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Bioconjugate Chem. 2010, 21, 2222–2226
Investigations on the Use of Fluorescence Dyes for Labeling Dendrimers: Cytotoxicity, Accumulation Kinetics, and Intracellular Distribution A. M. Scutaru,† M. Kru¨ger,† M. Wenzel,† J. Richter,‡ and R. Gust*,†,§ Institute of Pharmacy, Freie Universita¨t Berlin, Ko¨nigin Luise Str. 2 + 4, 14195 Berlin, Germany, Charite´ Campus Benjamin Franklin, Institute of Clinical Physiology, Hindenburgdamm 30, 12203 Berlin, Germany, and Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria. Received April 28, 2010
Fluorescent dyes (e.g., dansyl, fluoresceine isothiocyanate, or naphthalimide groups) are widely used as markers to study biological properties of drugs. In order to evaluate possible mediated cytotoxicity, we attached three molecules each to 1,3,5-tris(3-propylamino)benzene initially synthesized as core molecule for the design of dendrimers. Cytotoxic effects were only observed for the NO2-substituted naphthalimide conjugate. The intracellular distribution was visualized via confocal fluorescence microscopy and pointed to an accumulation in the endosome or nucleus, dependent on the cell line used.
INTRODUCTION Dendrimers have attracted considerable attention in the past two decades. The advantages of dendrimers, if compared with classical polymers, are their monodisperse geometry, the high terminal group density at their surface, as well as the possibility of a determined surface functionality. The biological properties of dendrimers, including in vitro and in vivo toxicity and biodistribution, have gained more and more importance in the past few years. Their unique physical and chemical properties make them ideal candidates for drug delivery systems (1-3). Drugs can be covalently attached at the surface of the molecule, or in the case of larger dendrimers, they can be enclosed as guest molecules. Both concepts ensure targeted release (4-11). For the use of macromolecular tumor therapy, it is important to control the accumulation of the carrier in the tumor tissue and the intercellular distribution. For this purpose, the carriers are miscellaneously labeled for detection through trace analysis. The most commonly used markers are radioactive elements such as 125I (12-16), 51Cr (17), and 111In (18-20), which can easily be detected in vivo and in vitro, e.g., by methods in radioactive trace analysis. Alternatively, fluorescence analysis and highresolution confocal fluorescence microscopy can be used to visualize the subcellular distribution if the molecule is labeled by fluorophors such as rhodamine (21), fluoresceine (15, 22-24), or Oregon Green (25, 26). The use of dendrimers as drug delivery systems implies no cytotoxic properties of the macromolecule with and without labeling. In a previous study, we synthesized various dendrimers starting from the 1,3,5-tris(3-propylamino)benzene core molecule (1, Scheme 1) (27). This G0 molecule did not influence the growth of hormone-dependent MCF-7 or hormone-independent MDA-MB 231 cells. Therefore, we decided to use it to study the influence of macromolecular-bound fluoresceine isothiocyanate (FITC), naphthalimide, and nitronaphthalimide fluorophores on cytotoxicity, the accumulation kinetics, and the * Corresponding author. Phone: +43 512 507 5245; Fax: +43 512 507 2940; E-mail:
[email protected]. † Freie Universita¨t Berlin. ‡ Institute of Clinical Physiology. § University of Innsbruck.
intracellular distribution in MCF-7 and MDA-MB-231 breast cancer cell lines.
