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Facile synthesis, cytotoxicity and bioimaging of Fe3+ selective fluorescent chemosensor
Bioorganic & Medicinal Chemistry 22 (2014) 2045–2051
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Facile synthesis, cytotoxicity and bioimaging of Fe3+ selective fluorescent chemosensor Muhammad Saleem a, Razack Abdullah a, Anser Ali b, Bong Joo Park b, Eun Ha Choi b, In Seok Hong a, Ki Hwan Lee a,⇑ a b
Department of Chemistry, Kongju National University, Gongju, Chungnam 314-701, Republic of Korea Department of Plasma Bioscience and Display, Kwangwoon University, 20 Kwangwoon-gil, Nowon-gu, Seoul 139-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 January 2014 Revised 20 February 2014 Accepted 24 February 2014 Available online 5 March 2014 Keywords: Turn on fluorescent chemosensor Bioimaging Cell permeability Toxicity assay KCN induced fluorescence quenching
a b s t r a c t The designing and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of alanine substituted rhodamine B derivative 3 having specific binding affinity toward Fe3+ with micro molar concentration level. Through fluorescence titration at 599 nm, we were confirmed that ligand 3 exhibited ratiometric fluorescence response with remarkable enhancement in emission intensity by complexation between 3 and Fe3+ while it appeared no emission in case of the competitive ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v) at neutral pH. However, the fluorescence as well as colorimetric response of ligand–iron complex solution was quenched by addition of KCN which snatches the Fe3+ from complex and turn off the sensor confirming the recognition process was reversible. Furthermore, bioimaging studies against L-929 cells (mouse fibroblast cells) and BHK-21 (hamster kidney fibroblast), through confocal fluorescence microscopic experiment indicated that ligand showed good permeability and minimum toxicity against the tested cell lines. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The design and synthesis of sensitive and selective fluorescent chemosensors for specific metals detection is an interesting topic due to their fundamental role in medical, environmental and biological applications.1,2 Fluorescent probes have potential to map the molecular details of biological processes related to metal ion transport, homeostasis, and participation in disease pathology.3,4 The use of optical sensors allows remote measurements and is therefore promising for environments where direct accessibility is hard and samples could be damaged when removed from their natural medium.5 Therefore, the development of selective chemosensors for quantification of environmentally and biologically important ionic species in solution and body fluid, especially for transition metal ions, has tremendously attracted a great deal of attention.6 As Fe3+ is one of the most essential trace elements in biological systems, is an ubiquitous metal in cells and plays a crucial role in a variety of vital cell functions and, humans in particular need quite large amounts of iron in the diet as it is a vital constituent of the red blood cell protein haemoglobin, which carries ⇑ Corresponding author. Tel.: +82 1085672819; fax: +82 41 856 8613. E-mail address: [email protected] (K.H. Lee). http://dx.doi.org/10.1016/j.bmc.2014.02.045 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
oxygen around the body.7,8 It is critically involved in oxygen metabolism and electron transfer processes in DNA and RNA synthesis by virtue of its facile redox chemistry and its high affinity for oxygen.9,10 Although iron is essential for the proper functioning of all living cells, conversely, excess amounts of iron ions in a living cell is detrimental,11,12 that can catalyze the production of reactive oxygen species (ROS) via the Fenton reaction, which can damage lipids, nucleic acids and proteins.13 The iron can also be damaging when it accumulates in the body causing hyperferremic and hypotransferrinemic disorder.14 Thus, the need for quantification of iron in clinical, medicinal, environmental and industrial samples has led to a number of methods for its measurement and one of these methods which offers simple, rapid and reliable tool is ion-selective sensors.15–18 A number of fluorescent molecular probes have been reported in recent years, enabling easy detection of ferric ions,19–26 but the major problems in fluorescent sensor are including low cell permeability, cellular toxicity and counter sensing toward competing ions. Bhalla et al.22 reported the fluorescence turn on chemosensor, however the reported ligand exhibited poor selectivity toward Fe3+ as competing ions Cu2+, Fe2+ and Ni2+ also showing considerable enhancement in the absorption signal, while the same receptor binding toward competing ions can be observed in
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Meng et al.23 article. Meanwhile, selectivity of a dual fluorescence chemosensor24 is a bit challenging for the investigation of particular metal ion. Another important dimension of a fluorescent chemosensor for practical applicability to eradicate the metal contamination from living systems is the live cell imaging study as well as cellular toxicity investigation has been found missing in numbers of recent reports.25,26 However, still there is ever-growing demand to the development of fluorescence probe for selective signaling of Fe3+ in aqueous solution with precise sensitivity as well as biocompatible for ferric ion determination within living cells. Therefore, we were motivated to design and synthesize a novel probe which can sense Fe3+ against environmental and biological samples with minimum cellular toxicity. The synthesized ligand exhibited good cell permeability for signaling of Fe3+ in living cells with colorimetric as well as fluorescent change for ferric ion detection in mixed aqueous organic solution. Furthermore, the adopted synthetic method was simple, provides good product yields, offer an advantage over other methods where complex chromatographic techniques are required for purification of the target compounds. 