Article pubs.acs.org/jmc
Folic Acid-Conjugated Europium Complexes as Luminescent Probes for Selective Targeting of Cancer Cells Silvio Quici,*,† Alessandro Casoni,† Francesca Foschi,† Lidia Armelao,‡ Gregorio Bottaro,‡ Roberta Seraglia,§ Cristina Bolzati,§,∥ Nicola Salvarese,⊥ Debora Carpanese,⊥ and Antonio Rosato⊥,▽ †
Istituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche, Via C. Golgi 19, 20133 Milano, Italy Istituto per l’Energetica e le Interfasi, Consiglio Nazionale delle Ricerche, e Consorzio Interuniversitario per la Scienza e Tecnologia dei Materiali, Dipartimento di Scienze Chimiche, Università di Padova, Via Marzolo 1, 35131 Padova, Italy § Istituto per l’Energetica e le Interfasi, Consiglio Nazionale delle Ricerche, Corso Stati Uniti 4, 35127 Padova, Italy ∥ Dipartimento di Scienze Farmaceutiche, Università di Padova, Via Marzolo 5, 35131 Padova, Italy ⊥ Dipartimento di Scienze Chirurgiche Oncologiche e Gastroenterologiche, Università di Padova, Via Giustiniani 2, 35128 Padova, Italy ▽ Istituto di Ricovero e Cura a Carattere Scientifico, Istituto Oncologico Veneto, Via Gattamelata 64, 35128 Padova, Italy ‡
S Supporting Information *
ABSTRACT: We report the synthesis of three optical probes (Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3) having a luminescent Eu complex (signaling unit) bonded in different positions to folic acid (FA), the folate receptor (FR) targeting unit. The structures of the two regioisomers Eu3+⊂1 and Eu3+⊂2 were assigned by mass spectrometric experiments. The optical properties and stability of these probes were assessed in phosphate-buffered saline, cell culture medium, rat serum, and cellular lysate, and results indicated that they are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells having high (SKOV-3), intermediate (OVCAR-3), low (IGROV-1), or null (A2780) expression of FRs. The internalized probe, evaluated in SKOV-3, IGROV-1, and A2780 cells, was in the order Eu3+⊂2 > Eu3+⊂1 > Eu3+⊂3. No internalization was observed for A2780 cells. Such results, together with those obtained in competition experiments of FA versus Eu3+⊂2 and FA or Eu3+⊂2 versus 3 H-FA, indicate that internalization is receptor-mediated and that Eu3+⊂2 shows high selectivity and specificity for FR.
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INTRODUCTION In modern medicine, and particularly in cancer, a pivotal role is played by theranostic systems containing a targeted therapeutic drug intimately coupled with an imaging moiety, which selectively accumulate within diseased cells and tissues and allow their monitoring before, during, and after therapy.1 The development of theranostics would not have been possible without the great advances made in the field of molecular imaging2 and in the synthesis of efficient targeting moieties.3 Over the years, several imaging techniques have been refined, including magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), and optical imaging (OI).4 Although the heterogeneity of tissues limits the transparency window of the human body and generally attenuates the signal intensity due to absorption and scattering by endogenous chromophores, fluorescence intensity imaging (FII) is ideally as sensitive as nuclear imaging, cost-effective, and widely available.5 Luminescence measurements show submicrometer spatial resolution and very fast (from nano- to millisecond) temporal resolution,6 making it possible to detect biological events at an early stage and © 2015 American Chemical Society
to more rapidly change therapeutic treatment of the disease on the basis of data obtained. For example, fluorescent imaging allows intraoperative detection of a tumor in real time, providing direct guidance for surgeons for an effective and complete resection of the tumor.7 Moreover, optical methods show their effectiveness when a simultaneous multispectral analysis of different probes during the same operation is required, a practice that would be impossible with other inherently monochromatic imaging methods.8 The use of luminescent complexes in cell imaging is particularly appealing, mostly because of the peculiar properties of luminescence emission from the metal excited states.9 In this context, a leading role is played by the luminescent lanthanide complexes (LLCs), which show a series of potential advantages: (1) narrow and intense bands that ensure high color purity, large Stokes shifts with no overlapping of excitation and emission spectra, and long emission lifetimes enabling time-resolved measurements; (2) increased loading of LLCs since they do not show luminescence self-quenching, which is a very strong drawback for Received: December 16, 2014 Published: January 20, 2015 2003
