Hybrid Imaging of Aspergillus fumigatus Pulmonary Infection with Fluorescent, 68Ga-Labelled Siderophores

Aspergillus fumigatus (A. fumigatus) is a human pathogen causing severe invasive fungal infections, lacking sensitive and selective diagnostic tools. A. fumigatus secretes the siderophore desferri-triacetylfusarinine C (TAFC) to acquire iron from the human host. TAFC can be labelled with gallium-68 to perform positron emission tomography (PET/CT) scans. Here, we aimed to chemically modify TAFC with fluorescent dyes to combine PET/CT with optical imaging for hybrid imaging applications. Starting from ferric diacetylfusarinine C ([Fe]DAFC), different fluorescent dyes were conjugated (Cy5, SulfoCy5, SulfoCy7, IRDye 800CW, ATTO700) and labelled with gallium-68 for in vitro and in vivo characterisation. Uptake assays, growth assays and live-cell imaging as well as biodistribution, PET/CT and ex vivo optical imaging in an infection model was performed. Novel fluorophore conjugates were recognized by the fungal TAFC transporter MirB and could be utilized as iron source. Fluorescence microscopy showed partial accumulation into hyphae. µPET/CT scans of an invasive pulmonary aspergillosis (IPA) rat model revealed diverse biodistribution patterns for each fluorophore. [68Ga]Ga-DAFC-Cy5/SufloCy7 and -IRDye 800CW lead to a visualization of the infected region of the lung. Optical imaging of ex vivo lungs corresponded to PET images with high contrast of infection versus non-infected areas. Although fluorophores had a decisive influence on targeting and pharmacokinetics, these siderophores have potential as a hybrid imaging compounds combining PET/CT with optical imaging applications.


Introduction
Fungal infections of humans are widespread and can appear in many different forms. Infections of the skin and nails are very common, like onychomycosis or oral thrush [1]. Most people will suffer from those at least once in their lifetime, but these infections are easily diagnosed and respectively curable. In contrast, severe systemic fungal infections kill about one and a half million people every year [2]. One of the most prominent examples is invasive pulmonary aspergillosis (IPA), an opportunistic infection of the lung mainly caused by Aspergillus fumigatus (A. fumigatus). Worldwide, an estimate of Therapeutic intervention is also highly challenging due to an increasing resistance of A. fumigatus to commonly used antifungals, and the rapid decline of the patient's health condition. Surgical procedures may be required to remove affected lung tissue [17]. Image guidance may help to visualize infected lung tissue intraoperatively. Chemical modification by introducing fluorophores into TAFC structure can provide novel hybrid imaging agents. TAFC can be in parallel radiolabelled with gallium-68 for PET diagnostic purposes as well as conjugated to a fluorophore for optically guided surgery, which has been successfully demonstrated for oncological applications [18][19][20]. A major challenge is to find the optimal fluorophore, which provides suitable pharmacokinetic properties and the correct wavelength [21]. The ideal detection window is between 650-850 nm, where the surrounding human tissue has a low intrinsic autofluorescence.
In this paper, we describe the chemical modifications of TAFC with different near infrared (NIR) dyes and the evaluation of their pharmacological properties. We also investigate possible hybrid imaging applications in vivo by using an IPA rat model with a µPET/CT and optical imaging system [16].

Synthesis of Fluorophore Conjugates
[Fe]FsC was used as a starting material to produce [Fe]DAFC as previously described [22]. All fluorescent dyes were used as carboxylic acid derivatives and activated with O-(7-azabenzotriazol-1- In this paper, we describe the chemical modifications of TAFC with different near infrared (NIR) dyes and the evaluation of their pharmacological properties. We also investigate possible hybrid imaging applications in vivo by using an IPA rat model with a µPET/CT and optical imaging system [16].