2. Experimental 2.1. Substrate and reagents Rhodamine B, BOC-D-Alanine, N,N0 -Dicyclohexylcarbodiimide (DCC), Trifluoroacetic acid (TFA) and NaHCO3 were purchased from Aldrich. Ethanol, methanol, 1,2-dichloroethane, acetonitrile, acetone, water, dimethyl sulfoxide, hexane and ethyl acetate (Samchun chemicals, Korea), and Sc(OTF)3, YbCl36H2O, InCl3, CeCl3, SmCl36H2O, CrCl36H2O, SnCl2, PbCl2, FeCl3nH2O, NiCl26H2O, CoCl26H2O, CuCl22H2O, BaCl22H2O, CaCl22H2O, CsCl, CuCl, KCl and KCN (Aldrich and Alfa Aesar) were used during experiment. The major chemicals utilized for biological studies includes MEM (minimum essential media, Wel Gene, Korea), FBS (fetal bovine serum, Bio west U.S.A), Tripsin (Thermo scientific, South Loga, Utah), PBS (Wel Gene, Korea) and MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, Sigma Aldrich, U.S.A]. 2.2. Instrumentations The reaction progress was monitored by thin layer chromatographic (TLC) analysis, and the Rf values were determined by employing pre-coated silica gel aluminum plates, Kieselgel 60 F254 from Merck (Germany), using dichloromethane: methanol, 9:1 as an eluent. TLC was visualized under a UV lamp (VL-4. LC, France). The FT-IR spectra were recorded in KBr pellets on a SHIMADZU FTIR-8400S spectrometer (Kyoto, Japan). Proton and carbon nuclear magnetic resonance (1H NMR & 13C NMR) spectra were recorded on a Bruker Avance 500 MHz spectrometer with TMS as an internal standard. The chemical shifts are reported as d values (ppm) downfield from the internal tetramethylsilane of the indicated organic solution. Peak multiplicities are expressed as follows: s, singlet, br s, broad signal, d, doublet, t, triplet, q, quartet and m, multiplet. The coupling constants (J values) are given in hertz (Hz). Mass spectra were recorded on the AB SCIEX Co. 4000 QTRAP LC/MS/MS System. Abbreviations are used as follows: DMSO-d6, Dimethyl sulfoxide-d6; FT-IR spectroscopy, Fourier transform infrared spectroscopy, DCC, N,N0 -Dicyclohexylcarbodiimide, TFA, Trifluoroacetic acid, BOC-D-Alanine, N-(tert-Butoxycarbonyl)-D-alanine, MC, Methylene chloride and DMAP, 4-Dimethylaminopyridine. 2.3. General procedure for the synthesis of ligand 3 The compound 1 was synthesized utilizing rhodamine B which was converted to BOC-protected alanine substituted rhodamine B
derivative 2 following the reported procedure.27,28 Briefly, the compound 1 (1 g, 2.06 mmol, 1 equiv), BOC-D-Alanine (0.78 g, 4.12 mmol, 2 equiv), N,N0 -dicyclohexylcarbodiimide (DCC, 1.7 g, 8.24 mmol, 4 equiv) and 4-dimethylaminopyridine (DMAP, 0.25 g, 2.06 mmol, 1 equiv) were stirred in methylene chloride (50 mL) for 3 h at room temperature, monitored by TLC. After consumption of starting material, the reaction mixture was filtered and filtrate after concentration on reduced pressure, mixed with saturated NaHCO3 solution and extracted on ethyl acetate to afford 2. The target compound 3 was extracted on ethyl acetate after stirring 2 in TFA:MC, 3:1, v/v for 3 h at room temperature and purified by column chromatography and crystallized on methanol. 2.4. 2-Amino-N-[2-{30 ,60 -bis(diethylamino)-3oxospiro(isoindoline-1,90 -xanthen)-2-yl}ethyl]propanamide (3) Off white powder; yield: 67%; Rf: 0.26 (dichloromethane: methanol, 9:1); 1H NMR (500 M Hz, DMSO-d6) d 7.81–7.78 (aromatic, 1H, m), 7.67 (aromatic, 1H, t, J = 7 Hz), 7.55–7.46 (aromatic, 2H, m), 7.05 (NH, 1H, br s), 7.00–6.98 (aromatic, 1H, m), 6.43–6.31 (aromatic, 5H, m), 3.35–3.29 (aliphatic, 8H, q, J = 10 Hz), 3.23– 3.10 (aliphatic, 4H, m), 2.84–2.79 (aliphatic, 1H, q, J = 8.5 Hz), 1.28 (NH, 2H, br s), 1.09–1.05 (aliphatic, 12H, t, J = 9 Hz), 1.02 (aliphatic, 3H, d, J = 8.5 Hz); 13C NMR (125 MHz, DMSO-d6) d 175.9, 175.4, 167.9, 167.1, 154.2, 153.7, 153.1, 153.0, 133.2, 130.4, 128.6, 124.0, 122.8, 108.6, 105.1, 97.7, 64.5, 50.6, 44.1, 37.5, 31.4, 22.5, 21.7, 14.4, 12.8; MS for C33H41N5O3 (ESI, m/z), 556.6 [M+H]+. 2.5. General procedure for spectroscopic assay A solution of ligand (300 lM) was prepared by dissolving 0.0056 g ligand in methanol (total volume 10 mL) then 3 mL of this solution was diluted to 10 mL with methanol to get 300 lM ligand stock solutions. Similarly, to prepare Fe3+ stock solution (300 lM), 0.0016 g of iron(III) chloride was dissolved in distilled water (total volume 10 mL), then 3 mL of this solution was diluted to 10 mL with distilled water to get 300 lM Fe3+ stock solution. All the metal ions solution was prepared similar as Fe3+ stock solution. For spectroscopic measurements, test solution of 1 mL was prepared with 780 lL of 40% aqueous methanol, 10 lL of ligand stock solution, 0.1 mL of buffer solution (10 mM) and 10 lL of Fe3+ stock solution. The resulting solution were mixed before measurement and final volume was fixed as 1 mL for UV–vis and Fluorescent measurement using [SCINCO] UV–vis Spectrophotometer ‘S-3100’ and FS-2 fluorescence spectrometer (SCINCO, Korea), respectively. 3. Results and discussions 3.1. Synthesis of ligand 3 In IR spectra, a peak in the range of 3222–3136 cm1 for NH stretching and a relatively strong peak with shoulder in the range of 3357–3285 cm1 for NH2 stretching vibration indicate the formation of ligand 3. Further confirmation was carried out by 1H NMR spectral analysis by the appearance of quartet and doublet at 2.86 ppm and 1.02 ppm, respectively, characteristic signal for alanine substitution. Meanwhile, the additional signal both in aliphatic and aromatic region of both 1H NMR and 13C NMR spectra justify the ligand 3 (Supporting information, Figs. S1 and S2). Furthermore, in the mass spectral analysis, the molecular ion peak [M+H]+ at m/z = 556 gives the correct mass of ligand 3 (Supporting information, Fig. S7). The reaction pathway adopted for the synthesis of 3 was outlined in Scheme 1.
