with FLUKA were compared with the literature data reported in [5] obtained with Geant4.
We found that FLUKA and Geant4 produce results in rather good agreement, with differences smaller than 15%. Despite further studies are needed, these results supports the possibility to use FLUKA as a Monte Carlo tool to investigate CR production for a wide range of isotopes.
Based on these results obtained in water, more complex scenarios will be considered. For instance we plan to investigate the yield of optical photons from a source inside different type of tissues, as well as located few mm below the skin of a small animal.

Conclusions

For different beta+ and beta- radioisotopes in water, the FLUKA predicitions of optical photon yields were compared with Geant4 results, and satisfactory agreement was found.
FLUKA simulations can be a valuable Monte Carlo tool to simulate and investigate Cerenkov light production for a wide range of isotopes and configurations. Based on the promising results obtained in water, more complex preclinical imaging scenarios will be considered.

References
[1] Spinelli AE and Boschi F 2015, “Novel biomedical applications of Cerenkov radiation and radioluminescence imaging”, Phys Med. 31(2):120-9
[2] Pogue BW and Wilson BC 2018, “Optical and x-ray technology synergies enabling diagnostic and therapeutic applications in medicine”, J Biomed Opt. 23(12):1-17
[3] Bohlen TT et al. 2014, “The FLUKA Code: Developments and Challenges for High Energy and Medical Applications”, Nuclear Data Sheets 120, 211-214
[4] Ferrari A et al. 2005, “FLUKA: a multi-particle transport code”, CERN-2005-10 (2005), INFN/TC_05/11, SLAC-R-773
[5] Mitchell GS et al. 2011, “In vivo Cerenkov luminescence imaging: a new tool for molecular imaging”, Phil. Trans. R. Soc. A 369, 4605-4619
Figure 1

Total number of Cerenkov photons per decay (wavelength range 400-800 nm), for different radioactive sources in water.

Keywords: Cerenkov imaging, optical imaging, Monte Carlo simulations
751

Photoactivatable formaldehyde donor with quantifiable fluorescence monitoring reveals tolerable threshold of formaldehyde in cells

Nicholas W. Pino1, Lukas P. Smaga1, Jefferson Chan1, 2

1 University of Illinois at Urbana-Champaign, Department of Chemistry, Urbana, United States of America
2 University of Illinois at Urbana-Champaign, Beckman Institute for Advanced Science and Technology, Urbana, United States of America

Introduction

Chemical tools that allow perturbation of complex biological systems in a controlled manner offer a powerful method for evaluation of the mechanisms of biological systems. While inhibitors of enzymes are amongst the most common of these chemical tools, small-molecule donors are an emerging field in which small signaling analytes can be delivered to cells, causing a phenotypic response.1 While many such tools have been made, none have been made to donate formaldehyde in a quantifiable manner. Here, we show the first photoactivatable formaldehyde donor with fluorescence monitoring, photoFAD-3.

Methods

In this work, a panel of photoactivatable formaldehyde donors were synthesized using a photocaged equivalent of formaldehyde masked as an asymmetric acetal. Upon photoactivation, one equivalent of formaldehyde and a fluorescent dye is released, allowing for fluorescence-based quantification of formaldehyde released. photoFAD was optimized through three generations, ultimately yielding a photostable, well-retained, and fast-reacting donor which lended itself to quantification in a novel and facile way. Here we also develop a generalizable workflow for the quantification of fluorescence activatable donors which we used to define concentrations ranges of tolerance, cytostasis, and toxicity of formaldehyde in live cells.

Results/Discussion

To establish an appropriate compound for quantifiable formaldehyde donation, we needed to satisfy 3 requirements: rapid release kinetics, cell retention, and photostability. Our first generation was a TokyoMagenta capped with a photocaged equivalent of formaldehyde with an acetal linkage. To ensure quick release kinetics, we used a 6-nitro[1,3]dioxole photocage which reacts quickly with UV light.2,3 To prevent leaking from the cells, we installed chloro groups at the 4’ and 5’ positions to reduce the pKa of the phenol producing a negative charge upon uncapping at pH 7.4, forcing retention. Finally, we installed a CF3 group to the bottom ring of the TokyoMagenta which improved photostability and fluorescence enhancement. With an optimized donor in hand, we used our own generalizable method to evaluate the concentration-dependent effects of HEK293 cells in a wound-healing assay. We found a tolerance of formaldehyde up to 2.2 μM but loss of wound healing as concentrations approached 4 μM.

Conclusions

Here, we reported the first photoactivatable formaldehyde donor equipped with the ability to be quantified by fluorescence using a bespoke quantification workflow. In this process, we were able to optimize the donor for appropriate release kinetics, cellular retention, and photophysical properties. We were able to show the tolerable threshold for formaldehyde in HEK293 cells. Future studies are ongoing on mechanisms of FA tolerance and toxicity.

AcknowledgmentThis work was supported the National Institutes of Health (R35GM133581). N.W.P. and acknowledges the Chemistry-Biology Interface Training Grant (T32 GM070421) and the Alfred P. Sloan Foundation for financial support. N.W.P. thanks Dr. Robert C. and Mrs. Carolyn J. Springborn for a graduate fellowship. Major funding for the 500 MHz Bruker CryoProbeTM was provided by the Roy J. Carver Charitable Trust (Muscatine, Iowa; grant no. 15-4521) to the
References
[1] Devarie-Baez, N. O.; Bagdon, P. E.; Peng, B.; Zhao, Y.; Park, C. M.; Xian, M. Light-Induced Hydrogen Sulfide Release from “Caged” Gem-Dithiols. Org. Lett. 2013, 15 (11), 2786–2789.
[2] Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. Development of a Fluorescein Analogue, TokyoMagenta, as a Novel Scaffold for Fluorescence Probes in Red Region. Chem. Commun. 2011, 47 (14), 4162–4164.
[3] Il’ichev, Y. V.; Schwörer, M. A.; Wirz, J. Photochemical Reaction Mechanisms of 2-Nitrobenzyl Compounds: Methyl Ethers and Caged ATP. J. Am. Chem. Soc. 2004, 126 (14), 4581–4595.
Structure of photoFAD-3 and image of activated donor in cells
photoFAD-3 contains a maked equivalent of formaldehyde which can be released via irradiation with UV light allowing for a quantifiable fluorescence readout which can report on the concentration of formaldehyde released. 
Keywords: Formaldehyde, Small molecule donor, photocage, fluorescence monitoring
752

