Ex Vivo Evaluations

Article pubs.acs.org/molecularpharmaceutics

Radiolabeled 111In-FGF‑2 Is Suitable for In Vitro/Ex Vivo Evaluations and In Vivo Imaging Alessandra Moscaroli,† Gabriel Jones,‡ Tessa Lühmann,‡ Lorenz Meinel,‡ Stephanie Wal̈ ti,§ Alain Blanc,† Eliane Fischer,† Manuel Hilbert,∥,⊥ Roger Schibli,†,§ and Martin Béhé*,† †

Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland Institute for Pharmacy and Food Chemistry, University of Wurzburg, 97074 Wurzburg, Germany § Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8092 Zurich, Switzerland ∥ Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland ‡

S Supporting Information *

ABSTRACT: Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an 111Inradiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT). 111In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as 125I-FGF-2. Furthermore, 111InFGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. 111In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (Kd: 0.6 ± 0.07 μM and 0.33 ± 0.03 μM, respectively) as assessed by isothermal titration calorimetry. The binding of 111In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC50 values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of 111In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore, 111In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development. KEYWORDS: fibroblast growth factor-2 (FGF-2), residue-specific radiolabeling, in vivo imaging, single photon emission tomography (SPECT)

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INTRODUCTION Fibroblast growth factor-2 (FGF-2) is a potent regulator of cell growth and differentiation, acting on cells of mesodermal, ectodermal, and endodermal origin and, thereby, mediating metabolic function of tissues leading to repair, regeneration, and potentially reactivated development.1−5 Actually, recombinant FGF-2 is a promising candidate for the regeneration of lost tissues and clinically administered against different cardiovascular disorders.5,6 However, the utilization of FGF-2 preparations has still several limitations (e.g., poor in vivo stability and rapid clearance), and numerous efforts are made to improve FGF-2 administration. This includes the development of biomaterial scaffolds or controlled release formulation for the systemic administration of FGF-2, among others.5,7 Two FGF-2 isoforms with overlapping but also distinct function are known: an 18 kDa protein referred to as cytosolic or low-molecular-weight FGF-2 (lo FGF-2) and the 20−34 kDa high-molecular-weight FGF-2 (hi-FGF-2).8 The cytosolic isoform of FGF-2, used here within, is secreted by the cells to © XXXX American Chemical Society

be stored in the extracellular matrix (ECM) through heparan sulfate proteoglycans (HSPGs) until its activation.9,10 During tissue repair, for example, FGF-2 is mobilized and released from the ECM by heparanases or by specific FGF-binding proteins.11 Afterward, FGF-2 binds with high affinity to the cell surface receptor tyrosine kinases (FGF receptor 1−3), activating different intracellular signaling pathways including the MAP kinase cascade.7,9,11−14 Radiolabeling of proteins is an important and widely used technique in biological research. In particular, the radioiodination with 125I is routinely used for in vitro and in vivo biological investigations.15,16 Most studies with radiolabeled FGF-2 in the field were conducted with 125I-FGF-2 as a tracer. However, the low gamma-ray (γ-ray) energy of 35 keV, the Received: October 11, 2016 Revised: January 26, 2017 Accepted: January 30, 2017

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DOI: 10.1021/acs.molpharmaceut.6b00913 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

