Supplementary Material
Improved PET Imaging of Tumors in Mice Using a Novel
18F-Folate
Conjugate with an Albumin-Binding Entity
Cindy R. Fischer1, Viola Groehn2, Josefine Reber3, Roger Schibli1,3, Simon M.
Ametamey1, Cristina Müller3,*
1
Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, Institute of Pharmaceutical
Sciences, ETH Zurich, 8093 Zurich, Switzerland
2
Merck & Cie, 8200 Schaffhausen, Switzerland
3
Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, Paul Scherrer Institute, 5232
Villigen-PSI, Switzerland
S1
HPLC Purification of Albumin-Binding [19F]FDG-Folate (3)
Albumin-binding [19F]FDG-folate (3) was purified by semi-preparative HPLC using a ACE-C18-HL
column (150 × 4.6 mm). A solution of 50 mM NH4HCO3 (solvent A) and methanol (solvent B) were
used as eluents with a gradient from 90 % A and 10 % B to 20 % A and 80 % B in 8 min at a flow rate
of 1 ml/min. The product peak appeared with a retention time of 6.6 min.
Production of [18F]Fluoride
No-carrier-added [18F]fluoride was produced via the
cyclotron (IBA) by irradiation of enriched
18
O(p,n)18F nuclear reaction at a Cyclone 18/9
18
O-water. [18F]-fluoride was immobilized on an anion-
exchange cartridge (QMA Light; Waters; preconditioned with 0.5 M K 2CO3-solution (5 ml) and H2O
(5−10 ml)) and eluted with a solution of Kryptofix K2.2.2 (5 mg) and K2CO3 (1 mg) in acetonitrile
(1.4 ml) and water (0.6 ml) into a 10 ml sealed reaction vessel. The [18F]fluoride (60 to 65 GBq) was
dried by azeotropic distillation of acetonitrile at 110 °C under vacuum with a stream of nitrogen. The
azeotropic drying process was repeated three times with 1 ml of acetonitrile.
HPLC Purification of Albumin-Binding [18F]FDG-Folate ([18F]3)
Semi-preparative radio-HPLC of [18F]3 was performed with a Gemini column (C18, 5 µm, 250 ×
10 mm, Phenomenex) at a flow rate of 3 ml/min. An aqueous solution of 50 mM NH4HCO3 (solvent
A) and acetonitrile (solvent B) were used as a solvent system using an elution program which started
with 80 % A and 20 % B (0 - 20 min) followed by a gradient from 80-30 % A and 20-70 % B (20 25 min) and ending with 30 % A and 70 % B (25 - 35 min). The product peak appeared with a
retention time of 19.3 min.
Cartridge Purification of Albumin-Binding [18F]FDG-Folate ([18F]3)
The HPLC-purified product fraction of [18F]3 in water (20 ml) was passed through a Sep-Pak column
(Sep-Pak C18 light; Waters; preconditioned with EtOH (5 ml) and H2O (5 ml)) and washed with water
(10 ml). The product [18F]3 was eluted with ethanol (1.0 ml) followed by evaporation at 80 °C under
vacuum.
S2
Analytical HPLC of Albumin-Binding [18F]FDG-Folate ([18F]3)
Analytical radio-HPLC of [18F]3 was performed with a reversed-phase column (Gemini C18, 5 μm,
4.6 × 250 mm, Phenomenex) using an aqueous solution of 50 mM NH4HCO3 (solvent A) and
acetonitrile (solvent B) as eluents. The elution program started with 80 % A and 20 % B (0 - 15 min)
followed by a gradient from 80-30 % A and 20-70 % B (15 - 20 min) and ended with 30 % A and
70 % B (20 - 25 min) at a flow rate of 1 ml/min. The product [18F]3 appeared with a retention time of
13.3 min and a radiochemical purity of 98 % (Fig. S1).
Fig. S1. Analytical radio-HPLC chromatogram of the albumin-binding [18F]FDG-folate ([18F]3).
S3
Determination of the Specific Activity
Specific radioactivity of the product [18F]3 was determined by analytical radio-HPLC from a
calibration curve obtained from different concentrations of the corresponding non-radioactive
reference compound (3) (Fig. S2).
Fig. S2. Calibration curve of the UV absorption (area under the curve) obtained with different
concentrations of compound 3 allowing determination of the specific activity of the albuminbinding [18F]FDG-folate ([18F]3) using HPLC.
Determination of the Distribution Coefficient (LogD7.4)
The distribution coefficient (logD7.4) of [18F]3 was determined by the shake flask method according to
the procedure previously reported for [18F]fluorodeoxyglucose-folate [1]. In brief, [18F]3 was dissolved
in a mixture of phosphate buffer (500 μl, pH 7.4) and n-octanol (500 μl) at 20 °C. The samples were
equilibrated for 15 min in an overhead shaker. The two phases were separated by centrifugation
(3 min, 5000 rpm). Aliquots of 50 μl of each of the phases were analyzed in a γ-counter (Wizard,
PerkinElmer). The partition coefficient was expressed as the ratio between the radioactivity
concentrations (cpm/ml) of the n-octanol and the buffer phase. Values represent the mean ± standard
deviation of ten determinations from two independent experiments.