DOI: 10.1021/jm501945w J. Med. Chem. 2015, 58, 2003−2014
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Journal of Medicinal Chemistry organic dyes; (3) preparation of different LLCs from the same ligand so that, depending on the antenna, they can be excited by the same wavelength; (4) very high chemical, thermal, kinetic, and photophysical stability; and (5) very low toxicity compared with the corresponding Gd3+ complexes of diethylenetriaminetetraacetic acid (DTTA) and 1,4,7,10-tetraazacyclododecane1,4,7-triacetic acid (DO3A), widely used in vivo as contrast agents for MRI.10 A second aspect that must be addressed in development of tumor-specific imaging agents is represented by the choice of the most appropriate carrier. Compared to other molecular vectors such as proteins, DNA, monoclonal antibodies, and peptides, commonly used for selective drug and/or imaging agent delivery in tumor cells and diseased tissues, folic acid (FA) shows some peculiar features: low cost, high stability in a range of temperature and pH, compatibility with aqueous and organic solvents, and easy conjugation synthetic protocols.11 In addition, this nonimmunogenic target ligand possesses high binding affinity (Kd = 100 pM) with folate receptors (FRs), whose expression in physiological conditions is largely restricted to cells important for the embryonic development and folate resorption (kidney). FRs are a family of glycoproteins (35−40 kDa) divided into three subtypes: FRα and FRβ, which are linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors, and FRγ, which is freely soluble since it lacks the GPI fragment.12 Among these, the FRα isoform is the most widely expressed on cell membranes of many epithelial tumors, with very low expression in normal tissues, whereas FRβ is upregulated on activated macrophages that are responsible for inflammatory and autoimmune diseases.13 The mechanism of transport of FRs, although not completely elucidated, seems to involve the activation of protein by the FA (or folate-conjugated) interaction and subsequent internalization through receptor-mediated endocytosis.14 FA consists of three distinct portions, namely, a pterin moiety, a p-aminobenzoate linker, and a glutamate residue. This molecular structure contains at least five possible sites for functionalization, which are potentially useful for preparation of folate conjugates (Figure 1). Nowadays, the choice of the conjugation site able to retain optimal receptor binding properties is still under discussion. Almost all the papers in the literature report the functionalization of carboxyl groups of the FA glutamic acid residue by reaction with an amino derivative. This reaction affords a mixture of two regioisomers (α- and γ-substituted) that, in some cases, is used as such in the cellular uptake experiments, since separation of the pure isomers can be quite difficult to achieve. Although many authors state that FA must be substituted in the γ-position and that it is essential to leave the α-carboxyl group free to maintain high recognition selectivity,15 other authors claim that the functionalization position has no effect on the binding ability of FA conjugates and disprove the necessity of a free carboxyl group on the glutamate moiety for FR binding.16 Only recently has a study of the crystal structure of FA bound to human FR evidenced a preferential interaction of pterin headgroup with the receptor’s binding pocket.17 Over the years, many FA-based conjugates have been synthesized and tested either in vitro or in vivo. These include protein toxins,18 low molecular weight drugs,19 radiolabeled drugs for imaging20 and therapy,21 liposome-entrapped drugs,22 and nanoparticles.5 It must be pointed out that, once linked to FA, almost any type of cargo can be specifically driven inside diseased cells overexpressing FRs. Among these, low molecular
Figure 1. Chemical structures of folic acid (arrows indicate possible sites of conjugation) and of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3.