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3.2. Ligand–metal chelation To understand the chelation mechanism of ligand–metal complex, NMR (1H NMR and 13C NMR) and mass spectra were recorded before and after addition of ferric ion into the ligand solution. As there was no characteristic signal loss from the ligand after ferric ion addition in both 1H NMR and 13C NMR spectra (Supporting information, Figs. S1–S4), verifying the ferric ion induced intramolecular rearrangement in the ligand molecule. This speculation was further confirmed by the addition of KCN in the ligand–metal complex solution, which dramatically reverse the reaction and the change can be easily observed by naked eye due to turning the color solution into colorless. This reversibility of reaction was also confirmed by 1H NMR and 13C NMR spectral analysis. There was same number of carbon and proton in aliphatic as well as aromatic region of both proton and carbon NMR spectrum after cyanide addition with slight distortion of multiplicities, confirm the reversible nature of the sensor molecule 3 (Supporting information, Figs. S5 and S6). Furthermore, mass spectrometric analysis was done to confirm the ferric ion attachment to the ligand; there was a characteristic signal at m/z 610 which gives the correct mass of ligand–iron complex (Supporting information, Fig. S8). On the basis of these observations, the proposed ligand–iron ligation mechanism was outline in Scheme 2. 3.3. Spectroscopic properties The spectroscopic properties of ligand were performed in aqueous/methanol (60:40, v/v) and the corresponding spectra are shown in Figure 1. Free probe 3 is colorless and non-fluorescence while upon addition of ferric ion into probe solution leads to tremendous increase in the fluorescence intensity with maximum emission at 599 nm as well as UV–vis absorption with absorption maxima of 557 nm. Furthermore, probe–metal complex trigger a color change of the solution for colorimetric detection of ferric ion. To find out the minimum detection ability of ligand 3, a fluorescence titration experiment was conducted by varying the concentration of
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ferric ion keeping constant ligand concentration. A linear relationship was observed on increasing ferric ion concentration up to 15 lM at maximum emission of 599 nm in aqueous/methanol (60:40, v/v) at pH 7.0. Further increasing the ferric ion concentration from 15 to 18 lM, there was slightly more increment in the fluorescent intensity of ligand solution. These results indicates that ferric ion triggered ligand ring opening reaction rate increased at higher ferric ion concentration as shown in the inset of Fig. 2. From this titration experiment, the minimum detection ability of ligand toward ferric ion was estimated to be about 3 lM as shown in Fig. 2. To find out the specificity of ligand 3 toward common metal ions, the sensing properties of ligand 3 were examined by addition of various metal ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v, pH 7.0.) at ambient temperature, into the ligand solution under same condition. The competing metal ions exhibited negligible fluorescence enhancement. By contrast, the most distinctive fluorescence intensity enhancement resulted upon ferric ion addition indicates that ligand 3 possesses selective ligation tendency toward ferric ions (Fig. 3). To understand the ligand–metal chelation reaction time, the fluorescent intensity of ligand–metal complex was measured with different time interval. The complexation reaction between ligand and ferric ion started immediately which can be visualized through naked eye by the solution color transformation from colorless to pink as well as fluorescent experiment. The fluorescence intensity of ligand–metal complex becomes stable within five minutes after metal addition as shown in Figure 4; provides quick sensing methodology for Fe3+ in natural physiological conditions. 3.3.1. Interfering ion effect In order to study the influence of other metal ions on ligand– iron binding, the competitive experiments were carried out in the presence of Fe3+ with other metal ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v, pH 7) at ambient temperature. The results indicates that competing metal ions did
Scheme 1. Synthesis of ligand 3: Reagents and conditions. (i) Compound 1, BOC-D-Alanine, DCC, DMAP, MC, 3 h, rt; (ii) TFA:MC, 3:1, v/v, 3 h, rt.