Conjugated Polymer Nanoparticles as Efficient Fluorescence Bioimaging Nanoplatforms by Means of Fluorination Substitution-Induced Polymer Chains Planarity Reduction

Panagiota Koralli2, Alkmini Negka2, Lida E. Vagiaki1, Antonia Dimitrakopoulou-Strauss2, Vasilis G. Gregoriou3, Christos L. Chochos1

1 National Hellenic Research Foundation, Institute of Chemical Biology, Athens, Greece
2 German Cancer Research Center (DKFZ), Clinical Cooperation Unit Nuclear Medicin, Heidelberg, Germany
3 National Hellenic Research Foundation, Athens, Greece

Introduction

Low-bandgap aqueous conjugated polymer nanoparticles (CPNs) have been prepared and applied in biological applications, especially in bio-imaging, due to their unique optoelectronic properties [1]. In this study, we try to develop new water-soluble conjugated polymers probes as fluorescent contrast agents utilizing the nanoprecipitation and the encapsulation methods. Under this scope, we designed and synthesized a series of CPs combining thiophene as the electron donating and quinoxaline as the electron deficient(TQs).The ability of the obtained probes to image cancer cells will be investigated

Methods

Alternating CPs were synthesized via metal catalyzed aromatic cross coupling Stille polymerization reaction.The average molecular weights per number and weight and the dispersity of the CPs were determined by SEC in CHCl3.All CPs were analyzed via 1H-NMR,UV-Vis and fluorescence spectroscopy.The formation of NPs was carried out via nanoprecipitation and encapsulation method.The UV-Vis measurements were performed in THF solution,and after the formation of NPs,in aqueous suspension.The shape of NPs was investigated with SEM & TEM,while DLS was exploited to elucidate the NPs size.The cellular cytotoxicity study will be performed in carcinoma cell lines to investigate the effect of the NPs.In order to investigate whether the CPNs are internalized by the cellular membrane, we will exploit a CLSM

Results/Discussion

The TQ series of polymers are comprising of thiophene derivatives as the electron donating units and quinoxaline derivatives as the electron withdrawing units with varying the number of fluoro atoms in the repeat unit.The DLS measurements indicate that all CPNs in aqueous suspension have a unimodal size distribution less than 80nm.TEM & SEM measurements showed that the CPNs have spherical shape.The optical properties of the CPNs show light absorption and fluorescence emission in the UV-Vis region,tunable depending on the structure, the position and the number of fluoro atoms on the polymer backbone.It is revealed a significant reduction of the fluorescence quantum yield in aqueous media derived from severe aggregation and charge transfer induced fluorescence quenching.However,with the proper rational design on the polymer backbone we can control the aggregation degree of the polymer chains at aqueous media resulting to enhanced fluorescence emission for the case of the T2fQf and T2fQ2f

Conclusions

We successfully synthesized a series of CPs with absorption and emission properties at the UV-Vis region and water-soluble CPNs formatted by two different methods, supporting their potential use as low-bandgap fluorescent probes for bio-imaging. The on-going tests for cytotoxicity and the ability of the probes to image cancer cells are of great importance. Overall, CPNs introduce a new generation of fluorescent probes for contrast agents

AcknowledgmentThis work was funding by the Helmholtz European partnering program for the cooperation between the German Cancer Research Center (DKFZ) and the National Hellenic Research Foundation (NHRF) to build the Athens Comprehensive Cancer Center (ACCC)
References
[1] Feng, L., Zhu, C., Yuan, H., Liu, L., Lv, F., & Wang, S., (2013), 'Conjugated polymer nanoparticles: preparation, properties, functionalization and biological applications', Chemical Society  Reviews, 42(16)
 
Synthesis of TQ conjugated polymers
Keywords: fluorescent probes, bioimaging, conjugated polymer nanoparticles
753

Design and synthesis of novel bodipy dyes

Olga Kirkilessi1, Kyriakos C. Prousis1, Theodora Calogeropoulou1

1 National and Hellenic Research Foundation, Institute of Chemical Biology, Athens, Greece

Introduction

Fluorescent dyes have emerged as a powerful tool for visualizing and labelling biomolecules in vitro and in vivo. Boron-dipyrromethenes (BODIPYs) play a pivotal role in fluorescent probe development due to their favorable physicochemical properties, their ease of synthesis and the wide range of applications in various research fields.1,2

Methods

Series of new mono- and di-substituted bodipy derivatives incorporating heterocyclic groups were synthesized. Starting from 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene the synthesis of the new dyes was effected through a sequence of condensations and cross-couplings reactions. The effect of the new substituents on the photochemical and photophysical properties of the new dyes was investigated. The spectroscopic properties of the new dyes were measured in different solvents while, the pH stability was assessed in various buffer solutions.