S4
Cell Culture
KB cells (human cervical carcinoma cell line, HeLa subclone: ACC-136) were purchased from the
German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The cells
were cultured as monolayers at 37 °C in a humidified atmosphere, containing 5 % CO2. Importantly,
the cells were cultured in a folate-free cell culture medium, FFRPMI (modified RPMI, without folic
acid, vitamin B12 and phenol red: Cell Culture Technologies GmbH, Gravesano/Lugano, Switzerland).
FFRPMI medium was supplemented with 10 % heat-inactivated fetal calf serum (FCS, as the only
source of folate), L-glutamine and antibiotics (penicillin/streptomycin/fungizone). Routine culture
treatment was performed twice a week with EDTA (2.5 mmol/l) in PBS.
In Vitro Cell Binding Affinity Assay
Cell binding affinity of the non-radioactive reference compound 3 was determined as previously
reported [1, 2]. In brief, triplicates of KB cell suspensions in phosphate buffered saline (PBS pH 7.4,
without, ice-cold, 7,000 cells in 240 µl) were incubated with [3H]-folic acid (10 l, 0.82 nM, 0.94
TBq/mmol, Moravek Biochemicals Inc.) and increasing concentrations of the non-radioactive
reference compound 3 or folic acid (5.0 × 10-6 to 5.0 × 10-12 M, 250 l) at 4 °C for 30 min. Nonspecific binding was determined in the presence of excess folic acid (10-3 M). After incubation, the
cell samples were centrifuged at 1000 rpm and 4 °C for 5 min and the supernatant was removed. The
cells were lysed by addition of 0.5 ml 1 N NaOH and transferred into scintillation tubes containing 5
ml scintillation cocktail (Ultima Gold; Perkin Elmer). Radioactivity was measured using a β-counter
(LS6500; Beckman), and the inhibitory concentrations of 50 % were determined from displacement
curves using GraphPad Prism 6.0 software. The relative binding affinities of non-radioactive reference
compound 3 was determined based on the binding affinity of folic acid which was set to 1.0. A value
equal to that of folic acid indicates an equal affinity for the FR, a value lower than 1.0 reflects weaker
affinity and a value higher than 1.0 reflects stronger affinity [3, 4].
S5
Cell Uptake and Internalization Studies
Cell uptake and internalization experiments were performed as previously described in the literature
for other folate radiotracers [5, 6]. In brief, KB cells were seeded in 12-well plates to grow over night
(~700,000 cells in 2 ml FFRPMI medium/well). The radiotracer [18F]3 (200 kBq per well) was added
to the cell monolayers. To determine the FR-specific binding of [18F]3 excess folic acid was
co-incubated to block FRs on the surface of KB cells. After incubation at 37 °C for 2 h, cells were
washed three times with PBS to determine total radiotracer uptake. In order to assess the fraction of
radiotracer which were internalized, KB cells were additionally washed with a stripping buffer
(aqueous solution of 0.1 M acetic acid and 0.15 M NaCl, pH 3 [7]) to release FR-bound radiotracer
from the cell surface. Cell lysis was accomplished by addition of aqueous 1 N NaOH (1 ml) to each
well. The cell suspensions were transferred to 4 ml-tubes for measuring in a -counter. In order to
standardize the measured radioactivity, protein concentration was averaged to a content of 0.3 mg
protein per well using a Micro BCA Protein Assay kit (Pierce, Thermo Scientific). The uptake and
internalized fraction of [18F]3 were calculated as percentage of total added radioactivity.
S6
References
1.
Fischer CR, Müller C, Reber J, et al. (2012) [18F]Fluoro-deoxy-glucose folate: a novel PET
radiotracer with improved in vivo properties for folate receptor targeting. Bioconjug Chem
23:805-813.
2.
Reber J, Struthers H, Betzel T, Hohn A, Schibli R, Müller C (2012) Radioiodinated folic acid
conjugates: evaluation of a valuable concept to improve tumor-to-background contrast. Mol
Pharm 9:1213-1221.
3.
Leamon CP, Parker MA, Vlahov IR, et al. (2002) Synthesis and biological evaluation of EC20:
a new folate- derived, 99mTc-based radiopharmaceutical. Bioconjug Chem 13:1200-1210.
4.
Reddy JA, Xu LC, Parker N, Vetzel M, Leamon CP (2004) Preclinical evaluation of
99m
Tc-
EC20 for imaging folate receptor-positive tumors. J Nucl Med 45:857-866.
5.
Müller C, Mindt TL, de Jong M, Schibli R (2009) Evaluation of a novel radiofolate in tumourbearing mice: promising prospects for folate-based radionuclide therapy. Eur J Nucl Med Mol
Imaging 36:938-946.
6.
Betzel T, Müller C, Groehn V, et al. (2013) Radiosynthesis and preclinical evaluation of 3'-aza2'-[18F]fluorofolic acid: a novel PET radiotracer for folate receptor targeting. Bioconjug Chem
24:205-214.
7.
Ladino CA, Chari RVJ, Bourret LA, Kedersha NL, Goldmacher VS (1997) Folatemaytansinoids: target-selective drugs of low molecular weight. Int J Cancer 73:859-864.
S7