weight FA conjugates (95%), whereas OVCAR-3 and IGROV-1 expressed the receptor of interest with intermediate (about 50−60%) and low (about 15−20%) intensity, respectively. A2780 results were completely negative (see Supporting Information). To test whether the three complexes affected cell growth, cytotoxicity of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 was carefully investigated for all ovarian cancer cell lines by the PerkinElmer ATPLite test, which estimates the concentration of viable cells on the basis of bioluminescence generated by reaction of adenosine triphosphate (ATP) with luciferase and D-luciferin (see Supporting Information). Results showed 100% survival for the SKOV-3 cell line up to 30 μM Eu3+⊂1 and Eu3+⊂2. Cell survival decreased to 80% at 100 μM and to 70% at 300 μM. A slight increase in cytotoxicity was observed in the case of Eu3+⊂3, which gave 100% survival up to 10 μM, with percentages decreasing to 95% at 30 μM, 80% at 100 μM, and 65% at 300 μM. A2780 cell line, which does not express FRs, showed 100% survival at all concentrations studied (Figure 7). Results of cytotoxicity of the three probes with OVCAR-3 and IGROV-1 cell lines are reported in Supporting Information. Cellular Internalization of Complexes and Competition Studies. To assess cellular internalization of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 by receptor-positive and -negative cell lines,
X = 1, 2, 3, and 4, 10−5 M in water. bMeasured at 310 nm. cThree independent measurements were carried out on each complex with an estimated error of ±20%. dEstimated error ±10%. eNumber of water molecules in the first coordination shell estimated by the Horrocks formula: qEu = 1.11(1/τH2O − 1/τD2O − 0.31).34 a
Photophysical data of the various complexes are summarized in Table 1. The pristine Eu3+⊂4 has a photoluminescence quantum yields (PLQY) of 7% despite the presence of a water molecule in the first coordination sphere. As a consequence of conjugation with folic acid, lower values have been detected for the PLQY of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3. It is likely that partial overlap between the absorption spectra of acetophenone and folic acid could affect the PLQY, since during excitation less photons are available for lanthanide sensitization. Lifetimes and number of coordinated water molecules are not affected by the presence of FA. Luminescence properties of the europium folate complexes were studied in phosphate-buffered saline (PBS), RPMI cell culture medium, and rat serum (RS) to evaluate their emission behavior and possible spectral interference from these media prior to in vitro evaluation studies (Figure 5). In all the spectra, the typical europium emission bands are clearly visible between 570 and 710 nm, and the different environments do not affect their shape. The spectra differ at λ < 550 nm where broad emission bands are present. These bands, having higher intensity in RS, are likely due to the organic moiety present in both RS and RPMI medium. It is worth noting that one can get rid of all these bands by performing time-gated luminescence experiments. Indeed, their intensity vanishes if emission is recorded over a 50−100 μs delayed time window after excitation. Comparable behavior has been observed for Eu3+⊂1. Chemical and Photophysical Stability of Bioconjugated Complexes. The in vitro chemical and photophysical stability of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 were determined by HPLC, monitoring over time the chemical purity with variation of the concentration of compounds after incubation in PBS, RPMI cell culture medium, and RS. Samples withdrawn at different times were analyzed by HPLC, the peaks were integrated, and the absolute peak areas of the complex were used to assess its
Figure 5. Emission spectra of 30 μM (a) Eu3+⊂2 and (b) Eu3+⊂3 in PBS, RPMI cell culture medium, and rat serum, excited at 310 nm. 2008
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Figure 6. Emission spectra of Eu3+⊂2 at different incubation times at 37 °C in (a) RPMI and (b) RS. Irrespective of the medium, the concentration of the complexes is 30 μM.
of each of the three Eu complexes, in a folic acid-deficient medium. At each time point, surface-bound complexes were removed by washing with glycine buffer. Cells containing the internalized complex were then lysed with Triton 5% and fluorescence intensity was measured. Finally, results were plotted against incubation time, according to calibration curves generated to quantify the amount of internalized complexes. All the measurements were carried out in triplicate according to the detailed procedure reported in Supporting Information. The values of uptake obtained for Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 in SKOV-3 cells are reported in Figure 8. In SKOV-3 cells, the internalized concentrations strongly increased with increasing concentrations of probes and incubation time, being 3.5, 6.0, and 1.9 nM at 30 μM concentration and 24 h time point, which correspond to 2.1 × 106, 3.6 × 106, and 1.1 × 106 molecules/cell for Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3, respectively. A similar trend, although with lower values of cellular uptake (e.g., 0.7, 2.0, and 0.7 nM at 30 μM and 24 h for Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3, respectively) was obtained with IGROV-1 cells line under the same experimental conditions (see Supporting Information). These amounts were in the same range as found for other FA derivatives published in literature.24,35 No internalization was observed for A2780 cells that do not express FRs, thus indicating that internalization of the tested derivatives was receptormediated. It is interesting to note that, in both SKOV-3 and IGROV-1 cells, the amounts of internalization for Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 were in the same range, suggesting that modification at different positions of the FA structure does not seem to significantly affect recognition by FRα. To demonstrate that internalization of the luminescent probes was specifically due to the presence of FRs, uptake of Eu3+⊂2 by SKOV-3 cells was repeated under the same conditions reported above but in the presence of increasing amounts of free FA as a competing binding substrate. The results reported in Figure 9 clearly show that uptake of Eu3+⊂2 strongly decreased upon addition of increasing amounts of FA and was practically absent in the presence of FA concentrations about 30 times higher than Eu3+⊂2 concentration. Competition uptake experiments were also carried out in SKOV-3 cells with either FA or Eu3+⊂2 as competitive substrate for 3H-FA. In this case, the uptake of 3H-FA, measured in counts per minute (cpm) with the TopCount NXT apparatus, sharply decreased when cells were incubated in the presence of increasing amounts of folic acid or Eu3+⊂2. As reported in Figure 10, the decrease of 3H-FA binding was the same with either FA or Eu3+⊂2 as competitor substrate for FR, thus
Figure 7. In vitro viability of ovarian tumor cell lines in response to (top) Eu3+⊂1, (middle) Eu3+⊂2, and (bottom) Eu3+⊂3. A2780 (blue bars) and SKOV-3 (red bars) cell survival was evaluated by ATPlite assay. Reported values are the mean of three independent experiments, estimated error ±5%.