Results/Discussion

Initially, we embarked on the synthesis of the mono-substituted analogues through a Knoevenangel condensation reaction of the corresponding aldehydes either at the α or at the β site of 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoro-boradiazaindacene. Subsequently, we extended the π-conjugation system of the bodipy core, through the synthesis of di-substituted derivatives, in order to generate red-shifted bodipy-dyes with improved optical properties. Specifically, the 2,6-disubstituted derivatives were generated through a Vilsmeier-Haack reaction at C2 and derivatization at C6, followed by condensation reactions of the C2-aldehyde to generate the different heterocyclic scaffolds. Conversely, the 3,5-disubstituted derivatives were obtained through a condensation reaction of the C3 and C5 methyl groups with aromatic aldehydes under microwave irradiation.

Conclusions

In conclusion, we recently designed and synthesized new fluorescent bodipy-dyes, bearing heterocyclic substituents at different positions of the bodipy scaffold. The new compounds exhibited a bathochromic shift compared to that of 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene. Studies on the optical properties, and potential biological applications of the new dyes are currently ongoing.

Acknowledgment

We acknowledge support of this work by the project “Bioimaging-GR” (MIS 5002755) which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure", funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund).

References
[1] Ni Y., Wu J., 'Far-red and near infrared BODIPY dyes: synthesis and applications for fluorescent pH probes and bio-imaging', Org. Biomol. Chem., 2014, 12, 3774
[2] Lakshmi V., Sharma R., Ravikanth M., 'Functionalized boron-dipyrromethenes and their applications', Reports in Organic Chemistry, 2016, 6, 1-24
Keywords: heterocyclic-substituted bodipy derivatives, synthesis and characterization, tuning photophysical properties
754

New cationic heptamethinecyanine-graphene hybrid materials

Kyriakos C. Prousis1, Ruben Canton-Vitoria2, Georgia Pagona2, Maria Goulielmaki1, Vassilis Zoumpourlis1, Nikos Tagmatarchis2, Theodora Calogeropoulou1

1 National Hellenic Research Foundation, Institute of Chemical Biology, Athens, Greece
2 National Hellenic Research Foundation, Theoretical and Physical Chemistry Institute, Athens, Greece

Introduction

Near-infrared (NIR) fluorophores have emerged as promising modalities for monitoring the levels of various biologically relevant species in cells and organisms [1,2]. The advantages of NIR probes include minimal interfering absorption and fluorescence from biological samples, inexpensive laser diode excitation, reduced scattering and enhanced tissue penetration depth [3]. Graphene has attracted considerable attention during the last years for potential applications in biosensing, biomedical diagnosis and therapy [4,5].

Methods

Our efforts were focused on the design and synthesis of new NIR dyes, exploiting the tricarbocyanine scaffold as template, taking advantage of the facile displacement of the chloro substituent, at the central vinyl carbon by various nucleophiles. Thus, appropriate linkers were introduced, which could in turn be covalently ligated to the exfoliated graphene surface either through amide bond formation or via in-situ generated aryl diazonium salt addition. Variation of the heteroatom linkage to the carbocyanine core (Nitrogen or Sulfur) enabled us to tune the photophysical properties of the synthesized NIR-fluorescent dyes.

Results/Discussion

Four new near-infrared fluorescent indolenine-based cationic heptamethinecyanine dyes were synthesized, which were further covalently conjugated to exfoliated graphene. The characterization of the hybrid materials was accomplished by complementary spectroscopic, thermal and microscopy imaging means. Specifically, Raman spectroscopy, attenuated-total-reflectance infra-red (ATR-IR) studies, thermogravimetric analysis (TGA) were employed to corroborate the successful covalent modification of exfoliated graphene. Cell viability studies using the RKO human colon adenocarcinoma cell line and WJ-MSC human umbilical cord mesenchymal stem cells revealed the absence of toxicity of the graphene-based dyads in both cell lines. Cellular uptake studies using confocal microscopy showed that graphene-NIR dyes hybrids are able to enter the cells with high efficacy.

Conclusions

In conclusion, we have synthesized four NIR indolenine-based cationic heptamethinecyanine fluorescent dyes and their hybrids with exfoliated graphene. Future studies will assess the suitability of the new cyanine-graphene hybrids as an optical imaging modality characterised by high cell viability or as a delivery platform for high loading efficiency of additional components for theranostic applications.

AcknowledgmentThis work was partially funded by the John S. Latsis Public Benefit Foundation. The sole responsibility for its content lies with its authors. In addition, partial funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 642742 is acknowledged. In addition, we acknowledge support of this work by the project “Bioimaging-GR” (MIS 5002755) which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure", is funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund). Τhe work of MG was supported by the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI PhD Fellowship grant (GA. no. 2400).
References
[1] Guo, Z., Park, S., Yoon, J., Shin, I. 2014Chem. Soc. Rev., 43, 16.
[2] Lade, U. B., Bhoyar, P. K., Hingankar, M. H., 2014WJPPS, 3, 2193. 
[3] Escobedo, J.O., Rusin, O.,  Lim, S., Strongin, R.M., 2010Curr. Opin. Chem. Biol., 14, 64.
[4] Zhang, H., Zhang, H., Aldalbahi, A., Zuo, X., Fan, C., Mi, X., 2017, Biosens. Bioelectron., 89, 96.
[5] Wang, Y., Li, Z., Wang, J., Li, J., Lin, Y., 2011, Trends Biotechnol., 29, 205.
Keywords: heptamethinecyanine fluorescent dyes, exfoliated graphene, cyanine-graphene hybrids
755

Fluorescent peptide-based smartprobe for human neutrophil elastase

Maria Rodriguez Rios1, Giulia Rinaldi2, Calum Robb2, Mark Bradley1, Adriano Rossi2

1 University of Edinburgh, Bradley Group / School of Chemistry, Edinburgh, United Kingdom
2 Centre for Inflammation and research/ University of Edinburgh, Rossi Group/ Queens Medical Research Insitute, Edinburgh, United Kingdom

Introduction

Lung diseases can result in ‘inflammation’, a protective response by the body as it responds to tissue damage caused by injury and infection. Human neutrophil elastase (HNE) is a serine protease implicated in the pathogenesis of acute and chronic inflammatory diseases and is secreted by neutrophils during inflammation to destroy bacteria. Herein we report a sensor providing an OFF/ON fluorescent signal targeting HNE in activated neutrophils and Neutrophil Extracellular Traps (NETs).

