Functional Hybrid Molecules for the Visualization of Cancer: PESIN‐Homodimers Combined with Multimodal Molecular Imaging Probes for Positron Emission Tomography and Optical Imaging: Suited for Tracking of GRPR‐Positive Malignant Tissue

Abstract We describe multimodal imaging probes for gastrin‐releasing peptide receptor (GRPR)‐specific targeting suited for positron emission tomography and optical imaging (PET/OI), consisting of PESIN (PEG3‐BBN7‐14) dimers connected to multimodal imaging subunits. These multimodal agents comprise a fluorescent dye for OI and the chelator ((1,4,7‐triazacyclononane‐4,7‐diyl)diacetic acid‐1‐glutaric acid) (NODA‐GA) for PET radiometal isotope labelling. Special focus was put on the influence of the used dyes on the properties of the whole bioconjugates. For this, several compounds with different fluorescent dyes and non‐dye carrying subunits were synthesized and investigated. As fluorescent dyes, dansyl, NBD, derivatives of fluorescein, coumarin and rhodamine as well as three pyrilium‐based dyes were employed. Considerable influence of the charge of the colored unit on hydrophilicity as well as in vitro target receptor binding was observed and classified. High radiochemical yields and purities were found during radiolabeling of the multimodal imaging subunits as well as their GRPR‐specific bioconjugates with 68Ga. Examinations of the photophysical properties of both molecule species displayed no loss or alteration of fluorescence characteristics.

Scheme S1 Depiction of the structures and syntheses of CC1 -CC5
For HPLC chromatography, a Dionex UltiMate 3000 system was used together with Chromeleon Software (Version 6.80). For analytical chromatography, a Chromolith Performance (RP-18e, 100-4.6 mm, Merck, Germany) and for semipreparative analyses, Chromolith (RP-18e, 100-10 mm, Merck, Germany) columns were used, respectively. For radioanalytical use, a Dionex UltiMate 3000 system equipped with a Raytest GABI Star radioactivity detector was used together with a Chromolith Performance (RP-18e, 100-4.6 mm, Merck, Germany) column. All operations were performed with a flow rate of 4 mL/min using H 2 O + 0.1% TFA and MeCN + 0.1% TFA as solvents. HR-ESI (high-resolution Electrospray Ionization) and MALDI (Matrix-Assisted Laser Desorption/Ionization) spectra were obtained with Finnigan MAT95Q and Bruker Daltronics Microflex spectrometers, respectively. γ-counting was performed using a 2480 Wizard gamma counter system from Perkin Elmer. A Cary 100 Bio system (Varian) was used to record the UV/Vis-Spectra together with 4 mL PMMA cuvettes from Sigma-Aldrich. Further, for absorbance and emission measurements, a Tecan Infinite M200 Microplate reader together with a Nunc Micro-Well 96 solid plate from ThermoFisher was used. Confocal fluorescence microscopy was performed on a Leica TCS SP8 confocal microscope with excitation lasers at λs of 488, 552 and 638 nm. Overlays of microscopies were generated directly with the operating software or afterwards using FIJI software (V1.50e).
2: 1 eq. 1 (1 mg) was dissolved in 1:1 MeCN:H 2 O + 0.1 % TFA and 2 eq. of SFB (0.94 mg), dissolved in MeCN + 0.1 % TFA, were added. The pH of the mixture was adjusted to 7.0 by adding phosphate buffer solution (0.5 M, pH = 7.2). The reaction mixture was stirred at 50°C for 30 minutes and afterwards, the reaction vessel was directly frozen with liquid nitrogen and freeze-dried overnight. The residue was dissolved in 1:1 MeCN:H 2 O + 0.1 % TFA, where a phase separation took place. The organic layer was subsequently purified by HPLC to give 2 as a white solid. (C 37

Radiochemistry
A solution of the respective multimodal imaging unit (MIU) 6a -13a (5 nmol) or multimodal imaging agent 6 -14 (5 nmol) in H 2 O (Tracepur quality, 1mM) was added to 90 -120 MBq of [ 68 Ga]GaCl 3 in a solution obtained by fractioned elution of an IGG 68 Ge/ 68 Ga generator system with HCl (0.1 M, 1.6 mL) and subsequent titration to pH 3.5 -4.2 by addition of sodium acetate solution (1.25 M, 50 -75 µL). All labeling experiments were performed by addition of 2 mg ascorbic acid to suppress radiolysis-induced product fragmentation. In the labeling experiments of the MIUs 6a -13a, 1 -4 mg TCEP x HCl were added and the pH was adjusted to 3.5 -4.2 by addition of sodium acetate solution (1.25 M). After reaction for 10 min at 45°C, the reaction mixtures were analyzed by analytical radio-HPLC. The radiolabeled products were found to be 95 -99 % pure and obtained in non-optimized molar activities of 90 -120 GBq/µmol.