SKOV-3, IGROV-1, and A2780 ovarian cancer cells were incubated for 1, 3, 18, and 24 h with increasing concentrations 2009
DOI: 10.1021/jm501945w J. Med. Chem. 2015, 58, 2003−2014
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Figure 9. Competitive inhibition of Eu3+⊂2 binding to FR on SKOV-3 by FA. Results show the mean values of three independent experiments.
Figure 10. Comparison of abilities of Eu3+⊂2 and FA to inhibit 3H-FA binding to SKOV-3 cells. Results show the mean values of three independent experiments.
Figure 8. Internalization curves of (top) Eu3+⊂1, (middle) Eu3+⊂2, and (bottom) Eu3+⊂3 by receptor-positive SKOV-3. Graphs show the amount of complex internalized at different concentrations (30 μM, 1 μM, and 100 nM) and time points (1, 3, 18, and 24 h). Mean values of three independent experiments are reported. Figure 11. HPLC profiles of SKOV-3 cell lysate, (A) blank, (B) Eu3+⊂2 at 24 h after internalization, and(C) Eu3+⊂2 in RPMI-1640 cell culture medium, after 24 h of incubation with SKOV-3 cells.
indicating that Eu3+⊂2 and free FA display a fully overlapping ability to inhibit the binding of radioactive folate to SKOV-3. Finally, to investigate the effects of the uptake process on chemical integrity of the complexes, HPLC analysis was conducted on Eu3+⊂2 before and after cellular incubation and internalization, and chromatographic profiles of the compound remaining in the medium after cell incubation and of the internalized compound recovered from the cytosolic fraction were evaluated. Results clearly indicate that Eu3+⊂2 remains stable over time and after cell exposure and internalization (Figure 11). No evidence of free FA and other species originated by hydrolysis of the amide bond in Eu3+⊂2 was observed in the postinternalization HPLC profiles, even after a 24 h incubation. Indeed, HPLC analysis of the Triton solutions obtained after cell lysis and centrifugation revealed the presence of the Eu3+⊂2 in
unmodified form. Other peaks present in the UV traces were attributed, by assessment, to cellular components and/or RPMI cell culture medium.