MATERIALS AND METHODS Synthesis. Compounds 1 and 3 were prepared according to the literature procedure (27). The other starting materials were purchased from commercial sources and were used without purification. Solvents were dried under standard conditions. 1H NMR spectra were taken on a Bruker Avance/DPX 400 MHz with TMS as internal standard. Melting points (uncorrected) were measured with a Bu¨chi Melting Point B-545. All column chromatography purifications were done by using silica gel 60 (0.063-0.1 mm, Merck). TLC was performed on silica gel 60 GF254 plates (Merck). Elemental analysis was carried out on a Vario EL (Hanau). EI spectra were recorded on a ThermoFisions VG Auto Spec (70 eV). The FAB spectra were measured on a CH-5, Varian MAT, Bremen. The ESI-TOF spectra were measured on an Agilent 6210 ESI-TOF, Agilent Technologies, Santa Clara, CA, USA. Solvent flow rate was adjusted to 4 µL/ min; spray voltage set to 4 kV. Drying gas flow rate was set to 15 psi (1 bar). All other parameters were adjusted for a maximum abundance of the relative [M+H]+. 1,3,5-Tris[N-(5-fluorescenyl)thiourea-propane-3,1-diyl]benzene (5). 1 × 3HCl (34.54 mg, 0.096 mmol) was dissolved in DMSO and treated with 0.16 mL of triethylamine (1.152 mmol). FITC was added to the solution, and the mixture was stirred for 24 h at room temperature in the dark. The solution was then poured in water and the pH value was adjusted to 4 with 5% hydrochloric acid. The color of the mixture changed from orange to yellow-green, and a solid precipitated. The compound was filtered off, washed with water, and then dried to afford an orange solid (100 mg, 73.4%); mp > 400 °C. 1H NMR (400 MHz, CD3OD): 1.98 (quin, 3J(H,H) ) 7.16 Hz, 6H, CH2), 2.68 (t, 3J(H,H) ) 7.18 Hz, 6H, CH2Ar), 3.61 (m, 6H, CH2NH), 6.55 (dd, 3J(H,H) ) 2.34 Hz, 8.64 Hz, 6H, FITC), 6.69 (d, 3J(H,H) ) 1.94 Hz, 9H, FITC), 6.71 (s, 3H, FITC), 6.99 (s, 3H, Ar), 7.11 (d, 3J(H,H) ) 8.22 Hz, 3H, FITC), 7.69 (d, 3J(H,H) ) 7.31 Hz, 3H, FITC), 8.12 (s, 3H NH). MS (ESI): calcd. for C78H60N6O15S3Na+, 1439.3171; found 1439.3118. Anal. (C78H60N6O15S3) C, H, N. 1,3,5-Tris{3-[1H-benzo[de]isoquinoline-1,3(2H)-dione]propylamino}benzene (7). 1 × 3HCl (359 mg, 1 mmol) was dissolved in 20 mL of dry ethanol and treated with 1.7 mL of dry
10.1021/bc1001906 2010 American Chemical Society Published on Web 11/04/2010
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Scheme 1. Synthesis of Labeled Conjugates 3, 5, 7, and 9a
a
Reagents and conditions: (a) DMSO, TEA, RT; (b) EtOH, TEA, reflux.
triethylamine. 1,8-Naphtalic anhydride (654 mg, 3.3 mmol) was added and the mixture was refluxed for 3 h in the dark. The precipitated solid was vacuumed off, washed with ethanol, and dried over P2O5. Yield: 650 mg (82%) as a colorless solid; mp ) 300 °C. 1H NMR (400 MHz, CDCl3): 1.97 (quin, 3J(H,H) ) 7.64 Hz, 6H, CH2), 2.55 (t, 3J(H,H) ) 8.28 Hz, 6H, CH2Ar), 4.20 (t, 3J(H,H) ) 7.38 Hz, 6H, CH2N), 6.77 (s, 3H, Ar), 7.70 (t, 3J(H,H) ) 7.61 Hz, 6H, naphthalimide), 8.16 (d, 3J(H,H) ) 7.61 Hz, 6H, naphthalimide), 8.55 (d, 3J(H,H) ) 7.61 Hz, 6H, naphthalimide). MS (EI, 80 eV, 300 °C); m/z (%): 789.4 (1.74) [M+], 398.9 (100.0) C27H28NO2, 197.9 (49.95), C12H7NO2. Anal. (C51H39N3O6) C, H, N. 1,3,5-Tris{3-[5-nitro-1H-benzo[de]isoquinoline-1,3(2H)dione]propylamino}benzene (9). 1 × 3HCl (200 mg, 0.56 mmol) was dissolved in 10 mL of dry ethanol and treated with 940 µL of dry triethylamine. 