ammonium acetate pH 5.5, the final product was analyzed by liquid chromatography−mass spectrometry (LC−MS) using a LCT Premier mass spectrometer (Waters AG, Dättwil, Switzerland). The analysis was carried out with a UP5BP1 column (150 mm × 2 mm; 5 μm) purchased from Interchim (Montluçon, France). The column was eluted using acetonitrile containing 0.1% formic acid, Ultrapure water (Millipore) containing 0.1% formic acid, and 2-propanol with a linear gradient starting with 2.5% acetonitrile, 2.5% 2-propanol, and 95% water up to 92.5% acetonitrile, 2.5% 2-propanol, and 5% water over 20 min, respectively. The protein concentration was determined with NanoPhotometer (Implen GmbH, Munich, Germany). Radiolabeling of FGF-2 with 111In. DTPA-FGF-2 and 111 InCl3 were mixed with a nuclide-to-protein ratio of 1:300. The reaction was performed in 0.5 M ammonium acetate pH 5.5 at 4 °C, overnight. The obtained product was analyzed by reversed-phase HPLC (Varian Prostar HPLC System, SpectraLab Scientific Inc., Canada) using a Dr. Maisch Reprospher (Dr. Maisch GmbH, Ammerbuch, Germany) 300 C18-TN column (4.6 cm × 150 mm; 5 μm). The column was eluted using acetonitrile containing 0.1% TFA and Ultrapure water (Millipore) containing 0.1% TFA and a linear gradient starting with 15% up to 95% acetonitrile over 15 min with a flow rate of 1 mL/min. Labeling of FGF-2 with natIn. natInCl3 was directly added in excess to DTPA-FGF-2 (ratio 4:1). The reaction occurred in 0.5 M ammonium acetate pH 5.5 at 4 °C, overnight. Finally, the completeness of the reaction was monitored by LC−MS (LCT Premier mass spectrometer, Waters AG, Dättwil, Switzerland), using a UP5BP1 column (150 mm × 2 mm; 5 μm) purchased from Interchim (Montluçon, France). The column was eluted using acetonitrile containing 0.1% formic acid, Ultrapure water (Millipore) containing 0.1% formic acid, and 2-propanol with a linear gradient starting with 2.5% acetonitrile, 2.5% 2-propanol, and 95% water up to 92.5% acetonitrile, 2.5% 2-propanol, and 5% water over 20 min, respectively. Isothermal Titration Calorimetry (ITC). The binding of FGF-2 and natIn-FGF-2 to low-molecular-weight heparin was measured using a MicroCal iTC200 calorimeter (GE Health Care, Freiburg im Breisgau, Germany), where the heparin solution was titrated into a solution of FGF-2 or natIn-FGF-2, respectively. The protein samples were dialyzed in 50 mM Naphosphate, 100 mM NaCl pH 6.8 containing 3 mM DTT buffer (ITC buffer). The syringe was loaded with lowmolecular-weight heparin (Enoxaparin, Clexane, Sanofi-Aventis SA, Vernier, Switzerland) diluted with the ITC buffer to 0.22 mM for FGF-2 and 0.30 mM for nat In-FGF-2. The corresponding protein solution was loaded into the cell: 66 μM FGF-2 and 57.2 μM natIn-FGF-2, respectively. The experiments were performed with an initial injection of 0.2 μL, followed by 19 injections of 2 μL with 150 s intervals. The solution in the syringe was stirred at 400 rpm with thermosetting at 25 °C. The data were analyzed using Origin 7 software (OriginLab Corporation, Northampton MA, USA), and the obtained binding curves were fitted to a one-site binding model. In Vitro Binding Experiments. NIH 3T3 cells (murine embryo fibroblasts, ATCC CRL-1658) were cultured at 37 °C in a 5% CO2 humidified atmosphere in DMEM culture medium (Dulbecco’s modified Eagle’s medium, Amimed, Bioconcept AG, Allschwil, Switzerland) supplemented with 10% fetal calf

long half-life of 60 days of 125I, and the high metabolic deiodination rate cause some limitation for the optimal in vivo imaging in small animals.17 In this study, we present an 111In-radiolabeled FGF-2 derivate for noninvasive imaging in small animals with single photon emission computed tomography (SPECT). Indium-111 is a radiometal used in the clinic for the diagnosis of certain types of cancer.18−21 Its suitable half-life of 2.8 days and a γ-ray energy of 171 and 245 keV permit the utilization of 111In-FGF2 for noninvasive in vivo imaging as well as for in vitro and postmortem experiments.17,22 We describe a strategy for the residue-specific radiolabeling of FGF-2 with 111In complemented by in vitro and in vivo characterization.