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CONCLUSIONS In conclusion, we described the preparation of three new luminescent probes, Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3, that are highly specific for cancer cells that overexpress membrane folate receptors. These probes are characterized by a luminescent europium complex as the signaling unit, linked in three different positions to the structure of folic acid that behaves as the 2010
DOI: 10.1021/jm501945w J. Med. Chem. 2015, 58, 2003−2014
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performed on a 15 cm × 4.6 mm Ascentis C-18 column and, for semipreparative HPLC purification, a 15 cm × 21.2 mm Ascentis C-18 column. HPLC-grade acetonitrile was purchased from VWR International and used without further purification. HPLC aqueous solvent was prepared with milli-Q water (18.2 MΩ·cm ionic purity). Optical rotations were measured on a PerkinElmer 241 polarimeter at 589 nm, with a 10 cm × 5 mL cell and c is in grams per 100 mL. Fourier transform infrared (FTIR) spectra were recorded on an Agilent Cary 630 infrared spectrophotometer and are reported as frequency of absorption (cm−1). High-resolution mass spectra were obtained with an electrospray iontrap mass spectrometer, ICR-FTMS Apex II (Bruker Daltonics), by the Centro Interdipartimentale Grandi Apparecchiature (CIGA), University of Milano. Assignment of the structure of the two regioisomers Eu3+⊂1 and Eu3+⊂2 was obtained by use of a LCQ Deca ion-trap instrument (ThermoFisher Scientific, San Jose, CA), operating in positive-ion mode. The entrance capillary temperature was 280 °C and the capillary voltage was 5 kV. Eu3+⊂1 and Eu3+⊂2 were first dissolved in Milli-Q water at a concentration of 1 mg/mL and then diluted 1:100 in water/methanol (50/50 v/v), containing 1% HCOOH. Water/ methanol solutions of each compound were introduced into ESI ion source by direct infusion at a flow rate of 8 μL/min. The He pressure inside the trap was kept constant. The pressure directly read by ion gauge (in the absence of the N2 stream) was 2.8 × 10−5 Torr. Tandem mass spectrometric (MS/MS) experiments were performed by resonant excitation of the ion of interest through a supplementary radio frequency (rf) voltage in the range 30−35% of its maximum value (5 V peak to peak). Considering that Eu has two isotopes (151Eu and 153Eu with relative abundance of 47.77% and 52.23%, respectively), as clearly shown in Figure S3 in Supporting Information, the ion chosen for the MS/MS experiments was that at m/z 1109, which is the monoisotopic one, containing only 12C, 14N, 1H, 16O, and 151Eu. Synthesis of Eu3+⊂1 and Eu3+⊂2 Complexes. To a stirred solution of folic acid (220 mg, 0.5 mmol) in 16 mL of anhydrous DMSO and 4 mL of pyridine over KOH were added the 10-[4-(3aminopropyloxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane1,4,7-triacetic acid europium complex (Eu3+⊂4)26 (378 mg, 0.55 mmol) and dicyclohexylcarbodiimide (DCC) (258 mg, 1.25 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was magnetically stirred at room temperature for 16 h, and the resulting solid was filtered through a glass frit. The filtrate was slowly dripped into wellstirred Et2O (150 mL), and the solid precipitate was filtered, washed four times with Et2O (10 mL) to remove traces of DMSO, and dried under vacuum until constant weight to afford 620 mg of pale yellow solid. Purification was carried out by semipreparative HPLC on an Ascentis C18 column (5 μm, 21.2 × 150 mm) by using as eluent a mixture of solvents [solvent A = H2O (with 0.1% trifluoroacetic acid, TFA) and solvent B = CH3CN (with 0.1% TFA), starting from 92% A/8% B to 86% A/14% B in 20 min, flow rate = 12 mL/min, T = 25 °C, UV detector λ = 310 nm] and afforded, after lyophilization, 100 mg of Eu3+⊂1 (18%) and 210 mg of Eu3+⊂2 (37.8%) (>95% purity by HPLC analysis), both as light yellow solids. Eu3+⊂1: [α]20 D −0.6 (c 0.4 H2O); IR (KBr) 3414.5, 3229.4, 1682.8, 1616.5, 1400.1, 1179.6 cm−1. Eu3+⊂2: [α]20 D −3.7 (c 0.4 H2O); IR (KBr) 3411.3, 3124.0, 1678.0, 1638.4, 1603.5, 1400.2, 1198.7 cm−1. Photophysical Measurements. Absorption spectra in the UV−vis region were obtained from a double-beam Cary 5E spectrophotometer with a spectral bandwidth of 1 nm. Luminescence spectra were recorded at room temperature with a Fluorolog-3 (Horiba Jobin Yvon) spectrofluorometer equipped with double-grating monochromator on both the excitation and emission sides coupled to a R928P Hamamatsu photomultiplier and a 450 W Xe arc lamp as the excitation source. The emission spectra were corrected for detection and optical spectral response of the spectrofluorometer through a calibration curve supplied by the manufacturer. The excitation spectra (PLE) were corrected for the spectral distribution of lamp intensity by use of a photodiode reference detector. Time-gated spectra have been acquired with the same instrument employing a pulsed Xe lamp as source. Absolute photoluminescence quantum yields have been calculated by corrected emission spectra obtained from an apparatus consisting of a Spectralon coated integrating sphere accessory (4″, F-3018, Horiba Jobin Yvon),