Methods

The probe is based on a multivalent scaffold combined with FRET-labelled peptides1,2. The first step on the synthesis was to synthesise the trimeric scaffold, following a previously described procedure1,2. The monomer was attached to a solid support (Chemmatrix resin) via a Rink linker and served as a scaffold for the synthesis of the FRET-peptide using fmoc solid phase peptide synthesis strategy, with 5-FAM and Methyl Red as fluorophore and quencher, respectively (FRET pair). Enzymatic testing and cell imaging measured the fluorescence increase upon HNE exposure and specificity in presence of other proteases and inhibitors.

Results/Discussion

The multivalent scaffold approach provides amplification of signal upon substrate (peptide) cleavage - the tribranched self-quenching capacity makes the probe super-silent under physiological conditions, and is specifically cleaved by HNE showing no cleavage by any related serine proteases as Cathepsin G and Proteinase 3. Kinetic assays showed a 46-fold increase in fluorescence upon cleavage with negligible background fluorescence levels. The probe successfully labeled activated neutrophils and NETs under inflammation-induced conditions and was successfully used in a novel flow cytometric assay method to detect activated neutrophils and NETs specifically. These probes have the potential to allow in vivo, in situ detection of inflammation deep within the lung, with a fluorescence-based microendoscopy system developed by Proteus to monitor lung conditions in real time3.

Conclusions

MR28, a tri-branched activatable fluorescent HNE sensor was synthesised and evaluated. The probe allowed the rapid, specific and sensitive analysis of HNE in activated neutrophils and NETs during inflammation-induced conditions.

Acknowledgment

EPSRC, Interdisciplinary Research Collaboration grant EP/R005257/1 for funding this work and the School of Chemistry, University of Edinburgh for funding.

References
[1] T. Craven, N. Avlonitis, N. McDonald, T. Walton, E. Scholefield, A. Akram, T. Walsh, C. Haslett, M. Bradley, and K. Dhaliwal, Sci Rep, 2018, 8, 1-10.
[2] N. Avlonitis, M. Debunne, T. Aslam, N. McDonald, C. Haslett, K. Dhaliwal, M. Bradley, Org Biomol Chem 2013, 11, 4414-4418.
[3] A. Akram, S. Chankeshwara, E. Scholefield, T. Aslam, N. McDonald, A. Megia-Fernandez, A. Marshall, B. Mills, N. Avlonitis, T. Craven, A. Smyth, D. Collie, C. Gray, N. Hirani, A. Hill, J. Govan, T. Walsh, C. Haslett, M. Bradley and K. Dhaliwal, Sci Trans Med, 2018, 10, 1-12.
Fluorescent peptide based SmartProbes
Tribranched peptide-based fluorescent probe and its activation (note quenching takes place in two ways – vertically (by the three fluorescein units) and horizontally (via the three quenchers)
Keywords: Smartprobe, FRET, HNE, inflammation, fluorescence
756

Polypyrrole nanoparticles for photoacoustic imaging

Vít Herynek1, Monika Paúrová2, Peter Keša1, Michal Babič2, Tomáš Heizer1, Luděk Šefc1

1 Charles University, Center for Advanced Preclinical Imaging, Prague, Czech Republic
2 Institute of Macromolecular Chemistry, Czech Academy of Science, Prague, Czech Republic

Introduction

Photoacoustic (PA) imaging is an emerging modality, which provides a supplemental information to standard ultrasound imaging. Although it might be used natively (e.g., reflecting oxygenation of hemoglobin), usage of specifically designed contrast agents substantially widens its capabilities.

Among others, polypyrrole has properties interesting also for PA imaging (1). We prepared polypyrrole (PPy) nanoparticles by polymerization at different conditions and tested them as a photoacoustic contrast agent in vitro.

Methods

Nanoparticles were synthetized by a chemical oxidative polymerization of pyrrole monomer (Py) with ammonium persulphate (ApS, (NH4)2S2O8). Molar ratio of Py and the oxidant ApS was set to 1:0.5, 1:1, 1:2, 1:3, and 1:5. Polymerization in a non-ionic stabilizer (polyvinylpyrrolidone) was realized during 24-hour incubation at room temperature and continuous stirring. The formed PPy nanoparticles were then washed by deionized water and repeated centrifugation.

Suspension was diluted to 6 concentrations (8, 6, 4, 3, 2, and 1 mg/mL). Aliquots of the suspensions were inserted into silicon tubes (inner diameter 0.5 mm), adjusted into a phantom and scanned on a multi-modal imaging platform combining ultrasound and photoacoustic imaging (wavelength range 680 - 970 nm and 1200 - 2000 nm).

Results/Discussion

Photoacoustic spectra of the nanoparticles prepared by polymerization at different molar ratios of Py and ApS are shown in Fig. 1. First three samples revealed a high PA signal (ratios Py:ApS = 1:0.5, 1:1, 1:2), while the rest two (with higher ratio of the oxidant) had signal very low in both wavelength ranges. This corresponds to the number of conjugated bounds in the polymer, which is higher at low ratio of the oxidant. High oxidant ratio leads to disruption of conjugation (both on short and long distance) by increasing the number of oxidated structures and subsequently to shift of the absorption spectra to shorter wavelengths.