3-Nitro-1,8-naphthalic anhydride (447.4 mg, 1.84 mmol) was added, and the mixture was refluxed for 3 h in the dark. The precipitated solid was vacuumed off, washed with ethanol, and dried over P2O5. Yield: 320 mg (61.8%) as a brown powder; mp ) 272 °C. 1H NMR (400 MHz, CDCl3): 1.97 (quin, 3J(H,H) ) 7.41 Hz, 6H, CH2), 2.53 (t, 3J(H,H) ) 7,93 Hz, 6H, CH2Ar), 4.20 (t, 3J(H,H) ) 7.44 Hz, 6H, CH2N), 6.74 (s, 3H, Ar), 7.92 (t, 3J(H,H) ) 7.67 Hz, 3H, naphthalimide), 8.40 (d, 3J(H,H) ) 8.14 Hz, 3H, naphthalimide), 8.75 (d, 3J(H,H) ) 6.46 Hz, 3H, naphthalimide), 9.10 (d, 3J(H,H) ) 2.10 Hz, 3H, naphthalimide), 9.24 (d, 3J(H,H) ) 2.13 Hz, 3H, naphthalimide). MS (FAB+, MNBA/DCM) m/z (%): 925.3 (30.23) [M+H]+, 699.4 (65.70) [M-C12H5N2O3]+. Anal. (C51H36N3O6) C, H, N. Cell Culture. The human MCF-7 and MDA-MB 231 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC). Cell line banking and quality control were performed according to the seed stock concept reviewed by Hay (28). The MCF-7 cells were maintained in Eagle’s minimal essential medium (EMEM) containing L-glutamine supplemented with NaHCO3 (2.2 g/L), sodium pyruvate (110 mg/L), gentamycin (50 mg/L), and 10% fetal calf serum (FCS, Gibco, Eggenheim, Germany) using culture flasks (75 cm2, Nunc) in a water-saturated atmosphere (5% CO2) at 37 °C. The MDA-MB-231 cells were maintained in McCoy’s 5a Medium and 5% FCS. The cells were
serially passaged weekly following trypsin treatment using a solution of trypsin (0.05%) and EDTA (0.02%). In Vitro Chemosensitivity Assay. The in vitro testing of the compounds for cytotoxic activity was carried out on exponentially dividing cancer cells according to a previously published microtiter assay (29). Briefly, in 96-well microtiter assay plates (Nunc), 100 µL of a cell suspension was plated into each well and incubated at 37 °C for 3 days (MCF-7) and 2 days (MDA-MB-231) in a water-saturated atmosphere (5% CO2). The desired test concentration was obtained by addition of an adequate volume of a stock solution of the respective compound to the medium. Sixteen wells were used for each test concentration and for the control, which contained the corresponding amount of solvent (0.1% v/v) respectively. After appropriate incubation periods, the medium was removed and the cells were fixed with a glutaraldehyde solution and stored at 4 °C. Cell biomass was determined by a crystal violet staining technique. The influence of the complexes on cell growth is expressed as the corrected T/C value according to the equation Cytostatic effect: %T/Ccorr ) [(T - C0)/(C - C0)] × 100 in which T (test) and C (control) are the optical density values at 578 nm of the crystal violet extract of the cells in the wells (that is, the chromatin-bound crystal violet extracted with 70% ethanol), and C0 is the density of the cell extract immediately before treatment. A microplate reader at 590 nm (Flashscan Analytik Jena AG) was used for the automatic estimation of the optical density of the crystal violet extract in the wells. The calculated %T/C values can be interpreted as follows: T/C > 80%: no antiproliferative effect 80% > T/C > 20%: antiproliferative effect 20% > T/C > 0%: cytostatic effect Cellular Uptake Studies. The cells were seeded in 6-well plates (Nunc). When the cells reached 50-60% confluence (approximately after 5 days of incubation), the medium was exchanged for serum-free EMEM/Mc Coy’s containing the compounds. The medium was removed after appropriate
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Figure 1. In vitro cytotoxicity of the nitronaphthalimide-conjugate 9.
Figure 2. 3D spectrum of compound 5; concentration: 1 µM in MeOH. Table 1. Excitation and Emission Wavelengths of the Fluorescence-Labeled Conjugates compound
fluorophor
λmaxex (nm)
λmaxem (nm)
3 5 7 9
dansyl fluoresceine isothiocyanate naphthalimide nitronaphthalimide
340 450 333 361
535 519 387 440
incubation periods, and the cells were washed with ice-cold PBS. After trypsination, the cells were harvested, washed two times with ice-cold PBS, and centrifuged (2000 g, 4 °C, 5 min) for storage at -18 °C until analysis. The cell pellet was homogenized by sonification in a Triton X-100 solution (1% (w/w)) and was adequately diluted for protein determination (30). The cellular concentrations of the compounds in the cells were determined as previously published (31).