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EXPERIMENTAL SECTION Chemicals and Reagents. The radioactive 111InCl3 was obtained from Mallinckrodt (Cham, Switzerland). Maleimide diethylenetriaminepentaacetic acid (maleimide DTPA) was purchased from CheMatech (Dijon, France). The natural nonradioactive indium (natIn) consists of indium-113 (4.3%) and indium-115 (95,7%). It is chemically equivalent to the radioactive indium-111, and it was utilized when radioactivity was not necessary for the experiment. natInCl3, deionized water (TraceSELECT quality), trifluoroacetic acid (HPLC grade), formaldehyde solution (ACS reagent), bovine serum albumin (BSA, lyophilized powder), and 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Buchs, Switzerland). Acetonitrile J.T. Baker (LC and LC−MS grade) was from P.H. Stehelin and CIE AG (Basel, Switzerland). Amicon Ultra centrifugal ultrafiltration devices (Amicon Ultra, 10 kDa cutoff filters) were from Merck Millipore (Merck & Cie, Schaffhausen, Switzerland). Heatinactivated fetal calf serum (FCS), L-glutamine, trypsin/EDTA, penicillin G, and streptomycin solutions were purchased from Gibco (LuBioScience GmbH, Lucerne, Switzerland). All other chemicals used for the preparations of buffer solutions are from Merck (unless noted otherwise). Expression and Purification of FGF-2. Except for the ITC measurements shown in the Supporting Information, where human FGF-2 (hFGF-2) was utilized, all the experiments described in this paper were performed with the recombinant murine FGF-2. The nomenclature nat/111In-FGF-2 or unlabeled FGF-2 related to the experiments refers always to murine FGF-2, while human FGF-2 has been specified with hFGF-2. Murine FGF-2 (mFGF-2) was expressed in Escherichia coli JM109 DE3 and purified as previously described.23,24 Concentration of FGF-2 was determined by UV absorbance at λ = 280 nm, using a molar extinction coefficient of 15930 M−1/cm. This value was calculated from the mFGF-2 amino acid sequence using the ProtParam tool on the ExPASy portal (http://web.expasy.org). Purified fractions, containing FGF-2, were lyophilized in phosphate buffered saline (pH 7.4) supplemented with 3 mM DTT as antioxidative agent and 20% (w/v) sucrose before storage at −80 °C. Conjugation of DTPA to FGF-2. For the conjugation, mFGF-2 was reconstituted in TraceSELECT water and transferred (Amicon Ultra, 10 kDa cutoff filter) to 0.5 M ammonium acetate solution pH 7. Maleimide DTPA was dissolved in 0.5 M ammonium acetate solution pH 7 and directly added to the FGF-2 solution (protein-to-chelator ratio 1:2.2). The reaction mixture was incubated for 1 h at 37 °C, under gentle agitation. After the sample was rebuffered in 0.5 M B


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0.91% (w/v) nonessential amino acids) and incubated at 37 °C and 5% CO2 in humidified atmosphere. After 24 h, the growth medium was exchanged against assay medium (DMEM supplemented with 0.5% FCS, 0.52% (w/v) BSA, 1.82 mM Lglutamine, 91 U/mL penicillin G, 88 μg/μL streptomycin, and 0.91% (w/v) nonessential amino acids). After a further 24 h, the cells were incubated with the wild type FGF-2 or natIn-FGF2 (0.1−200 ng/mL in 200 μL of assay medium/well). Cells incubated without FGF-2 supplementation were the control group. 92 h postseeding, 50 μL of MTT staining solution (5 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C and 5% CO2. The medium was carefully aspirated before addition of 100 μL of dimethyl sulfoxide to solubilize the formed formazan crystals. The plates were incubated in the dark at room temperature for 90 min. Finally, the absorbance was determined at a wavelength of λ = 570 nm on a Spectramax 250 microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). To quantify the cell viability, the absorbance of the samples was normalized to the absorbance of the control group (0% proliferation). The viability of the cells compared to the control group was plotted as a percentage versus the protein concentration. The data were analyzed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). In Vivo Experiments. All animal experiments were approved by the cantonal authorities for animal experiments (permission number 75531). All the experiments were conducted in accordance with the Swiss laws of animal protection. For the biodistribution study, 5-week old female healthy CD1 nu/nu mice (Charles River Laboratories, Sulzfeld, Germany) were injected into the tail vein with 150 kBq 111 In-FGF-2 (35 pmol, 100 μL of PBS pH 7.4). Groups of 4 mice were sacrificed at 1, 4, 24, and 48 h postinjection (p.i.). For the blocking experiment, an additional group of mice was utilized (n = 4). This experiment was performed at 4 h p.i. by the injection of an excess of unlabeled DTPA-FGF-2 (5.7 nmol and 0.57 nmol, respectively, in 100 μL of PBS) directly before the administration of the radiolabeled derivative. Selected organs and tissues (blood, heart, lung, spleen, kidneys, pancreas, stomach, intestines, liver, muscle, and bone) were collected, weighed, and counted for activity by γ-counter (Packard Cobra II Auto, gamma, PerkinElmer AG, Schwerzenbach, Switzerland). The measured outcome was expressed as a percentage of injected activity normalized per the weight of the corresponding organ (% IA/g tissue), permitting comparisons between the different organs. SPECT/CT experiments were performed using a 4-head multiplexing multipinhole camera (NanoSPECT/CTplus, Bioscan Inc., Poway, CA, USA). A 5-week-old female healthy CD1 nu/nu mouse (Charles River Laboratories, Sulzfeld, Germany) was intravenously (tail vein) injected with 17 MBq 111In-FGF-2 (approximately 3.9 nmol, 100 μL of PBS). CT scans were performed with a tube voltage of 45 kV and a tube current of 145 μA. SPECT scan at 4 h postinjection was obtained with an acquisition time of 20−90 s per view, resulting in a total scanning time of 20−45 min. SPECT images were reconstructed using HiSPECT software (Scivis GmbH, Gö ttingen, Germany). The images were processed with InVivoScope software (Bioscan Inc., Poway, CA, USA). In Vitro Autoradiography. For autoradiography studies, a 5-week old CD1 nu/nu mouse (Charles River Laboratories, Sulzfeld, Germany) was euthanized. Liver and kidneys were immediately removed and embedded in TissueTek O.C.T.