Interestingly, although the signal increases with concentration of the nanoparticles at low concentrations, the signal significantly drops at the highest concentration (Fig. 2). We hypothesize that high absorption of light limits penetration in the sample and the observed signal is produced in a surface layer of the sample only.

Conclusions

We synthetized and tested polypyrrole nanoparticles as a promising substance for photoacoustic imaging. Variations in preparation led to different photoacoustic properties corresponding to nanoparticle chemical structure. The study represents a basis for optimization of nanoparticles for in vivo imaging.

AcknowledgmentCzech Science Foundation - Project No. 18-05200S, Ministry of Education, Youth and Sports of the Czech Republic - Czech-BioImaging LM2015062, National Sustainability Program II (Project BIOCEV-FAR LQ1604)
References
1 Gürbüz O et al. Structural, optical and electrical properties of polypyrrole in an ionic liquid. Polym Bull 2017;74:2625
Fig. 1: Photoacoustic spectra of the polypyrrole nanoparticles
Polypyrrole nanoparticles prepared by oxidative polymerization with different molar ratios of the oxidant. Spectra were acquired in two wavelength ranges, 680 - 970 nm (A) and 1200 - 2000 nm (B). Note different scale of the graphs.
Fig. 2: Concentration dependence of the photoacoustic signal
Signal intensity of the polypyrrole particles prepared by oxidation at molar ratio 1:2 and measured at selected wavelenghts.
Keywords: Polypyrrole nanoparticles, Photoacoustic imaging, conjugation
757

Near Infrared Absorbing Conjugated Polymer Nanoparticles (λmax >900 nm) as New Photoacoustic Contrast Agents

Aristea Pavlou1, Alkmini Negka2, Panagiota Koralli2, Antonia Dimitrakopoulou-Strauss2, Vasilis G. Gregoriou3, Christos L. Chochos1

1 National Foundation of Research, Institute of Biology,Medicinal Chemistry & Biotechnology, Athens, Greece
2 German Cancer Research Center, Clinical Cooperation Unit Nuclear Medicine,, Heidelberg, Germany
3 National Hellenic Research Foundation, Athens, Greece

Introduction

Conjugated Polymers (CPs) are proven to have unique optoelectronic properties due to the π-conjugated backbone.[1] Strategies, such as nanoprecipitation or encapsulation, were proven to be very effective in preparing conjugated polymer nanoparticles (CPNPs), which are operable in aqueous. [2],[3],[4]
This feature study focuses on the recent progress in the preparation of new near infrared absorbing CPs and nanoparticles for use as photoacoustic contrast agents.

Methods

Alternating conjugated polymers were synthesized via metal catalyzed aromatic cross coupling Stille polymerization reaction. The average molecular weights per number and weight and the dispersity of the polymers were determined by size exclusion chromatography in CHCl3. All polymers were analyzed via 1H-NMR spectroscopy and UV-Vis spectroscopy. The formations of NPs were carried out via nanoprecipitation and/or by utilizing amphiphilic polymer(PLGA-b-mPEG). The shape of the NPs was investigated with Transmission Electron Microscopy while Dynamic Light Scattering was exploited to elucidate the NPs size. The formations of NPs were carried out via encapsulation and the quantum yield was calculated by photoluminescence (PL) spectroscopy.

Results/Discussion

The synthesized donor-acceptor (D-A) CPs comprised tetraphenyl ot tetrathienyl substituted-indacenodithiophene derivatives and thiadiazoloquinoxaline biphenyl substituted. These two near infrared absorbing polymers exhibit an absorption maximum at 993 nm, in chloroform solution due to the strong intramolecular charge transfer (ICT) between the indacenodithiophene and thiadiazolo-quinoxaline units.
As expected, the amphiphilic CPNPs appear to be more stable as compared to the nanoprecipitated hydrophobic CPNPs, a procedure showed in Scheme 1. The DLS measurement indicates that the nanoparticles formed by encapsulation in different composition ratio, showed that in 7ml H2O, the two with good repeatable results are 1:5 and 1:9 (CP:PLGA-b-mPEG). Also, the CPNPs sizes at these composition ratios are approximately 100nm. The compatible particle size that these CPNPs demonstrate, combined with the absorption in the NIR-I window, qualifies them to be an ideal probe for biological applications.

Conclusions

Both the conjugated and the amphiphilic-encapsulated polymer NPs display a promising size and morphology, a NIR I-shifted optical absorption and low cytotoxicity levels. Additionally, on-going studies will define the photoacoustic effect of the CPNPs. To conclude, in this study we introduce a new organic candidate (CPNPs) for the photoacoustic imaging that shows low toxicity and optimal optical properties in the NIR I region.

AcknowledgmentThis work was funding by the Helmholtz European partnering program for the cooperation between German Cancer Reasearch Center (DKFZ) and National Hellenic Research Foundation (NHRF) to built the Athens Comprehensive Cancer Center (ACCC)
References
[1] D. Ding, J. Liu, G. Feng, K. Li, Yong H, B. Liu, “Bright Far-Red/Near-Infrared Conjugated Polymer Nanoparticles for In Vivo Bioimaging”, Small, 2013, 10, DOI: 10.1002/smll.201300171
[2] Kai Li, Bin Liu, “Polymer encapsulated conjugated polymer nanoparticles for fluorescence bioimaging, J. Mater. Chem., 2012, 22, 1257, DOI: 10.1039/c1jm14397b
[3] Feng, L., Zhu, C., Yuan, H., Liu, L., Lv, F., & Wang, S. (2013), “Conjugated polymer nanoparticles: preparation, properties, functionalization and biological applications”, Chemical Society Reviews, 42(16), 6620, DOI:10.1039/c3cs60036j
[4] Jie Liu, Junlong Geng, “Conjugated polymer nanoparticles for photoacoustic vascular imaging” Polym. Chem., 2014,5, 2854-2862, DOI: 10.1039/C3PY01587D
[5] Kanyi Pu, Adam J. Schuhendler, “Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice” Nature Nanotechnology 9, 233–239, 2014
Scheme 1
General display of nanoprecipitation and encapsulation
Keywords: conjugated polymer nanoparticles, photoacoustic, optical imaging, near infrared
758