Scheme 2. The proposed spirolactam ring opening mechanism of ligand 3 into 4 upon ferric ion addition in aqueous/methanol (60:40, v/v) at pH 7.0 and corresponding KCN triggered reversibility of complex 4 back to 3.
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Figure 1. (A) Fluorescence emission spectrum of ligand 3 (3 lM) in the presence and absence of Fe3+ (6 lM) in aqueous/methanol (60:40, v/v) at pH 7.0, (B) UV–visible absorption spectrum of ligand 3 (6 lM) in the presence and absence of Fe3+ (12 lM) in aqueous/methanol (60:40, v/v) at pH 7.0.
Figure 2. The fluorescence titration of ligand 3 (3 lM) at emission maxima of 599 nm as a function of ferric ion concentration (3–18 lM, 1–6 equiv), the inset described the fluorescence enhancement at maximum emission of 599 nm; F/Fo is determined as a ratio between the maximum fluorescence intensity (F, after Fe3+ addition) and minimum fluorescence intensity (Fo, free ligand solution in absence of Fe3+).
Figure 3. Fluorescence emission spectrum of ligand (3 lM) in the presence of Fe3+ (18 lM, 6 equiv) and competing ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) (18 lM, 6 equiv) in aqueous/ methanol (60:40, v/v) at pH 7.0.
not interfere ferric ion chelation with ligand as there was no or negligible effect on the fluorescence intensity of ligand–metal complex in the presence of various metal ions. However, KCN
Figure 4. Effects of reaction times on the fluorescent intensity of ligand (3 lM) at maximum emission of 599 nm in the presence of Fe3+ (5–9 lM) in aqueous/ methanol (60:40, v/v) at pH 7.0.
Figure 5. The fluorescence intensity contrast bars in order to investigate the interference effect of other metal ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v) at ambient temperature on the detection ability of ligand for Fe3+, red bars represent the fluorescence response of ligand (3 lM) to Fe3+ (18 lM) in the presence of interfering metal ions (18 lM) and black bars represent the fluorescence intensity of ligand (3 lM) with competing ions (18 lM) in the absence of Fe3+.
reverse the ligand–metal complex to the backward direction and caused the complete quenching of fluorescence as shown in Figure 5.
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Figure 6. Fluorescence intensity variation at maximum emission of 599 nm, with alteration of pH from 2 to 11, in MeOH/Tris–HCl buffer solution.
by spontaneous measurement of UV-visible absorption spectra. The results clearly showed that organic solvent exert continuous increment on the absorption intensity of probe–metal complex formation up to 40%, with further increase in concentration of organic solvent; there was a negligible effect on the peak intensity. These results inspired us to use 40% aqueous organic media for further measurements. Conversely, acetonitrile exhibited reverse effect on the ligand–metal complexation reaction. In acetonitrile solvent system, there was no UV-visible absorption as shown in Figure 7. This may be due to strong affinity of ferric ion toward cyanide ion. To confirm this, we have further investigated the ligand–iron complexation in the presence of KCN, which cause complete quenching of UV-visible absorption and this experiment support our expectation of reversible nature of ligand in the presence of cyanide ion. While, there was no absorption signal for ligand in the given range. The effect of different organic solvent on the fluorescence emission and UV-visible absorption spectra and corresponding intensity values are tabulated in Table 1. The Stokes shift was calculated by Eq. 1.29
ðtA tFÞ ¼
1 1 kA kF
107
ð1Þ
3.4. Bioimaging applications of ligand 3 in L-929 and BHK-21 cell lines
Figure 7. Variation of absorption intensity of ligand 3 upon two equivalent addition of ferric ion at absorption maxima of 557 nm with different aqueous/organic solvent ratio.
3.3.2. pH effect The effect of pH was evaluated in MeOH/Tris–HCl buffer in the pH range of 2–11 as shown in Figure 6. The probe works well in neutral pH condition which is more suitable for ferric ion detection in the biological fluid and living cells under neutral pH range. However, the sensor molecules are sensitive to acid with pH less than 5 that bring spirolactam ring opening of the ligand by the activation of ligand spirolactam carbonyl while there was no acid induced ring opening in MeOH/Tris–HCl buffer under pH span of 5–11. This property of ligand makes it a reliable source for ferric ion detection in natural water system and in living cells. 3.3.3. Effect of media Solvent showed direct effect on the ligand–iron chelation. A suitable ratio of aqueous organic solvent was finding out by varying the concentration of methanol into aqueous solution followed
3.4.1. Bioimaging applications of ligand 3 in L-929 cells (mouse fibroblast cells) To explore the sensing performance of the ligand 3 toward Fe3+ in biological samples, fluorescence imaging experiments were performed using L-929 (mouse fibroblast cells) cells on confocal fluorescent microscope following the reported procedure30 with slight modification. Briefly, L-929 cells were incubated with ligand (5 lM) in complete MEM (minimum essential media) for 3 h at 30 °C, and very week fluorescence was observed. The samples were washed with PBS three times and then treated with various concentrations of Fe3+ ranging from 5 to 25 lM for 30 min at 37 °C, which displayed bright intracellular fluorescence depending upon the Fe3+ concentrations. The results of confocal fluorescence microscopic experiments against L-929 (mouse fibroblast cells) are given in Figure 8. 3.4.2. Bioimaging applications of ligand 3 in BHK-21 cells (hamster kidney fibroblast) To investigate the practical applicability of ligand 3 toward several cells of living system, the different cell lines were utilized including BHK-21 cells (hamster kidney fibroblast). The confocal fluorescent microscopic experimental results for BHK-21 (hamster kidney fibroblast) are shown in Figure 9; suggesting extensive applicability of synthesized ligand over a wide biological cell lines. 3.5. Cytotoxicity assay Furthermore, the ligand cytotoxicity was accessed after 24 h treatment to the cells by MTT assay, a frequently used method in
Table 1 Solvent effect on the spectroscopic properties of ligand upon treating with ferric ion