RESULTS AND DISCUSSION Synthesis. The synthesis of the dansyl derivative 3 (formulas of the conjugates; see Scheme 1) was already described in a previous paper (27). For the synthesis of the FITC-derivative 5, the core molecule 1 × 3HCl was dissolved in DMSO and treated with triethylamine to provide the free base. Subsequently, FITC was added and the mixture was stirred in the dark at room temperature. The pure product 5 was obtained with 73% yield. To obtain 7 and 9, 1 and the respective naphthalic anhydride (6, respectively, 8) were refluxed for 3 h in ethanol under protection from light. The final products were precipitated from the reaction mixture. In Vitro Cytotoxicity. The antiproliferative activity of the conjugates was determined in tests on the human MCF-7 and MDA-MB-231 breast cancer cell lines. Cisplatin used as a reference influenced the cell growth in a concentration-depend-
Figure 3. Accumulation kinetics of (A) dansyl conjugate 3 and (B) FITC conjugate 5.
ent manner and caused almost 100% inhibition in both cell lines at the highest concentration (5 µM). The antiproliferative effect (at concentrations of 1, 5, and 10 µM) expressed as T/C corr values (T, treated group; C, control group) was correlated with the incubation time and documented for the free core molecule 1 (27) as well as the dansyl, fluoresceine isothiocyanate, and naphthalimide conjugates 3, 5, and 7. No influence on the growth of MCF-7 and MDA-MB-231 cells could be observed (data not shown). Interestingly, introduction of a nitro group to the naphthalimide in 7 strongly increased the cytotoxicity of the conjugate. As depicted in Figure 1, the effects of 9 were more pronounced
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Figure 4. Fluorescence microscopy images of 5: (A) MCF-7 cells; (B) MDA-MB-231 cells.
at the MDA-MB-231 cell line up to an incubation time of 72 h. The recuperation of the cells until the end of the test equals the effects at both cell lines. A possible explanation for these antitumor properties could be an involvement of the nitro group in intracellular redox reactions (32). These findings documented the suitability of the dansyl, fluoresceine isothiocyanate, and the naphthalimide residue as fluorescence marker and require the determination of their spectroscopical properties after binding to 1. Fluorophor Characterization. The fluorescence of the synthesized conjugates was characterized by using a Hitachi F-4500 fluorescence spectrophotometer. All compounds showed only one excitation maximum. As an example, the 3D fluorescence spectrum of 5 is depicted in Figure 2. The emission maximum of conjugates 3, 5, 7, and 9 depended on the dye used (Table 1). Only 3 and 5 fulfill the requirement for accumulation studies (λmax em > 500 nm) and can be used for accumulation studies. Accumulation Kinetics. In order to quantify the cellular uptake, MCF-7 and MDA-MB-231 cells were incubated with 5 µM of the dye conjugates 3 and 5, respectively, and the accumulation of the substance expressed as nmol/mg protein was fluorimetrically estimated. Possible inactivation reactions of the compounds due to protein binding were minimized by growing the cells in FCS-free EMEM. FCS is necessary for cell growth, but the short-term incubation did not change cell morphology and cell growth. As depicted in Figure 3A, the dansylated conjugate 3 was accumulated to a higher degree in the tumor cells than 5 (Figure 3B). Furthermore, the kinetics differed significantly. In MDAMB-231 cells, a fast accumulation of 3 occurred with saturation after 20 h. After 24 h, the intracellular content amounted to 56 nmol/mg protein. In the MCF-7 cells, the saturation was also reached after 20 h, but the intracellular amount of the dye was much lower (13 nmol/mg protein). Exchange of the dansyl residue by fluoresceine isothiocyanate (3 f 5) reduced the accumulation kinetics. At both cell lines, no saturation was observed during the incubation time of 24 h. Furthermore, the intracellular amount of the conjugate 5 was considerably reduced. Intracellular Localization. Conjugates 3, 5, 7 and 9 were incubated with either MCF-7 or MDA-MB-231 cells at a concentration of 10 µM for 24 h to visualize the intracellular distribution. Confocal fluorescence microscopy (Zeiss LSM 510) shots of the adnated cells clearly showed an accumulation of the fluorescence labeled conjugates in cell compartments. Surprisingly, the intracellular distribution differs depending on the cells used. The dye was mainly determined in the endosomes
of MCF-7 cells, while the conjugates accumulated in MDAMB-231 cells at the nucleus. Fluorescence microscopy images of 5 were depicted as examples in Figure 4.
CONCLUSION Attachment of fluorescence dyes to the core molecule 1 did not increase the cytotoxicity but determined the intracellular distribution dependent on the cell type. Only the NO2-bearing naphthalimide derivative 9 reduced the growth of MCF-7 and MDA-MB-231 cells, probably due to the interference in intracellular redox processes.
ACKNOWLEDGMENT The presented study was supported by grants Gu285/4-1, Gu285/5-1, and Gu285/5-2, as well as the SFB 765 from the Deutsche Forschungsgemeinschaft. Supporting Information Available: Elemental analyses of the new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
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