Synthesis of Fluorophore Conjugates
[Fe]FsC was used as a starting material to produce [Fe]DAFC as previously described [22]. All fluorescent dyes were used as carboxylic acid derivatives and activated with O-(7-azabenzotriazol-1yl)-1,1,3,3-tetramethyluronium-hexafluorophosphat (HATU) for conjugation to the free amine of [Fe]DAFC in DMF. After reaction (<2 h) at ambient temperature and under light exclusion, quantitative conjugation was reached and the reaction solution was purified by preparative RP-HPLC to give a coloured solid powder after lyophilisation. Identity was confirmed by MALDI-TOF MS. For detailed chemical information, see supplementary material.
For radiolabelling, iron was removed from the complex by incubation with a 1000-fold excess of ethylenediaminetetraacetic acid (EDTA) at pH 4 and subsequent purification by preparative RP-HPLC as previously described [23].

Distribution Coefficient (Log D)
Log D was determined by measuring the distribution of each compound between octanol and PBS buffer. For this purpose, radiolabelled compound was dissolved with PBS to 1 mL (~9 µM). Aliquots of 50 µL were added to 450 µL PBS and 500 µL octanol, shaken for 20 min with 1400 rpm at RT (MS 3 basic vortexer, IKA, Staufen, Germany) followed by centrifugation for 2 min at 4500 rpm (Eppendorf Centrifuge 5424, Eppendorf AG, Hamburg, Germany). Hereafter, 200 µL of each phase were collected and measured in a 2480 automatic Gamma counter Wizard 2 3" (PerkinElmer, Waltham, MA, USA). Log D value was calculated using Excel. (n = 3, six technical replicates)

Protein Binding
Radiolabelled compound was diluted with PBS to 1 mL (~9 µM) and 50 µL of that solution was added to 450 µL serum or 450 µL PBS as a control. After 30, 60 and 120 min incubation at 37 • C, aliquots of 25 µL were analysed by size exclusion chromatography using MicroSpin G-50 columns (Sephadex G-50, GE Healthcare, Vienna, Austria) according to the manufacturer's protocol. Hereafter, column and eluate were measured separately in the gamma counter to calculate percentage of free-(column bound) and protein-bound (eluate) fraction.

Uptake and Competition Assay
Uptake assays were performed as previously published [14]. Briefly, 180 µL of A. fumigatus culture in iron-depleted and iron-replete media were added in 96-well MultiScreen Filter Plates HTS (1 µm glass fiber filter, Merck Millipore, Darmstadt, Germany) and pre-incubated for 15 min with either PBS or [Fe]TAFC (blocking solution). Subsequently, radiolabelled compound (final concentration approximately 90 nM) was added and incubation continued for 45 min at room temperature. Dry filters were measured in the gamma counter.
Competition assays were performed in the same way with slight modifications. Fungal cultures were pre-incubated with iron-labelled fluorophore conjugates for 15 min and the uptake value of [ 68 Ga]Ga-TAFC into hyphae was determined after another 45 min of incubation.

Growth Promotion Assay
To investigate the ability of A. fumigatus to use iron containing siderophores for its metabolism, a mutant strain (∆sidA/∆ftrA) lacking two genes, sidA and ftrA was point inoculated (10 4 conidia) in 24-well plates, containing 0.5 mL of aspergillus minimal media agar with [Fe]siderophore concentrations ranging from 0.1-50 µM. Plates were incubated for 48 h at 37 • C in a humidity chamber and visually assessed afterwards [14].

Fluorescence Microscopy
Fluorescence microscopy was performed for [Fe]DAFC-Cy5 and -SulfoCy5 allowing an excitation wavelength below 700 nm. Fungal cultures were prepared in µ-Slide 8 Well chambered coverslips (ibidi GmbH, Martinsried, Germany). Each well was inoculated with 5000 Spores in 200 µL of minimal Biomolecules 2020, 10, 168 5 of 14 medium and incubated at 37 • C in a humidified chamber. A. fumigatus (ATCC 46645) was cultivated for 14 h and A. terreus (ATCC 3633) for 48 h to obtain well developed germlings and young hyphae without extensive germling or vegetative hyphal fusion. For microscopy, a Leica TCS SP5 II inverted confocal laser scanning microscope was used with HeNe laser (10 mW: 633 nm) as excitation light source: AOBS (Acousto-Optical Beam Splitter) (640-690 nm) and Leica HyD detector. Fluorophores were used at a final concentration of 2.5 µM and incubated for 10-40 min. Excitation laser intensity during imaging was kept to a minimum to reduce photobleaching and phototoxic effects to the cells while still achieving good signal-to-noise ratios. Images were recorded with a maximum resolution of 1024 × 1024 pixels and saved as TIFF. Some images represent a z-Stack that is labelled in the particular picture description. Apart from brightness adjustments and cropping using the ImageJ 1.52a opensource software platform (Wayne Rasband, NIH, Bethesda, MD, USA), images were not subjected to further manipulation.