a b c
S. No.
Solvent
F/Foa
Emission maxima (nm)
Ab
Absorption maxima (nm)
Stokesshift (cm1)
1 2 3
MeCN: H2O (40:60, v/v) MeOH: H2O (40:60, v/v) EtOH: H2O (40:60, v/v)
3.27 181 174
599 599 599
0.001 0.475 0.471
557 557 557
—c 1258 1258
F/Fo = Fluorescence intensity of 3 lM ligand at emission maxima 599 nm, in the presence and absence of 6 lM Fe3+. A = Absorption signal intensity of 6 lM ligand at absorption maxima 557 nm, in the presence of 12 lM Fe3+. — = Absorption and emission intensity in case of MeCN solvent was quenched, so, Stokes shift was not calculated in aqueous MeCN media.
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Figure 8. Confocal fluorescence microscopic images for L-929 cells; (A1-A3) L-929 cells incubated with probe (5 lM); (B1-B3) L-929 cells incubated with probe (5 lM) in the presence of (5 lM) ferric ion; (C1-C3) L-929 cells incubated with probe (5 lM) in the presence of (10 lM) ferric ion; (D1-D3) L-929 cells incubated with probe (5 lM) in the presence of (25 lM) ferric ion. A1-D1: Bright field images; A2-D2: fluorescence images: A3-D3: merged images.
biology for cells toxicity. The results showed no toxicity for both BHK-2 (hamster kidney fibroblast) and L-929 (mouse fibroblast cells) cells as shown in Figure 10. The non-toxic behavior of ligand
3 at low doses and its ability to track the change in Fe3+ level in living cells suggest its possibility to use in biological system for Fe3+ detection.
Figure 9. Confocal fluorescence microscopic images for BHK-21 cells; (A1-A3) BHK-21 cells incubated with probe (5 lM); (B1-B3) BHK-21 cells incubated with probe (5 lM) in the presence of (5 lM) ferric ion; (C1-C3) BHK-21 cells incubated with probe (5 lM) in the presence of (10 lM) ferric ion; (D1-D3) BHK-21 cells incubated with probe (5 lM) in the presence of (25 lM) ferric ion. A1-D1: Bright field images; A2-D2: fluorescence images: A3-D3: merged images.
M. Saleem et al. / Bioorg. Med. Chem. 22 (2014) 2045–2051
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Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2010-0027963). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.045. References
Figure 10. Cell viability of BHK-21 and L-929 cells cultured in complete media with 5 lM of ligand 3 and the control cells were cultured in the medium without ligand.
4. Conclusion In summary, alanine substituted rhodamine B derivative 3 was successfully prepared and its sensing capability for various metal ions were examined. The ligand exhibited high selectivity for Fe3+ over various metal ions including Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+ in aqueous/methanol (60:40, v/v) at neutral pH. The ligand was sensitive enough to detect the micro molar concentration of ferric ion with turn-on colorimetric as well as fluorescence change that was unaffected by the presence of other common coexisting metal ions, however, the recognition process was reversible confirmed by addition of KCN. Furthermore, confocal fluorescence microscopic experiments utilizing L-929 cells (mouse fibroblast cells) as well as BHK-21 (hamster kidney fibroblast), showed that the ligand exhibited good biocompatibility for cell imaging study with precise cell permeability and low toxicity against the tested cell lines. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0015056) and the National Research Foundation of
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Facile synthesis, cytotoxicity and bioimaging of Fe3+ selective fluorescent chemosensor Muhammad Saleem a, Razack Abdullah a, Anser Ali b, Bong Joo Park b, Eun Ha Choi b, In Seok Hong a, Ki Hwan Lee a,⇑ a b
Department of Chemistry, Kongju National University, Gongju, Chungnam 314-701, Republic of Korea Department of Plasma Bioscience and Display, Kwangwoon University, 20 Kwangwoon-gil, Nowon-gu, Seoul 139-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 January 2014 Revised 20 February 2014 Accepted 24 February 2014 Available online 5 March 2014 Keywords: Turn on fluorescent chemosensor Bioimaging Cell permeability Toxicity assay KCN induced fluorescence quenching
a b s t r a c t The designing and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of alanine substituted rhodamine B derivative 3 having specific binding affinity toward Fe3+ with micro molar concentration level. Through fluorescence titration at 599 nm, we were confirmed that ligand 3 exhibited ratiometric fluorescence response with remarkable enhancement in emission intensity by complexation between 3 and Fe3+ while it appeared no emission in case of the competitive ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v) at neutral pH. However, the fluorescence as well as colorimetric response of ligand–iron complex solution was quenched by addition of KCN which snatches the Fe3+ from complex and turn off the sensor confirming the recognition process was reversible. Furthermore, bioimaging studies against L-929 cells (mouse fibroblast cells) and BHK-21 (hamster kidney fibroblast), through confocal fluorescence microscopic experiment indicated that ligand showed good permeability and minimum toxicity against the tested cell lines. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The design and synthesis of sensitive and selective fluorescent chemosensors for specific metals detection is an interesting topic due to their fundamental role in medical, environmental and biological applications.1,2 Fluorescent probes have potential to map the molecular details of biological processes related to metal ion transport, homeostasis, and participation in disease pathology.3,4 The use of optical sensors allows remote measurements and is therefore promising for environments where direct accessibility is hard and samples could be damaged when removed from their natural medium.5 Therefore, the development of selective chemosensors for quantification of environmentally and biologically important ionic species in solution and body fluid, especially for transition metal ions, has tremendously attracted a great deal of attention.6 As Fe3+ is one of the most essential trace elements in biological systems, is an ubiquitous metal in cells and plays a crucial role in a variety of vital cell functions and, humans in particular need quite large amounts of iron in the diet as it is a vital constituent of the red blood cell protein haemoglobin, which carries ⇑ Corresponding author. Tel.: +82 1085672819; fax: +82 41 856 8613. E-mail address: [email protected] (K.H. Lee). http://dx.doi.org/10.1016/j.bmc.2014.02.045 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
oxygen around the body.7,8 It is critically involved in oxygen metabolism and electron transfer processes in DNA and RNA synthesis by virtue of its facile redox chemistry and its high affinity for oxygen.9,10 Although iron is essential for the proper functioning of all living cells, conversely, excess amounts of iron ions in a living cell is detrimental,11,12 that can catalyze the production of reactive oxygen species (ROS) via the Fenton reaction, which can damage lipids, nucleic acids and proteins.13 The iron can also be damaging when it accumulates in the body causing hyperferremic and hypotransferrinemic disorder.14 Thus, the need for quantification of iron in clinical, medicinal, environmental and industrial samples has led to a number of methods for its measurement and one of these methods which offers simple, rapid and reliable tool is ion-selective sensors.15–18 A number of fluorescent molecular probes have been reported in recent years, enabling easy detection of ferric ions,19–26 but the major problems in fluorescent sensor are including low cell permeability, cellular toxicity and counter sensing toward competing ions. Bhalla et al.22 reported the fluorescence turn on chemosensor, however the reported ligand exhibited poor selectivity toward Fe3+ as competing ions Cu2+, Fe2+ and Ni2+ also showing considerable enhancement in the absorption signal, while the same receptor binding toward competing ions can be observed in
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Meng et al.23 article. Meanwhile, selectivity of a dual fluorescence chemosensor24 is a bit challenging for the investigation of particular metal ion. Another important dimension of a fluorescent chemosensor for practical applicability to eradicate the metal contamination from living systems is the live cell imaging study as well as cellular toxicity investigation has been found missing in numbers of recent reports.25,26 However, still there is ever-growing demand to the development of fluorescence probe for selective signaling of Fe3+ in aqueous solution with precise sensitivity as well as biocompatible for ferric ion determination within living cells. Therefore, we were motivated to design and synthesize a novel probe which can sense Fe3+ against environmental and biological samples with minimum cellular toxicity. The synthesized ligand exhibited good cell permeability for signaling of Fe3+ in living cells with colorimetric as well as fluorescent change for ferric ion detection in mixed aqueous organic solution. Furthermore, the adopted synthetic method was simple, provides good product yields, offer an advantage over other methods where complex chromatographic techniques are required for purification of the target compounds. 2. Experimental 2.1. Substrate and reagents Rhodamine B, BOC-D-Alanine, N,N0 -Dicyclohexylcarbodiimide (DCC), Trifluoroacetic acid (TFA) and NaHCO3 were purchased from Aldrich. Ethanol, methanol, 1,2-dichloroethane, acetonitrile, acetone, water, dimethyl sulfoxide, hexane and ethyl acetate (Samchun chemicals, Korea), and Sc(OTF)3, YbCl36H2O, InCl3, CeCl3, SmCl36H2O, CrCl36H2O, SnCl2, PbCl2, FeCl3nH2O, NiCl26H2O, CoCl26H2O, CuCl22H2O, BaCl22H2O, CaCl22H2O, CsCl, CuCl, KCl and KCN (Aldrich and Alfa Aesar) were used during experiment. The major chemicals utilized for biological studies includes MEM (minimum essential media, Wel Gene, Korea), FBS (fetal bovine serum, Bio west U.S.A), Tripsin (Thermo scientific, South Loga, Utah), PBS (Wel Gene, Korea) and MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, Sigma Aldrich, U.S.A]. 