serum, 1% L-glutamine, 100 U/mL penicillin G, and 100 μg/ mL streptomycin. The cells were cultured as monolayers and harvested by trypsinization with trypsin/EDTA. The cells were seeded in 6-well plates (1 × 106 cells/well) to grow overnight for the saturation binding assay. A constant amount of 2.5 kBq 111 In-FGF-2 (0.5 pmol) was added to the cells, which permits the proper signal for the analysis by γ-counter. Total binding was determined by the addition of an increasing amount of nat In-FGF-2 to the cells directly after the addition of 111In-FGF2 to reach the final FGF-2 concentrations of 0.5−700 nM/well. In this way, the amount of γ radiation could be minimized. For each nat/111In-FGF-2 concentration, triplicates were made. The nonspecific binding was analyzed blocking the binding sites by the simultaneously addition of the unlabeled FGF-2 diluted in 0.1% bovine serum albumin in PBS, pH 7.4 (final concentration 0.8 μM/well). The influence of enoxaparin on the binding of 111 In-FGF-2 was analyzed by adding an increasing amount of enoxaparin (0.01−1000 nM) together with 2.5 kBq 111In-FGF2 (0.5 pmol) to the cells. The cells were incubated with the growth factor and enoxaparin for 2 h at 4 °C under gentle stirring. At the end, the cells were washed twice with 1× PBS pH 7.4 and lysed by the addition of 1 M NaOH. The cells were collected in RIA tubes, and the activity was estimated by γcounter (Packard Cobra II Auto, gamma, PerkinElmer AG, Schwerzenbach, Switzerland). The data were analyzed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). Internalization Assay. The internalization kinetics of 111InFGF-2 was determined using NIH3T3 cells. The cells were cultured as monolayers and harvested by trypsinization with trypsin/EDTA. The cells were seeded in 6-well plates (1 × 106 cells/well) to grow overnight for the internalization assay. A constant amount of 2.5 kBq 111In-FGF-2 (0.4 pmol and 1 pmol, respectively) was added to the cells, which permits the proper signal for the analysis by γ-counter. Directly afterward, the binding sites were blocked by the addition of unlabeled FGF-2 diluted in 0.1% bovine serum albumin in PBS, pH 7.4 (final concentration 0.8 μM/well), to determine the nonspecific binding. To investigate the influence of enoxaparin on the internalization and the binding of 111In-FGF-2, an increasing amount of enoxaparin was added to the corresponding cells (0−1000 nM/well), each assessed in triplicate. The cells were incubated with the growth factor and enoxaparin for 2 h at 37 °C. After incubation, the supernatant was aspirated and the wells were washed with 1 mL of PBS. Both the supernatant and wash fractions were pooled and used to determine the unbound fraction. In order to collect the surface-bound fraction, the cells were incubated at room temperature with 1 mL of glycine buffer pH 2.6 for 5 min. At the end, the cells were washed twice and lysed by the addition of 1 M NaOH; this fraction corresponded to the internalized fraction. All three samples were separately collected in RIA tubes, and the activity was estimated by γ-counter (Packard Cobra II Auto, gamma, PerkinElmer AG, Schwerzenbach, Switzerland). The data were analyzed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). MTT Proliferation Assay. Bioactivity of FGF-2 and natInFGF-2 was determined in NIH 3T3 cells by means of a colorimetric proliferation assay (MTT assay) as described before.23 After trypsinization, the NIH3T3 cells were harvested and seeded in 96-well tissue culture plate in cell growth medium (DMEM supplemented with 10% FCS, 1.82 mM Lglutamine, 91 U/mL penicillin G, 88 μg/μL streptomycin, C