The Effect of Fatty Acids and BSA Purity on Fluorescent Gold Nanoclusters

Daniel Jirák1, 2, Pavlína Andrýsková3, Klára Jiráková2, Šárka Michetschlägerová2, Martin Kubala3, Karolína Machalová Šišková3

1 Institute for Clinical and Experimental Medicine, Department of Computed Tomography, Magnetic Resonance and Clinical Experimental Spectroscopy, Prague, Czech Republic
2 Technical University of Liberec, Faculty of Health Studies, Liberec, Czech Republic
3 Palacký University Olomouc, Faculty of Science, Olomouc, Czech Republic

Introduction

The aim of this project is to develop a novel type of fluorophores based on fluorescent gold nanoclusters (AuNCs). Here we present results about the first comparative study investigating the effect of presence / absence / abundance of fatty acids (namely palmitic acid, PA) or other substances (like glycoproteins and globulins) in the protein (bovine serum albumin, BSA) on synthesis and properties of the final AuNCs. The most popular template (BSA) and microwave (MW)-assisted synthesis of AuNCs were intentionally chosen.

Methods

Gold nanoclusters AuNCs were prepared by the reduction of HAuCl4 using BSA of different purity: 96% BSA (96BSA), 98% BSA (98BSA), and fatty acids free BSA (df98BSA). Fluorescence of NCs was recorded spectrofluorimeter. Emission – excitation 3D maps were measured in the ranges: excitation 250 – 420 nm and emission 300 – 800 nm. Fluorescence decays of AuNCs were measured on a fluorometer using pulsed laser diode. The quantum yield (QY) was measured using tryptophan. Changes in the protein structure were evaluated by ATR-IR Nicolet with diamond crystal at the range of 4000 – 400 cm-1 and the resolution of 4 cm-1. Hydrodynamic size and zeta potential of NCs were characterized by dynamic light scattering. AuNCs cytotoxicity was assessed using Alamar Blue.

Results/Discussion

The 3D excitation-emission spectral maps show a reduced fluorescence intensity at ex/em 280/340 nm (attributed to residual albumin peak) with the increasing content of PA in dfBSA. Simultaneously, the emission maximum of AuNCs (around 650 nm) becomes more insensitive with the PA concentration increase. The changes in the secondary structure caused by PA addition into dfBSA revealed substantial differences in amide I, amide II regions (namely, in peak positions and their relative intensities). Moreover, the peak 879 cm-1 (stemming from Tyr residues) increased in intensity with the increasing amount of PA. The presence of an increased amount of other impurities could probably lead to a lower emission of the AuNCs. Fluorescence lifetime values of AuNCs, re-measured after three months confirmed that fluorescence decay characteristics are unaltered. Cytotoxicity testing stated that AuNCs are not significantly influencing cells viability.

Conclusions

The content of PA and/or other substances in BSA plays an utmost important role in the AuNCs formation and in fluorescence lifetime and quantum yield. The accessibility of cysteine and tyrosine residues is most probably different in 96BSA vs 98BSA vs df98BSA, which consequently influences the AuNCs formation and properties as it was determined by several spectroscopic and experimental techniques. AuNCs were non toxic and long term stable.

Acknowledgment

The study was supported by the Grant Agency of the Czech Republic (grant no. 19-03207S) and by the Ministry of Health of the Czech Republic (CZ-DRO, IN 00023001).

Keywords: Au nanocluster, fluorescence, fatty acid, palmitic acid
759

Redox-sensitive cyanine dyes for GSH sensing

Isabelle Heing-Becker1, Rainer Haag1, Kai Licha1

1 Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany

Introduction

Cyanine dyes present versatile structural platforms which can be used to modulate their spectroscopic properties, solubility and conjugatibility. Responsive dyes are especially useful because their fluorescence is turned on by an external trigger, e.g. by pH-changes or ROS. Little has been done in the area of glutathione (GSH) responsive dyes, which could be used e.g. for live cell imaging to characterize the intracellular uptake pathway of dye-labeled conjugates, which is essential to understand the mode of action of new drugs, or to visualize areas or tissues with high levels of GSH.

Methods

Water-soluble and organo-soluble indodicarbocyanine (Cy5) dyes were synthesized from suitable indolenine precursors, resulting either in symmetric or in asymmetric, conjugatable dyes. A Suzuki reaction and subsequent oxidation were performed to introduce a quinone moiety into the dye. The spectroscopic characterization included absorption and fluorescence spectra in buffer solution and methanol. Reactions of the quinone containing dyes with GSH were carried out and the fluorescence was measured before and after the reaction to test their sensitivity towards GSH.

Results/Discussion

Different synthetic strategies were explored to introduce the quinone moiety into the new symmetric or asymmetric Cy5 dyes, where different reaction conditions are required for the organo-soluble and water-soluble dyes. Introduction of the quinone into the conjugated π-system of the dyes results in significant quenching of their fluorescence. Exposure to GSH (5 – 10 mM) increased the fluorescence by a factor of 2 – 10, depending on the dye, its concentration and the reaction time, thereby making them suitable as GSH-sensitive fluorophores. While the symmetric dyes can e.g. be encapsulated into polymeric structures and released upon exposure to a certain trigger, the asymmetric dyes enable direct covalent binding to drugs.