2.2. Instrumentations The reaction progress was monitored by thin layer chromatographic (TLC) analysis, and the Rf values were determined by employing pre-coated silica gel aluminum plates, Kieselgel 60 F254 from Merck (Germany), using dichloromethane: methanol, 9:1 as an eluent. TLC was visualized under a UV lamp (VL-4. LC, France). The FT-IR spectra were recorded in KBr pellets on a SHIMADZU FTIR-8400S spectrometer (Kyoto, Japan). Proton and carbon nuclear magnetic resonance (1H NMR & 13C NMR) spectra were recorded on a Bruker Avance 500 MHz spectrometer with TMS as an internal standard. The chemical shifts are reported as d values (ppm) downfield from the internal tetramethylsilane of the indicated organic solution. Peak multiplicities are expressed as follows: s, singlet, br s, broad signal, d, doublet, t, triplet, q, quartet and m, multiplet. The coupling constants (J values) are given in hertz (Hz). Mass spectra were recorded on the AB SCIEX Co. 4000 QTRAP LC/MS/MS System. Abbreviations are used as follows: DMSO-d6, Dimethyl sulfoxide-d6; FT-IR spectroscopy, Fourier transform infrared spectroscopy, DCC, N,N0 -Dicyclohexylcarbodiimide, TFA, Trifluoroacetic acid, BOC-D-Alanine, N-(tert-Butoxycarbonyl)-D-alanine, MC, Methylene chloride and DMAP, 4-Dimethylaminopyridine. 2.3. General procedure for the synthesis of ligand 3 The compound 1 was synthesized utilizing rhodamine B which was converted to BOC-protected alanine substituted rhodamine B
derivative 2 following the reported procedure.27,28 Briefly, the compound 1 (1 g, 2.06 mmol, 1 equiv), BOC-D-Alanine (0.78 g, 4.12 mmol, 2 equiv), N,N0 -dicyclohexylcarbodiimide (DCC, 1.7 g, 8.24 mmol, 4 equiv) and 4-dimethylaminopyridine (DMAP, 0.25 g, 2.06 mmol, 1 equiv) were stirred in methylene chloride (50 mL) for 3 h at room temperature, monitored by TLC. After consumption of starting material, the reaction mixture was filtered and filtrate after concentration on reduced pressure, mixed with saturated NaHCO3 solution and extracted on ethyl acetate to afford 2. The target compound 3 was extracted on ethyl acetate after stirring 2 in TFA:MC, 3:1, v/v for 3 h at room temperature and purified by column chromatography and crystallized on methanol. 2.4. 2-Amino-N-[2-{30 ,60 -bis(diethylamino)-3oxospiro(isoindoline-1,90 -xanthen)-2-yl}ethyl]propanamide (3) Off white powder; yield: 67%; Rf: 0.26 (dichloromethane: methanol, 9:1); 1H NMR (500 M Hz, DMSO-d6) d 7.81–7.78 (aromatic, 1H, m), 7.67 (aromatic, 1H, t, J = 7 Hz), 7.55–7.46 (aromatic, 2H, m), 7.05 (NH, 1H, br s), 7.00–6.98 (aromatic, 1H, m), 6.43–6.31 (aromatic, 5H, m), 3.35–3.29 (aliphatic, 8H, q, J = 10 Hz), 3.23– 3.10 (aliphatic, 4H, m), 2.84–2.79 (aliphatic, 1H, q, J = 8.5 Hz), 1.28 (NH, 2H, br s), 1.09–1.05 (aliphatic, 12H, t, J = 9 Hz), 1.02 (aliphatic, 3H, d, J = 8.5 Hz); 13C NMR (125 MHz, DMSO-d6) d 175.9, 175.4, 167.9, 167.1, 154.2, 153.7, 153.1, 153.0, 133.2, 130.4, 128.6, 124.0, 122.8, 108.6, 105.1, 97.7, 64.5, 50.6, 44.1, 37.5, 31.4, 22.5, 21.7, 14.4, 12.8; MS for C33H41N5O3 (ESI, m/z), 556.6 [M+H]+. 2.5. General procedure for spectroscopic assay A solution of ligand (300 lM) was prepared by dissolving 0.0056 g ligand in methanol (total volume 10 mL) then 3 mL of this solution was diluted to 10 mL with methanol to get 300 lM ligand stock solutions. Similarly, to prepare Fe3+ stock solution (300 lM), 0.0016 g of iron(III) chloride was dissolved in distilled water (total volume 10 mL), then 3 mL of this solution was diluted to 10 mL with distilled water to get 300 lM Fe3+ stock solution. All the metal ions solution was prepared similar as Fe3+ stock solution. For spectroscopic measurements, test solution of 1 mL was prepared with 780 lL of 40% aqueous methanol, 10 lL of ligand stock solution, 0.1 mL of buffer solution (10 mM) and 10 lL of Fe3+ stock solution. The resulting solution were mixed before measurement and final volume was fixed as 1 mL for UV–vis and Fluorescent measurement using [SCINCO] UV–vis Spectrophotometer ‘S-3100’ and FS-2 fluorescence spectrometer (SCINCO, Korea), respectively. 3. Results and discussions 3.1. Synthesis of ligand 3 In IR spectra, a peak in the range of 3222–3136 cm1 for NH stretching and a relatively strong peak with shoulder in the range of 3357–3285 cm1 for NH2 stretching vibration indicate the formation of ligand 3. Further confirmation was carried out by 1H NMR spectral analysis by the appearance of quartet and doublet at 2.86 ppm and 1.02 ppm, respectively, characteristic signal for alanine substitution. Meanwhile, the additional signal both in aliphatic and aromatic region of both 1H NMR and 13C NMR spectra justify the ligand 3 (Supporting information, Figs. S1 and S2). Furthermore, in the mass spectral analysis, the molecular ion peak [M+H]+ at m/z = 556 gives the correct mass of ligand 3 (Supporting information, Fig. S7). The reaction pathway adopted for the synthesis of 3 was outlined in Scheme 1.