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Figure 1. (A) Schematic representation of the conjugation of one maleimide DTPA moiety to one FGF-2 molecule. The specific thiol−maleimide chemistry reaction permits a residue-directed radiolabeling of FGF-2. (B) The mass spectra show the conjugation of two chelators to FGF-2: mFGF2 (Mw = 17667 Da), FGF-2 coupled with two maleimide DTPA molecules (Mw = 18697 Da), and FGF-2 chelated with natIn (Mw = 18921 Da). (C) The radiolabeling quality control performed by RP-HPLC showed a labeling yield of 98% with a specific activity for 111In-FGF-2 of 5 MBq/ nmol.

Figure 2. ITC analysis of FGF-2 (A) and natIn-FGF-2 (B). The measurements were performed at 25 °C. For FGF-2 the extrapolated Kd was 0.60 ± 0.07 μM, while for DTPA-FGF-2 a Kd value of 0.33 ± 0.03 μM was obtained. The N value (stoichiometry of the binding reaction) was not altered between the two FGF-2 derivatives showing a ligand to protein ratio of 0.29 and 0.24, respectively. This indicates that one molecule of enoxaparin bound to approximately three to four molecules of FGF-2 and DTPA-FGF-2, respectively. (C) Saturation binding curve for the binding of nat/111InFGF-2 to fibroblast cells. The estimated Kd value was 103.8 ± 8 nM. R2 = 0.99. (D) By the exogenous addition of low-molecular-weight heparin (enoxaparin) a competition binding curve was obtained. The IC50 was 17.95 ± 0.65 nM. R2 = 0.99.

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Figure 3. (A, B) Internalization assay performed with NIH3T3 cells. The cells were incubated with the growth factor and enoxaparin for 2 h at 37 °C. The percentage of the membrane-bound fraction is represented in black and the internalized fraction in gray (mean ± standard deviation, n = 3). ** indicates p-value 95% homology (amino acid sequences listed in the

In-FGF-2, mean % to unblocked group ± SD (n = 4), blocking at 4 h p.i.

5.7 nmol of DTPA-FGF-2 149.28 164.31 120.59 70.10 169.43 231.81 186.45 341.98 102.31 92.83 84.05 54.47 258.63 163.50

± ± ± ± ± ± ± ± ± ± ± ± ± ±

21.04** 40.10* 79.46 14.03* 17.73** 65.80* 51.29* 69.92** 4.87 43.55 24.96 12.09** 68.90* 31.32*

0.57 nmol of DTPA-FGF-2 94.25 104.97 83.35 56.85 83.35 96.99 52.97 116.44 78.67 93.91 85.59 44.46 105.16 64.42

± ± ± ± ± ± ± ± ± ± ± ± ± ±

22.28 32.72 20.19 17.44** 26.09 32.41 29.20 39.41 16.16 43.83 32.31 7.39** 26.58b 39.19

a

The influence of the preinjection of a 160-fold and a 16-fold higher amount of DTPA-FGF-2, respectively, on 111In-FGF-2 uptake was analyzed 4 h p.i. The uptake values were normalized to the mouse group that received only the radiotracer and represented as a percentage (%). Significance: *p < 0.05, **p < 0.01. bn = 3 mice.