Conclusions

New Cy5 dyes were synthesized and equipped with a fluorescence quenching moiety that loses its quenching effects when exposed to GSH in physiological concentrations. Modifications of the dye structure afford differently soluble dyes and enable attachment to molecules either via encapsulation or covalent binding, e.g. for intracellular pathway tracking.

AcknowledgmentThe Dahlem Research School and the Studienstiftung des deutschen Volkes are acknowledged for funding.
References
[1] K. Licha, U. Resch-Genger, Comprehensive Biomedical Physics, Elsevier, Oxford, 2014, 85-109.
 
[2] A. C. Benniston, W. Clegg et al., Eur. J. Org. Chem. 2008, 2705-2713.
 
[3] C.-X. Yin, R. M. Strongin et al., Angew. Chem. Int. Ed. 2017, 56, 13188-13198.
Keywords: cyanines, fluorescence, molecular imaging, glutathione, redox-sensitive
760

Evidence for Natural Anticancer Properties of Near-Infrared Emissive Conjugated Polymer Nanoparticles Towards Cancer Theranostics

Alkmini Negka1, Panagiota Koralli1, Maria Goulielmaki2, Lida E. Vagiaki2, Aristea Pavlou2, Giannis Antoniou3, Panagiotis Keivanidis3, Dimitris Moschovas4, Apostolos Avgeropoulos4, Aristotelis Xenakis2, Vassilis Zoumpourlis2, Antonia Dimitrakopoulou-Strauss1, Vasilis G. Gregoriou5, Christos L. Chochos2

1 German Cancer Research Center, Clinical Cooperation Unit Nuclear Medicine, Heidelberg, Germany
2 National Hellenic Research Foundation, Institute of Chemical Biology, Athens, Greece
3 Cyprus University of Technology, Department of Mechanical Engineering and Materials Science and Engineering, Limassol, Cyprus
4 University of Ioannina, Department of Materials Science Engineering, Ioannina, Germany
5 National Hellenic Research Foundation, Athens, Germany

Introduction

Bioimaging provides multiple methods for examining biological processes and provides the potential for diagnosis and therapy1. For this reason, it is highly desirable to increase excellent imaging agents such as the conjugated polymers nanoparticles2. Herein, we examine the potential use of novel low bandgap water soluble conjugated polymers NPs as fluorescent contrast agents. Under this scope, we synthesized two CPs; consisting of thiadiazoloquinoxaline biphenyl substituted-bithiophene and thiadiazoloquinoxaline bimethyl substituted-bithiophene, BTTDQ-Ph and BTTDQ-Me, respectively.

Methods

Alternating conjugated polymers were synthesized via metal catalyzed aromatic cross coupling Stille polymerization reaction and the nanoparticles were prepared via nanoprecipitation method.
The Mw and the PDI of the polymers were determined by SEC. All polymers were analyzed via 1H-NMR and UV-Vis spectroscopy in solution, solid state and aqueous NPs. The morphology of NPs was investigated with FESEM while the NPs size and stability were measured via DLS and ζ potential. The quantum yield was calculated by PL spectroscopy. The cytotoxicity study was performed in MSC-WJ as normal reference cell line while WH165 and HCT116 were used as cancer cell lines. In order to investigate whether the CP NPs are internalized by the cellular membrane, a Confocal Fluorescence Microscopy was used.

Results/Discussion

The absorption spectra of the BTTDQ-Ph and BTTDQ-Me copolymers in tetrahydrofuran (THF) solution  shown that both copolymers exhibit the same absorption maxima at the low-wavelength peak (360 nm), whereas the high-wavelength transition is detected at 650 nm for the BTTDQ-Ph and at 595 nm for the BTTDQ-Me. The emission spectra of the BTTDQ-Ph and BTTDQ-Me in solution using THF as solvent were recorded. Both NPs demonstrate different behaviors upon incubation with increasing concentrations at cancer and stem cells. BTTDQ-Ph NPs is well tolerated in all concentrations up to 0,5μg/ml by WJ-MSC. Similarly, in HCT116 only an average reduction of 20% was noted, while WM164 were more sensitive with a reduction in cell viability approaching 40% in the same concentrations.
Similar results were obtained after incubation with BTTDQ-Me NPs. These results show an anti-cancer effect of selected concentrations of the BTTDQ-Ph and BTTDQ-Me, with no toxicity against non-malignant stem cells.

Conclusions

We effectively synthesized CP NPs fluorescent materials via nanoprecipitation, which can serve as an alternative to standard fluorescence probes for bioimaging in vitro. The average hydrodynamic radius and size of nanoparticles were ideal for bio-applications. The cytotoxicity analysis confirmed the biocompatibility of the NPs in aqueous solution and highlighted their anticancer potential regarding the viability of WM164 and HCT116 cell lines.

Acknowledgment

This work was funding by the Helmholtz European partnering programm for the cooperation between German Cancer Research Center (DKFZ) and National Hellenic Research Foundation (NHRF) to built the Athens Comprehensive Cancer Center (ACCC)

Keywords: fluorescent probes, bioimaging, conjugated polymer nanoparticles
761

Fluorescent Fungal Probes: Multiplexed Imaging of Microorganisms

Assel Baibek1, Muhammed Ucuncu1, Bryn Short2, Gordon Ramage3, Mark Bradley1

1 The University of Edinburgh, School of Chemistry, Edinburgh, United Kingdom
2 The University of the West of Scotland, Institute of Healthcare, Policy and Practice, Paisley, United Kingdom
3 The University of Glasgow, Oral Sciences Research Group, Glasgow, United Kingdom

Introduction

Current methods of detection of infection are based on culturing and are inherently slow. This delay point of care diagnostics and subsequent treatment, be it for patents in in intensive care or those in developing countries. Optical molecular imaging using target specific fluorescent probes can be used for the rapid diagnosis of infection.1 In this study, we used the membrane targeting, and clinically used antifungal polyene Amphotericin B (AmB) to develop probes that allowed the rapid and selective detection of fungi.