Log D determination
For 68 Ga-6/6a -68 Ga-13/13a, the water/1-octanol partition coefficient (log D ) was determined by adding 5 µL of the respectively 68 Ga-labeled compound (0.8 -1.2 MBq) to a mixture of phosphate buffer (0.05 M, pH 7.4, 795 µL) and 1-octanol (800 µL). The mixtures were intensively shaken for 5 minutes on a vibrating plate. After subsequent centrifugation at 13 000 rpm for 5 min, 125 µL were taken from each phase and measured in a γ-counter. The log D values were calculated from three or four independent experiments, each performed in triplicate.
For 14, the water/1-octanol partition coefficient (log D ) was determined by semipreparative HPLC. For this purpose, 10 µL solution (c = 5 x 10 -4 mol/L) of the substance were added to a mixture of 1 mL 1-octanol and 990 µL phosphate buffered solution (0.05 M, pH 7.4) and the mixture was vigorously shaken for 5 minutes. After centrifugation, the phases were separated and both phases were analyzed by semipreparative HPLC. The log D was determined by three separate measurements, each experiment performed in triplicate.

Competitive receptor binding assay
Stably GRPR-transfected Human Embryonic Kidney 293 cells (HEK-GRPR) were cultured at 37°C in Dulbecco's Modified Eagle's Medium (DMEM, high glucose, GlutaMax-I, 500 mL) supplemented with 10 % FCS (50 mL), 1.5 % Geniticin (8.25 mL) and 1 % PenStrep (5.5 mL) in a humidified atmosphere containing 5 % CO 2 . The medium was exchanged every two or three days and cells were split at >75 % confluence. In vitro binding affinities were determined via competitive displacement experiments which were performed at least trice, each experiment performed in triplicate. A Millipore Multiscreen punch kit and Millipore 96 well filter plates (pore size 1.2 µm) were used. The plates were incubated with PBS/BSA (1%) solution (each well 200 µL) for one hour before use. HEK-GRPR cells were harvested and suspended carefully in Opti-MEM I (GlutaMAX I) medium. 50 µL of a cell suspension containing 10 5 cells were seeded in each well. To this, a total volume of 50 µL was added to each well, containing 25 µL (0.012 kBq/µL) of the GRPR-specific radioligand [ 125 I]-Tyr 4 -bombesin (81.4 GBq/µmol) and 25 µL of the respective competitor 6 -14 or endogenous bombesin (BBN, used as reference compound). The competitor was added in 11 increasing concentrations ranging from 0.25 -500 nM for 6 -14 or 0.1 -250 nM for BBN, whereat the twelfth well contained no competitor to ensure 100 % binding of the radioligand. After one hour of incubation at ambient temperature, the solution was filtrated and the filters were washed with cold PBS (3 times). The filters were collected and measured by γ-counting. The 50 % inhibitory concentration (IC 50 ) values of 6 -14 and bombesin were calculated by fitting the obtained data via a nonlinear regression analysis using GraphPad Prism Software (v5.04).

Confocal Fluorescence Microscopy
Since the HEK-GRPR do not exhibit any adhered characteristics to glass plates, the GRPRpositive human tumor cell line PC-3 was used for the initial confocal fluorescence microscopy experiments instead. PC-3 cells were cultured at 37°C in RPMI 1640 medium supplemented with 10% FCS, 1% L-Glutamine and 1% PenStrep in a humidified atmosphere containing 5% CO 2 . The medium was exchanged every two or three days and cells were split at >75% confluence. Fluorescence microscopy was performed on a Leica TCS SP8 confocal microscope with laser excitation wavelengths of 405 and 638 nm. Overlays of microscopies were generated directly with the operating software or afterwards using FIJI software (v1.50e). PC-3 cells (4 x 10 4 ) were seeded two days prior to the measurements in six-well culture plates, each well was equipped with cover slips for microscopy. Medium was exchanged after 24h. After 2 days, the medium was removed and the cells were washed carefully with PBS. Then, 1 mL of a 10 μM solution of 6 in antibiotic-free medium was added, followed by incubation for 4h. After washing of each cover slip with PBS thrice, they were consequently put reversed on object slides which were before prepared with a drop (10μL) of DAPI antifade solution and then the respective cover slide was sealed and fixed with clear coat on the object slides.