In Vivo Characterisation
All animal experiments were conducted in compliance with the Austrian and Czech animal protection laws and with approval of the Austrian Ministry of Science (BMWFW-66.011/0161-WF/V/3b/2016), the Czech Ministry of Education Youth and Sports (MSMT-21275/2016-2) and the institutional Animal Welfare Committee of the Faculty of Medicine and Dentistry of Palacky University in Olomouc.

Invasive Pulmonary Aspergillosis Model in Rats
Female Lewis rats of 2-3 months age were treated with the immunosuppressant cyclophosphamide (Endoxan, Bayter, 75 mg/kg i.p.) 5 days and 1 day before A. fumigatus inoculation to induce neutropenia. To avoid bacterial superinfections, animals repeatedly (5 days, 1 day before and on the day of inoculation) received antibiotic teicoplanin (Targocid, Sanofi, 35 mg/kg-5 days before i.m. or 25 mg/kg i.m.-1 day before and on the day of inoculation) and additional antibiotics were administered by drinking water (Ciprofloxacin, 2 mM, Polymyxin E, Colomycin, 0.1 mM) for the duration of the experiment. Fungal infection was established by intratracheal application of 100 µL of A. fumigatus spores (10 9 CFU/mL A. fumigatus 1059 CCF) using TELE PACK VET X LED system equipped with a flexible endoscope (Karl Stroz GmbH & Co. KG, Tuttlingen, Germany) only [24].

Micro PET/CT and Optical Imaging
In vivo PET/CT images were acquired with an Albira PET/SPECT/CT small animal imaging system (Bruker Biospin Corporation, Woodbridge, CT, USA). Female Lewis rats were retro-orbitally (r.o.) injected with radiolabelled fluorophore conjugate in a dose of 5-10 MBq corresponding to~2 µg of DAFC-conjugate per rat. Animals were anaesthetized with isoflurane (Forene ® , Abbott Laboratories, Abbott Park, IL, USA) (2% flow rate) and positioned prone head first in the Albira system before the start of imaging. Static PET/CT imaging was carried out 45 min p.i. for all tested compounds. PET/CT imaging of infected animals was performed three days after the inoculation with A. fumigatus spores. A 10-min PET scan (axial FOV 148 mm) was performed, followed by a triple CT scan (axial FOV 3 × 65 mm, 45 kVp, 400 µA, at 400 projections). Scans were reconstructed with the Albira software (Bruker Biospin Corporation, Woodbridge, CT, USA) using the maximum likelihood expectation maximization (MLEM) and filtered backprojection (FBP) algorithms. After reconstruction, acquired data were viewed and analysed with PMOD software (PMOD Technologies Ltd., Zurich, Switzerland). 3D images were obtained using VolView software (Kitware, Clifton Park, NY, USA). After PET/CT imaging, the animals were sacrificed by exsanguination and lungs were collected for ex vivo fluorescence imaging.

Synthesis of Fluorophore Conjugates
Fluorescent dye coupling was straightforward with reaction times under 2 h. Yields for iron-containing and iron-free compounds varied between 60-80% with a high chemical purity of >95%.

Radiolabelling
The mild labelling conditions at room temperature and short time of 10 min allowed high radiochemical yields for all compounds, respectively. Both radio-ITLC and radio-RP-HPLC showed radiochemical purity over >95%. (representative HPLC radiochromatograms are shown Figure S2)

Distribution Coefficient and Protein Binding
Log D of fluorophore conjugates showed a high dependence on the number of sulfate groups in their chemical structure.