Methods

Amphotericin B was conjugated to a range of fluorophores through amide bond coupling on either the amino or carboxylic acid sites of the molecule to investigate their effect on labelling. Fluorescent labelling properties of the probes were evaluated on planktonic C. albicans and C. auris (including clinically isolated strains and AmB-tolerant strain), C. auris biofilms using confocal microscopy. The Cy5-based probe was used for the highly specific detection of fungi in the multiplex visualisation of fungi and bacteria mixture in the presence of bacteria specific probes.

Results/Discussion

The synthesised probes showed selective labelling of fungi as single stains and as mixtures. The probes also showed labelling of fungal biofilms, where penetration of the external molecules is challenging due to the extracellular matrix. After confirming that the probes showed selective labelling of fungi in the presence of bacteria, Cy5 conjugated AmB was used with previously reported Gram-positive and Gram-negative bacteria specific probes for multiplex visualization of microorganisms in complex mixture in three colours.

Conclusions

Having in hand a tool for rapid detection and distinguishing of different types of microorganisms a single sample will be able to be used to provide information to enable specific/correct treatment of the patient.

References
[1] Akram, A.R., Bradley, M., Dhaliwal K., 2018, ‘In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A’, Sci. Transl. Med., Vol 10, Issue 464
Schematic illustration of fungal cell labelling by Amphotericin B based probes.
Keywords: Smartprobes, Fungal infection, Amphotericin B, Multiplexed Imaging, Fungal Biofilm
762

Fluorescent organic nanoparticles demonstrating high FRET efficiency for their use as bioimaging probes

Judit Morla-Folch1, 3, Guillem Vargas-Nadal1, Tinghan Zhao3, Cristina Sissa2, Antonio Ardizzone1, Siarhei Kurhuzenkau2, Mariana Kober1, Mehrun Uddin3, Anna Painelli2, Jaume Veciana1, Kevin D. Belfield3, Nora Ventosa1

1 Institut de Ciència de Materials de Barcelona (ICMAB-CSIC)-CIBER-BBN, Nanomol Group, Credanyola del Vallés, Spain
2 Università di Parma, Dipartamento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Parma, Italy
3 New Jersey Institute of Technology, Department of Chemistry and Environmental Science, Newark, United States of America

Introduction

In the very recent years, FRET-based nanoparticle for biosensing, bioimaging, and theragnostic applications have experienced an unprecedented upsurge of interest. One of the advantages of FRET over single fluorophores as optical reporters is the relatively larger gap between the excitation and emission maxima and thus can significantly reduce the background while imaging. The use of nanoparticles as nanocarriers for fluorophores such as organic dyes offers an interesting strategy to bring them in aqueous media showing photostability, biocompatibility and generally, higher brightness.

Methods

To overcome the challenges for obtaining a suitable fluorescent nanoparticle based on FRET, our approach consists in using highly stable and monodisperse quatsome nanovesciles (QSs) as carriers, and integrating hydrophobic carbocyanine dye in the vesicle membrane. Quatsomes are a new class of nanoscopic unilamellar vesicles made by surfactants and sterols, such as CTAB and cholesterol in an equimolar ratio. Based on our previous works, QSs have already demonstrated high photostability and biocompatibility when encapsulating organic dyes, showing an enormous potential as bioimaging probes. Carbocyanine dyes were selected as the fluorophores because of their hydrophobicity and high excitation coefficients, the FRET pair is composed by the donor DiI and the acceptor DiD.

Results/Discussion

The photophysical properties of quatsomes loaded with DiI and DiD simultaneously, were studied showing FRET efficiencies values up to 80%. When FRET occurs, donor quantum yield decreases significantly. To further confirm the occurrence of FRET, time-resolved fluorescence measurements were carried out elucidating a dramatic decrement on donor lifetime in presence of the acceptor, asserting the presence of FRET. The stability of dye-loaded QS has been investigated monitoring the FRET emission and efficiency on time, showing no appreciable changes in their optical properties for at least 8 months. In addition to stability, the high brightness displayed and the low photodegradation quantum yield demonstrate their distinguished appropriateness for bioimaging purposes. To further ascertain the potentiality of QS with FRET as bioimaging probes, cell viability tests, and cell imaging were conducted in commonly employed cancer and normal cell lines.

Conclusions

We anticipate this novel fluorescent organic nanoparticle with tremendous FRET efficiencies, stable during long periods of time, and biocompatible to gain a vast impact and set of applications in different fields, including bioimaging. This kind of nanostructures can be easily functionalized with targeting groups, representing a very promising platform, especially for theragnostic nanomedicine.

References
[1] Ferrer-Tasies, L. et al. 2013. 'Quatsomes: Vesicles Formed by Self-Assembly of Sterols and Quaternary Ammonium Surfactants.' Langmuir 29, 6519–6528.
[2] Cabrera, I. et al. 2013. 'Multifunctional Nanovesicle-Bioactive Conjugates Prepared by a One-Step Scalable Method Using CO2-Expanded Solvents.' Nano Lett. 13, 3766–3774.
[3] Antonio, A. et al. 2018. 'Nanostructuring Lipophilic Dyes in Water Using Stable Vesicles, Quatsomes, as Scaffolds and Their Use as Probes for Bioimaging.' Small 14, 1703851. 
[4] Cabrera, I. et al. 2016. 'α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration'. Adv. Healthc. Mater. 5, 829–840. 
QS with FRET

Cryo-TEM image of QS and schematic representation of QS membrane organization (top). Emission spectra of QS containing the lipophilic dyes (down). 

Keywords: Fluorescent organic nanoparticles, bioimaging, FRET, nanovesicles