Figure 5. SPECT/CT images of a CD1 nu/nu healthy mouse. The in vivo scan was performed 4 h after the injection of 17 MBq 111In-FGF-2 (3.9 nmol). The majority of the activity was detected in liver and kidneys, while marginal uptake was localized in the retina, salivary glands, and pituitary glands. These results correlate with the ex vivo data.

autoradiography (Figure 6). In particular, we aimed to analyze if the binding of exogenously administered 111In-FGF-2 is matrix- or receptor-modulated.32 The experiment was performed with cryosections obtained from a healthy mouse. Tissue sections were incubated with 10 nM 111In-FGF-2 (1 kBq) for 2 h on ice. Furthermore, the influence of heparin on 111 In-FGF-2 binding was analyzed. The addition of enoxaparin (50 nM) resulted in a complete displacement of 111In-FGF-2 in the tissue. This effect was dosedependent and was stronger than the effect of addition of a 100-fold higher amount of unlabeled FGF-2 (1 μM).

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DISCUSSION Radiolabeled proteins are widely used in biological research, and radioiodination with 125I is a routinely applied technique for in vitro and in vivo biological investigations, since tracers with a high specific activity can be produced and the gammarays can be directly detected by a scintillation counter.15,16 The biological data on FGF-2 obtained from the current literature G


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Figure 6. In vitro autoradiography with cryosections of kidney (A) and liver (B). The specificity of 111In- FGF-2 binding was analyzed in the presence of heparin. The blocking of the binding sites was performed by the addition of 1 μM FGF-2 before the experiment.

and in the liver (Figure 5). Small amounts of the tracer were detected in the pituitary gland, in the salivary glands, and in the retina, which are known to express high levels of FGF receptors.32,41 This outcome was confirmed by the biodistribution data obtained, in which 111In-FGF-2 exerted a fast clearance from the blood with a rapid accumulation in both the liver and the kidneys with a maximum at 4 h p.i. (Figure 4). Beyond this, a relevant amount of radioactivity in the adrenal glands, in the spleen, and in the retina was detected. The fast clearance of intravenously administered 125I-FGF-2 and its rapid accumulation in these organs has also been described in various publications before.32,41−45 However, it remains to be clarified to what extent the distribution of exogenously administered 111In-FGF-2 is matrix- or receptor-specific. The preinjection of a 160-fold higher amount of unlabeled DTPA-FGF-2 significantly decreased the uptake of 111In-FGF-2 in the spleen (p < 0.05) and the adrenal glands (p < 0.01), suggesting a specific binding in these organs. A significant increase in the accumulation of the radiotracer was observed in different organs like the kidneys, the retina, the salivary glands, and the intestines (Table 1). The accumulation in the salivary glands was visible by SPECT analysis, in which an equal amount of 111In-FGF-2 was injected (Figure 5). Proangiogenic molecules such as FGF-2 exert biological effects including vasodilatation and increased vessels’ permeability, in particular in small capillaries.46,47 According to this, a 10-fold lower amount of DTPA-FGF-2 was preinjected before 111In-FGF-2, to evaluate the influence of higher tracer concentrations on the organism and, thus, on the experiment. Preinjection of DTPAFGF-2 caused a significant decrease of 111In-FGF-2 activity in the adrenal glands and in the spleen (p < 0.01), while the uptake was in the same range for all the other organs (Table 1). This outcome supported the hypothesis of a biological influence of 111In-FGF-2 on the vasculature at higher concentrations and confirmed the specificity of the binding in the spleen and the adrenal glands. On the other hand, the systemically increased uptake of 111In-FGF-2 in the organs after the administration of DTPA-FGF-2 is likely to be related to the saturation of the binding sites in the blood (e.g., heparin) resulting in a higher amount of free circulating 111In-FGF-2