Utilization of Siderophore-Conjugates by A. Fumigatus
To measure utilization of the synthesized siderophore conjugates, a previously described bioassay was employed (14). Therefore, the A. fumigatus ΔsidA/ΔftrA mutant strain was used, which lacks both siderophore biosynthesis and reductive iron assimilation. Consequently, this strain is able to grow only in the presence of recognized siderophores or ferrous iron concentrations ≥3 mM.

Utilization of Siderophore-Conjugates by A. Fumigatus
To measure utilization of the synthesized siderophore conjugates, a previously described bioassay was employed (14). Therefore, the A. fumigatus ∆sidA/∆ftrA mutant strain was used, which lacks both siderophore biosynthesis and reductive iron assimilation. Consequently, this strain is able to grow only in the presence of recognized siderophores or ferrous iron concentrations ≥3 mM. Control with [Fe]TAFC and [Fe]DAFC showed an induction of growth already at 0.1 µM and sporulation at 10 µM (Figure 3). [Fe]DAFC-IRDye supported growth to a slightly lower degree but did not support sporulation even at highest concentration.
[Fe]DAFC-SulfoCy7 supported growth to a similar degree as [Fe]DAFC-IRDye at 1 µM but showed a growth inhibitory effect at higher concentrations, as seen for [Fe]DAFC-Cy5, although not as severe.

Fluorescence Microscopy
Fluorescence microscopy revealed a significant difference in the uptake of [Fe]DAFC-Cy5 and [Fe]DAFC-SulfoCy5 (Figure 4), which possess chemically similar structures (Figure 1). For [Fe]DAFC-Cy5, rapid uptake into hyphae of A. fumigatus with pronounced visualisation of subcellular organelles was observed 5 min after compound application, which remained visible for more than two hours. In contrast, no uptake of [Fe]DAFC-Cy5 was found by A. terreus, which lacks uptake of [Fe]TAFC (14). Remarkably, the Cy5 carboxylic acid dye alone showed rapid hyphal uptake in both A. fumigatus and A. terreus. These data demonstrate that the linking of DAFC imparts the feature of

Fluorescence Microscopy
Fluorescence microscopy revealed a significant difference in the uptake of [Fe]DAFC-Cy5 and [Fe]DAFC-SulfoCy5 (Figure 4), which possess chemically similar structures (Figure 1). For [Fe]DAFC-Cy5, rapid uptake into hyphae of A. fumigatus with pronounced visualisation of subcellular organelles was observed 5 min after compound application, which remained visible for more than two hours. In contrast, no uptake of [Fe]DAFC-Cy5 was found by A. terreus, which lacks uptake of [Fe]TAFC (14). Remarkably, the Cy5 carboxylic acid dye alone showed rapid hyphal uptake in both A. fumigatus and A. terreus. These data demonstrate that the linking of DAFC imparts the feature of specific uptake by A. fumigatus to Cy5; [Fe]DAFC-Cy5 is specifically and actively imported, most likely by MirB, into hyphae of A. fumigatus. Growth is reflected by whitish-mycelia, while sporulation is reflected by the green colour, which arises from the green conidial-specific pigment. The last row shows controls of agar without siderophores: W = sterile water; S = Spores.
In contrast, no uptake was observed for [Fe]DAFC-SulfoCy5 or the -SulfoCy5 carboxylic acid dye alone by A. fumigatus or A. terreus by fluorescence microscopy. In contrast, no uptake was observed for [Fe]DAFC-SulfoCy5 or the -SulfoCy5 carboxylic acid dye alone by A. fumigatus or A. terreus by fluorescence microscopy.    In contrast, no uptake was observed for [Fe]DAFC-SulfoCy5 or the -SulfoCy5 carboxylic acid dye alone by A. fumigatus or A. terreus by fluorescence microscopy.