targeting unit. All compounds were carefully purified and underwent a complete chemical and photophysical characterization in different media: water, PBS, RPMI cell culture medium, and rat serum. In vitro studies have delivered important information regarding chemical and photophysical stability as well as biochemical properties of these compounds. In particular, these complexes turned out to be very stable in all conditions investigated, and no biotransformation and no significant interaction with serum protein occurred. HPLC profiles of the complexes collected after serum incubation perfectly matched those of the native compounds. Spectroscopic analysis of the three new luminescent probes Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 was also performed to determine the luminescence properties of the europium folate complexes before and after media incubation. The collected data clearly demonstrated that the probes remain intact and the fluorescence signal was retained. Cytotoxicity of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 was tested for SKOV-3, OVCAR-3, IGROV-1, and A2780 human ovarian cancer cells featuring high, medium, low, and null expression of folate receptors, respectively. Results indicate that cell viability was not affected unless extremely high concentrations of the compounds were reached. Cellular uptake of these probes was strictly receptordependent, being critically correlated to FR expression. Moreover, it was a function of conjugation site, as observed in SKOV-3 cells where the binding activity follows the order Eu3+⊂2 > Eu3+⊂1 > Eu3+⊂3. Notably, the results of competition experiments in the presence of FA or 3H-FA showed selectivity and specificity of Eu3+⊂2 probe for the FR comparable to that of FA, thus suggesting that the conjugated moiety did not likely affect FA binding properties. Finally, Eu3+⊂2 proved to be chemically and photophysically stable after the internalization process.
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EXPERIMENTAL SECTION
Materials and Methods. All available chemicals and solvents were purchased from commercial sources and were used without any further purification. Glycine, hydrochloric acid, Triton X-100, and folic acid were purchased from Sigma−Aldrich (St. Louis, MO). 3H-Folic acid (14.3 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). SKOV-3, OVCAR-3, IGROV-1, and A2780 human ovarian cancer cell lines were cultured in RPMI 1640 medium without folic acid (Life Technologies) supplemented with 2 mM L-glutamine (Gibco BRL), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; PAA Laboratories, Linz, Austria), 150 units/mL streptomycin (BristolMyers Squibb Italia, Rome, Italy), 200 units/mL penicillin (Pharmacia and Upjohn, Milan, Italy) and 10% (v/v) heat-inactivated fetal calf serum (FCS; Gibco BRL, Paisley, U.K.), hereafter referred to as complete medium. Thin-layer chromatography (TLC) was performed on 0.25 mm silica gel precoated plates, Si 60-F254 (Merck, Darmstadt, Germany), visualized by 254 nm UV light and cerium ammonium molybdate (CAM) staining. Column chromatography was conducted by use of silica gel Si 60, 230−400 mesh, 0.040−0.063 mm (Merck). Melting points were determined on a Buchi B450 apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on a Bruker Avance 400 (400 and 100.6 MHz, respectively) or Bruker Fourier 300 or Bruker AMX 300 instrument (recorded at 300.13 MHz for 1H and 75.00 MHz for 13C); chemical shifts are indicated in parts per million (ppm) downfield from SiMe4, with residual proton [CHCl3 = 7.26 ppm, (CH3)2SO = 2.50 ppm, HOD = 4.80 ppm] and carbon [CDCl3 = 77.0 ppm, (CD3)2SO = 40.45 ppm] solvent resonances as internal reference. Protons and carbon assignments were achieved by 13C attached proton test (APT), 1H−1H correlation spectroscopy (COSY), and 1H−13C heteronuclear correlation experiments. Coupling constants values J are given in hertz. All detailed synthetic procedures and spectra are reported in Supporting Information. Chemical purity of the three optical probes, estimated by HPLC, was ≥95%. HPLC analyses were 2011