Supporting Information). In particular, the sequences involved in the binding to the FGF receptors and to the heparin are evolutionarily stable.36 Therefore, no relevant pharmacokinetic and pharmacological differences between the murine and the human variant are expected. The in vitro results obtained from the binding assays showed Kd values for the binding of 111In-FGF-2 to the cell-expressed HSPGs in line with previously reported studies performed with 125 I-FGF-2, in which the in vitro binding to the receptor, to the heparin chains, and to HSPGs was described.28,37−39 According to the ITC measurements, one molecule of enoxaparin binds to three or four molecules of FGF-2 and natIn-FGF-2, respectively, which is in agreement with published studies describing the binding properties of the wild-type FGF-2 to heparin (Figure 2).38 ITC measurements performed with the human FGF-2, DTPA-hFGF-2, and natIn-hFGF-2 revealed Kd values in the same range as for mFGF-2 and murine nat In-FGF-2 (Supporting Information, p 8). All together, these in vitro experiments indicated that the binding and the biological properties of mFGF-2 and hFGF-2 were not affected by the labeling procedure and confirm the robustness of the developed protocol. The internalization of 111In-FGF-2 into the fibroblasts as well as the inhibiting effect of enoxaparin directly depended on the radiotracer concentration (Figure 3A,B). The results suggested the modulation of 111In-FGF-2 bioavailability to the cell by the ECM (HSPGs) rather than by the FGF receptors, a hypothesis already postulated by other independent studies.13,40 More precisely, at a lower concentration (0.4 nM 111In-FGF-2), still 8% of 111In-FGF-2 was internalized by the cells, despite the addition of 1000 nM enoxaparin (Figure 3A). This indicated a binding to the receptor since the interaction between 111InFGF-2 and the cell was too strong to be disrupted by the addition of enoxaparin. In contrast, at higher 111In-FGF-2 concentrations, the addition of 500 nM enoxaparin completely displaced the tracer (Figure 3B), suggesting an interaction of the tracer with the membrane-bound HSPGs, maybe as a consequence of the saturated receptor. SPECT/CT imaging performed in healthy mice showed that the highest amount of the protein accumulated in the kidneys H


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Grünberg for valuable comments and suggestions. This research was supported by the Swiss Competence Center for Materials Science and Technology CCMX (Grant 0-21108-11).

and, thus, to an increased bioavailability in the organs. These results confirm on the one hand the comparable in vivo behavior of 111In-FGF-2 to the native protein, and, on the other hand, they represent the complexity of FGF-2 signaling previously observed by the in vitro internalization experiments. Finally, in vitro autoradiography experiments performed in kidney and liver sections suggested that the binding of 111InFGF-2 in these tissues prevalently depends rather on the interaction with the matrix than on the interaction with the receptors, since the addition of heparin displaced the tracer. This outcome is in line with previously conducted ex vivo experiments (Figure 6).32