Discussion
Hybrid imaging of fungal infections can be a very useful tool allowing not only diagnosis of IPA using PET/CT, but also precise localization of infected tissues during surgery or endoscopy by means of optical probes. Different hybrid imaging approaches have already been developed for oncological applications [25,26], but are currently very limited in the field of infection. First steps have been made to establish this method for bacterial infections [27] but so far this approach has not been investigated for fungal infections. Based on the sophisticated TAFC-dependent mechanism of A. fumigatus to acquire iron from the human host, the exchange of iron by gallium-68 resulted in successful PET imaging in an IPA rat model [14,16,28], the overall approach of using radiolabelled siderophores for molecular imaging is described in [29]. The specificity of [ 68 Ga]Ga-TAFC for PET imaging of A. fumigatus has been shown by Petrik et al. in comparison with various organisms (fungi/bacteria) and also human lung cancer cells [24]. Doyle et al. modified [Fe]FsC with the fluorescent dye NBD (6-(N-(7-nitrobenz-2-oxa,1,3-diazol-4-yl)amino)Hexanoate) for fluorescent microscopy of A. fumigatus showing the feasibility of modifying siderophores retaining recognition by pathogenic fungi [30]. This study combines the advantages of both radioactive and fluorescent labelling in a hybrid imaging compound for fungal infection imaging. We had previously reported on the use of fusarinine C as a scaffold for hybrid imaging probes for tumor targeting, with [22] forming the basis for the modifications reported here.
Starting from [Fe]DAFC modification with different fluorophores using a HATU coupling strategy to activate carboxylic acid resulted in high yields in a very short time. After removing the iron from the complex, 68 Ga-labelling in high radiochemical purity over 95% and high molar radioactivity was achieved for in vitro and in vivo experiments. Previous studies showed that different modifications, depending on size and charge of the functional groups, influence the uptake properties in A. fumigatus [14]. Fluorescent dyes used in this study were all charged from +1 to −3 with high molecular weight compared to the [Fe]DAFC molecule. This had a decisive influence on target interaction as well as pharmacokinetics. Competition assays showed blocking of [ 68 Ga]Ga-TAFC uptake into hyphae indicating recognition of the conjugates by the MirB transporter. Even so, uptake assays resulted in a high unspecific binding, probably related to cell wall interactions due to charge and/or lipophilicity of the fluorophores. These findings could be clarified by employing growth assay using the A. fumigatus ∆sidA/ ∆ftrA mutant strain, requiring external  [33] or ferrichrome with naphthalimide or quantum dots to visualize cellular structures [34]. However, these compounds have only been used for microscopy and not for hybrid imaging applications.
In vivo µPET/CT images of the conjugates in an IPA rat model confirmed in vitro data. As expected, excretion of each compound could be connected to the Log D and protein binding values. Lipophilic compounds like [ 68 Ga]Ga-DAFC-Cy5/ATTO 700 or [ 68 Ga]Ga-DAFC-SulfoCy7 showed mainly hepatobiliar excretion, whereas hydrophilic conjugates [ 68 Ga]Ga-DAFC-SulfoCy5/IRDye 800CW were primarily eliminated through the kidneys. In line with the rapid uptake of [Fe]DAFC-Cy5 in microscopy studies, visualization of the infected lung area was also clearly observed for [ 68 Ga]Ga-DAFC-Cy5. Moreover, lack of accumulation of [ 68 Ga]Ga-DAFC-SulfoCy5 in the IPA model corresponded to growth assays and microscopy.
To complete these findings, ex vivo optical images of lungs showed the same pattern as µPET/CT scans. Except for [ 68 Ga]Ga-DAFC-SulfoCy5, where a slightly higher signal in the non-infected control was detected, all other infected lungs showed a more intense signal than respective non-infected controls.
Overall, our results show that functional modification of DAFC as a mimic for the A. fumigatus specific siderophore TAFC is possible, and that the introduction of fluorescent dyes provides reliable hybrid imaging compounds for IPA. Recognition by the target and pharmacokinetics were highly dependent on the fluorophore applied. Furthermore, in addition to novel hybrid imaging applications, these compounds also provide insight into the structure related target interaction and uptake characteristics to design potentially antifungal siderophore conjugates.

Conclusions
Overall, this study shows that the modification of TAFC with various fluorescent dyes is possible and they are still recognized by A. fumigatus. Radiolabelling is easily achievable with high stability in vivo and in vitro. This work reveals insight into the structure related properties of modified siderophores, especially with respect to their combination with antifungal drugs and species-specific fluorescence microscopy applications. This opens up new possibilities for applications to combine PET with optical imaging (e.g., surgery) as a hybrid imaging agent in IPA with A. fumigatus.