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Journal of Medicinal Chemistry fitted in the fluorometer sample chamber. Three independent measurements were carried out on each complex with an estimated error of ±20%. Luminescence lifetimes (error ±10%) in the microsecond−millisecond scales were measured by a pulsed xenon lamp with variable repetition rate and elaborated with standard software fitting procedures. Stability Studies in Phosphate-Buffered Saline, RPMI Medium, and Rat Serum. HPLC was used to evaluate the chemical purity and stability of the complexes and was measured on a Beckman System Gold instrument equipped with a programmable solvent model 126 and a scanning detector module 166. Analyses were carried out on a RP Symmetry300 C4 guard column (5 μm, 3.9 × 20 mm) and a RP Symmetry300 C4 column (5 μm, 4.6 × 150 mm). Solvents: A = H2O (with 0.1% TFA); B = CH3CN (with 0.1% TFA). Gradient: 0−8 min, % B = 0 (isocratic); 8−23 min, %B = 20 (gradient); 23−33 min, %B = 65 (gradient); 33−38 min, %B = 65 (isocratic); 38−39 min, %B = 0 (gradient); 39−40 min, %B = 0 (isocratic). Flow rate = 1 mL/min; UV detector λ = 305 nm. Retention times: Eu3+⊂1 21.14 ± 0.11 min; Eu3+⊂2 22.88 ± 0.18 min; Eu3+⊂3 23.79 ± 0.06 min. Evaluation Procedures for in Vitro Stability. For each compound, a stock solution was prepared by dissolving Eu3+⊂1, Eu3+⊂2, or Eu3+⊂3 in DMSO (1 mM) and used to achieve a final concentration of 30 μM. In vitro stability of the complexes was determined by HPLC, monitoring over time the variation of chemical purity (as variation of concentration) of the compounds in the medium considered, by the following procedure: in an Eppendorf vial, an aliquot of the complex stock solution (30 μL) was added to (a) PBS (970 μL, 0.02 M; pH 7.4), (b) rat serum (970 μL), or (c) RPMI-1460 medium (970 μL). The resulting mixtures were incubated at 37 °C for 24 h. At 0, 45, 90, 135, 180, and 240 min and 24 h, samples (100 μL) of the reaction mixture were withdrawn and analyzed by HPLC. For serum stability, 50 μL of the incubation mixture was quenched with PBS (450 μL of 0.02 M; ratio 1/10) and analyzed by HPLC (injection volume 100 μL). The peaks were integrated and the absolute peak areas of the complex were used to assess its concentration (micromolar) by means of calibration curves. All the experiments were conducted in triplicate (n = 3). The results are expressed as average ± standard deviation and plotted versus incubation time. Cell Staining and Flow Cytometric Analysis. FRα expression on cancer cell lines was investigated by flow cytometry. Cells (3 × 105) were stained for 30 min at 4 °C with an anti-human-FRα IgG1 monoclonal antibody (mAb; clone MOv18, LifeSpan BioSciences, Seattle, WA) and then with a goat anti-mouse IgG1-R-PE mAb (SouthernBiotech, Birmingham, AL) for 30 min at 4 °C. The samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and collected data were evaluated with FlowJo software (TreeStar Inc., Olten, Switzerland). Stability of Eu3+⊂2 after Cell Exposure (Uptake and Internalization). SKOV-3 cells were seeded at a density of 6 × 106 in a T25 sterile culture flask (Sarstedt, Germany) and incubated for 3 or 24 h with 100 μM Eu3+⊂2 complex in 2 mL of complete medium at 37 °C and 5% CO2. At each time point the medium was removed and collected, and the uptake was halted by rinsing cells three times with ice-cold 1× PBS. Thereafter, membrane-bound ligand was removed by incubating the cells at room temperature for 5 min in 1 mL of ice-cold glycine buffer (pH 2.8). The cells were lysed with Triton 5% and centrifuged (1400 rpm, 22 °C, 7 min) to eliminate cell debris and obtain the supernatant containing the internalized complex. As control, the cells were incubated only with complete medium and treated in the same manner. Culture media and cell lysates were analyzed by HPLC. As controls, the following samples were further analyzed by HPLC: (1) 1× PBS solution of Eu3+⊂2, retention time = 22.57; (2) 1× PBS solution of folic acid, retention time = 11.57 min; (3) 1× PBS solution of Eu3+⊂4, retention time = 4.52; and (4) 5% Triton solution in sterile water. Cytotoxicity Assay. Cytotoxicity of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 was carefully investigated for ovarian cancer cells having different folate receptor expression, namely, SKOV-3 (97% FR), OVCAR-3 (55% FR), IGROV-1 (17% FR), and A2780 (does not contain FR) by the ATPlite test. The ATPlite test measures adenosine triphosphate (ATP), which is a marker of cells viability since it is present in living cells but sharply decreases in dying cells. This assay measures the bioluminescence
generated by reaction of ATP with luciferase and D-luciferin, which is proportional to ATP concentration according to the following equation: ATP + D‐luciferin + O2 luciferase