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(1) Ornitz, D. M.; Itoh, N. The Fibroblast Growth Factor Signaling Pathway. Wiley Interdiscip. Rev. Dev. Biol. 2015, 4 (June), 215−266. (2) Facchiano, A.; Russo, K.; Facchiano, A. M.; De Marchis, F.; Facchiano, F.; Ribatti, D.; Aguzzi, M. S.; Capogrossi, M. C. Identification of a Novel Domain of Fibroblast Growth Factor 2 Controlling Its Angiogenic Properties. J. Biol. Chem. 2003, 278 (10), 8751−8760. (3) Nugent, M.; Iozzo, R. V. Fibroblast Growth Factor-2. Int. J. Biochem. Cell Biol. 2000, 32 (2), 115−120. (4) Itoh, N.; Ornitz, D. M. Fibroblast Growth Factors: From Molecular Evolution to Roles in Development, Metabolism and Disease. J. Biochem. 2011, 149 (2), 121−130. (5) Beenken, A.; Mohammadi, M. The FGF Family: Biology, Pathophysiology and Therapy. Nat. Rev. Drug Discovery 2009, 8 (3), 235−253. (6) Sonmez, A. B.; Castelnuovo, J. Applications of Basic Fibroblastic Growth Factor (FGF-2, bFGF) in Dentistry. Dent. Traumatol. 2014, 30 (2), 107−111. (7) Wenk, E.; Murphy, A. R.; Kaplan, D. L.; Meinel, L.; Merkle, H. P.; Uebersax, L. The Use of Sulfonated Silk Fibroin Derivatives to Control Binding, Delivery and Potency of FGF-2 in Tissue Regeneration. Biomaterials 2010, 31 (6), 1403−1413. (8) Kardami, E.; Detillieux, K.; Ma, X.; Jiang, Z.; Santiago, J.-J.; Jimenez, S. K.; Cattini, P. A. Fibroblast Growth Factor-2 and Cardioprotection. Heart Failure Rev. 2007, 12 (3−4), 267−277. (9) Soulet, F.; Bailly, K.; Roga, S.; Lavigne, A.-C.; Amalric, F.; Bouche, G. Exogenously Added Fibroblast Growth Factor 2 (FGF-2) to NIH3T3 Cells Interacts with Nuclear Ribosomal S6 Kinase 2 (RSK2) in a Cell Cycle-Dependent Manner. J. Biol. Chem. 2005, 280 (27), 25604−25610. (10) Cross, M.; Claesson-Welsh, L. FGF and VEGF Function in Angiogenesis: Signalling Pathways, Biological Responses and Therapeutic Inhibition. Trends Pharmacol. Sci. 2001, 22 (4), 201−207. (11) Abuharbeid, S.; Czubayko, F.; Aigner, A. The Fibroblast Growth Factor-Binding Protein FGF-BP. Int. J. Biochem. Cell Biol. 2006, 38 (9), 1463−1468. (12) Padera, R.; Venkataraman, G.; Berry, D.; Godavarti, R.; Sasisekharan, R. FGF-2/fibroblast Growth Factor Receptor/heparinlike Glycosaminoglycan Interactions: A Compensation Model for FGF-2 Signaling. FASEB J. 1999, 13 (13), 1677−1687. (13) Beenken, A.; Mohammadi, M. The FGF Family: Biology, Pathophysiology and Therapy. Nat. Rev. Drug Discovery 2009, 8 (3), 235−253. (14) Dailey, L.; Ambrosetti, D.; Mansukhani, A.; Basilico, C. Mechanisms Underlying Differential Responses to FGF Signaling. Cytokine Growth Factor Rev. 2005, 16 (2), 233−247. (15) Caplan, S.; Baniyash, M. Radioiodination of Cellular Proteins. In Current Protocols in Cell Biology; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2002; Chapter 7, Unit 7.10. (16) Bailey, G. S. Labeling of Peptides and Proteins by Radioiodination. In Basic Protein and Peptide Protocols; Humana Press: NJ, 1994; Vol. 32, pp 441−448. (17) Saha, G. B. Fundamentals of Nuclear Pharmacy; Springer New York: New York, NY, 2010. (18) Wållberg, H.; Ståhl, S. Design and Evaluation of Radiolabeled Tracers for Tumor Imaging. Biotechnol. Appl. Biochem. 2013, 60 (4), 365−383. (19) Brechbiel, M. W. Bifunctional Chelates for Metal Nuclides. Q. J. Nucl. Med. Mol. Imaging 2008, 52 (2), 166−173. (20) Fani, M.; Maecke, H. R. Radiopharmaceutical Development of Radiolabelled Peptides. Eur. J. Nucl. Med. Mol. Imaging 2012, 39 (S1), 11−30.

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CONCLUSION In conclusion, we described a novel bioactive 111In-radiolabeled FGF-2 derivative suitable for in vivo molecular SPECT imaging. The facile radiolabeling strategy presented herein may be of general relevance for the FGF superfamily and allows evaluating the in vivo distribution and the pharmacokinetic parameters of FGF-2 in healthy mice, which opens exciting future applications. On the one hand, SPECT analysis with 111InFGF-2 could be performed for the elucidation of in vivo regulatory mechanisms of FGF-2 signaling, in molecular research. On the other hand, the utilization of 111In-FGF-2 could be extended to in vivo analysis of different FGF-2 containing pharmaceutical formulations in developmental or preclinical stages.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00913. Protein digestion and LC−MS analysis, binding assay study, study of 111In-FGF-2 behavior in blood plasma, internalization experiment, and ITC measurements (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E- mail: [email protected]. Phone: +41-56 310 28 17. Fax: +41-56 310 28 49. ORCID

Lorenz Meinel: 0000-0002-7549-7627 Martin Béhé: 0000-0002-1110-2665 Present Address ⊥

M.H.: RICB, Chemical Biology, Basel, Switzerland.

Author Contributions

A.M. and M.B. designed the research; T.L., L.M., E.F., and R.S. were involved in the design and in the analysis of the experiments; A.M., G.J., and S.W. performed the experiments; M.H. and A.B. were involved in the experiments; A.M. and M.B. analyzed the data; A.M. and M.B. wrote the paper; and L.M., T.L., M.B., and R.S. revised the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Olga Gasser and Christine de Pasquale for technical assistance and Prof. Michel O. Steinmetz, Prof. Viola Vogel, Prof. Sabine Werner, and Dr. Jürgen I


Article

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