⎯⎯⎯⎯⎯⎯⎯⎯ → oxyluciferin + AMP + PPi + CO2 + light 2+ Mg
The procedure described below for SKOV-3 was followed for each of the cell lines in the study. SKOV-3 cells were seeded (8 × 103 cells/well) in triplicate in 96-well flat-bottom plates (Viewplates, PerkinElmer Life Science, Waltham, MA). After 24 h, media containing Eu complexes at scalar concentrations (ranging from 300 to 0.03 μM) were added. After 72 h of culture in the presence of the complexes, supernatants were eliminated and cell viability was estimated by ATP dosing, by use of the ATPlite kit (PerkinElmer Life Science) and an automated luminometer (TopCount NXT, PerkinElmer Life Science, Boston, MA), according to manufacturer’s instructions. Cell viability was calculated as follows:
%cell survival =
cps(sample) − cps(reference) × 100 cps(control) − cps(reference)
where cps = counts per second, cps(sample) is the experimental value, cps(reference) is the value of medium alone, and cps(control) is the value of untreated cells. Three independent experiments were performed for each cell line. Cellular Uptake. Cellular uptake of Eu3+⊂1, Eu3+⊂2, and Eu3+⊂3 has been investigated for SKOV-3 and IGROV-1 ovarian cancer cell lines, which show 97% and 17% FR expression, respectively. SKOV-3, IGROV-1, and A2780 cells were seeded at a density of 1 × 106 cells/well in 96-well flat-bottom plates (Sarstedt, Germany), and incubated for 1, 3, 18, or 24 h with each of the three Eu complexes at different concentrations (30 μM, 1 μM, or 100 nM) in a final volume of 50 μL/well of complete medium at 37 °C and 5% CO2. At each time point the medium was removed, and the uptake was halted by rinsing cells three times with ice-cold 1× PBS. Thereafter, membrane-bound ligand was removed by incubating the cells with 100 μL of ice-cold glycine buffer (pH 2.8) for 5 min at room temperature. Cells were finally lysed with 5% Triton and the plates were analyzed with VictorX4 (PerkinElmer Life Science). As control, cells were incubated with complete medium only and treated in the same manner. Titration curves were generated by adding fixed concentrations of the three complexes to the cell lysates obtained as above. In each experiment, determinations were performed in triplicate and experiments were repeated three times. Competition Studies. For competition experiments, the procedures outlined above were followed with a few modifications. For Eu3+⊂2 displacement, SKOV-3 cells were incubated for 24 h with 30 μM complex and different concentrations of free folic acid, starting from 960 μM at 1:2 dilutions. As control, cells were incubated with Eu3+⊂2 alone. For 3H-folic acid displacement, SKOV-3 cells were incubated for 24 h with scalar concentrations of Eu3+⊂2 or free folic acid (ranging from 1600 to 50 nM) in the presence of 400 nM 3H-folic acid. As control, cells were incubated with 3H-folic acid alone. After cell lysis, supernatants were recovered and transferred in 96-well GF/C UniFilter plates (PerkinElmer Life Science), to be counted in the liquid scintillation counter TopCount NXT. For both competition assays, determinations were performed in triplicate and experiments were repeated three times.
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ASSOCIATED CONTENT
* Supporting Information S
Additional text and 48 figures with experimental details, detailed synthetic procedures, NMR spectra of all new compounds, photophysical characterization, HPLC stability studies of the complexes, evaluation procedures for in vitro stability, and in vitro viability of ovarian tumor cells in response to the complexes. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail
[email protected]. 2012
DOI: 10.1021/jm501945w J. Med. Chem. 2015, 58, 2003−2014
Article
Journal of Medicinal Chemistry Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research has been supported by Italian MIUR through FIRB RBAP114AMK “RINAME”.
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ABBREVIATIONS USED FA, folic acid; FR, folate receptor; MS, mass spectroscopy; PBS, phosphate-buffered saline; MRI, magnetic resonance imaging; SPECT, single photon emission computed tomography; PET, positron emission tomography; OI, optical imaging; FII, fluorescence intensity imaging; LLC, luminescent lanthanide complex; DTTA, diethylenetriaminetetracetic acid; DO3A, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; GPI, glycosylphosphatidylinositol; PLE, photoluminescence excitation; PLQY, photoluminescence quantum yield; RS, rat serum; ATP, adenosine triphosphate; cpm, counts per minute; CAM, cerium ammonium molybdate
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