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3.2. Ligand–metal chelation To understand the chelation mechanism of ligand–metal complex, NMR (1H NMR and 13C NMR) and mass spectra were recorded before and after addition of ferric ion into the ligand solution. As there was no characteristic signal loss from the ligand after ferric ion addition in both 1H NMR and 13C NMR spectra (Supporting information, Figs. S1–S4), verifying the ferric ion induced intramolecular rearrangement in the ligand molecule. This speculation was further confirmed by the addition of KCN in the ligand–metal complex solution, which dramatically reverse the reaction and the change can be easily observed by naked eye due to turning the color solution into colorless. This reversibility of reaction was also confirmed by 1H NMR and 13C NMR spectral analysis. There was same number of carbon and proton in aliphatic as well as aromatic region of both proton and carbon NMR spectrum after cyanide addition with slight distortion of multiplicities, confirm the reversible nature of the sensor molecule 3 (Supporting information, Figs. S5 and S6). Furthermore, mass spectrometric analysis was done to confirm the ferric ion attachment to the ligand; there was a characteristic signal at m/z 610 which gives the correct mass of ligand–iron complex (Supporting information, Fig. S8). On the basis of these observations, the proposed ligand–iron ligation mechanism was outline in Scheme 2. 3.3. Spectroscopic properties The spectroscopic properties of ligand were performed in aqueous/methanol (60:40, v/v) and the corresponding spectra are shown in Figure 1. Free probe 3 is colorless and non-fluorescence while upon addition of ferric ion into probe solution leads to tremendous increase in the fluorescence intensity with maximum emission at 599 nm as well as UV–vis absorption with absorption maxima of 557 nm. Furthermore, probe–metal complex trigger a color change of the solution for colorimetric detection of ferric ion. To find out the minimum detection ability of ligand 3, a fluorescence titration experiment was conducted by varying the concentration of
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ferric ion keeping constant ligand concentration. A linear relationship was observed on increasing ferric ion concentration up to 15 lM at maximum emission of 599 nm in aqueous/methanol (60:40, v/v) at pH 7.0. Further increasing the ferric ion concentration from 15 to 18 lM, there was slightly more increment in the fluorescent intensity of ligand solution. These results indicates that ferric ion triggered ligand ring opening reaction rate increased at higher ferric ion concentration as shown in the inset of Fig. 2. From this titration experiment, the minimum detection ability of ligand toward ferric ion was estimated to be about 3 lM as shown in Fig. 2. To find out the specificity of ligand 3 toward common metal ions, the sensing properties of ligand 3 were examined by addition of various metal ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v, pH 7.0.) at ambient temperature, into the ligand solution under same condition. The competing metal ions exhibited negligible fluorescence enhancement. By contrast, the most distinctive fluorescence intensity enhancement resulted upon ferric ion addition indicates that ligand 3 possesses selective ligation tendency toward ferric ions (Fig. 3). To understand the ligand–metal chelation reaction time, the fluorescent intensity of ligand–metal complex was measured with different time interval. The complexation reaction between ligand and ferric ion started immediately which can be visualized through naked eye by the solution color transformation from colorless to pink as well as fluorescent experiment. The fluorescence intensity of ligand–metal complex becomes stable within five minutes after metal addition as shown in Figure 4; provides quick sensing methodology for Fe3+ in natural physiological conditions. 3.3.1. Interfering ion effect In order to study the influence of other metal ions on ligand– iron binding, the competitive experiments were carried out in the presence of Fe3+ with other metal ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v, pH 7) at ambient temperature. The results indicates that competing metal ions did
Scheme 1. Synthesis of ligand 3: Reagents and conditions. (i) Compound 1, BOC-D-Alanine, DCC, DMAP, MC, 3 h, rt; (ii) TFA:MC, 3:1, v/v, 3 h, rt.
Scheme 2. The proposed spirolactam ring opening mechanism of ligand 3 into 4 upon ferric ion addition in aqueous/methanol (60:40, v/v) at pH 7.0 and corresponding KCN triggered reversibility of complex 4 back to 3.
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Figure 1. (A) Fluorescence emission spectrum of ligand 3 (3 lM) in the presence and absence of Fe3+ (6 lM) in aqueous/methanol (60:40, v/v) at pH 7.0, (B) UV–visible absorption spectrum of ligand 3 (6 lM) in the presence and absence of Fe3+ (12 lM) in aqueous/methanol (60:40, v/v) at pH 7.0.
Figure 2. The fluorescence titration of ligand 3 (3 lM) at emission maxima of 599 nm as a function of ferric ion concentration (3–18 lM, 1–6 equiv), the inset described the fluorescence enhancement at maximum emission of 599 nm; F/Fo is determined as a ratio between the maximum fluorescence intensity (F, after Fe3+ addition) and minimum fluorescence intensity (Fo, free ligand solution in absence of Fe3+).
Figure 3. Fluorescence emission spectrum of ligand (3 lM) in the presence of Fe3+ (18 lM, 6 equiv) and competing ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) (18 lM, 6 equiv) in aqueous/ methanol (60:40, v/v) at pH 7.0.
not interfere ferric ion chelation with ligand as there was no or negligible effect on the fluorescence intensity of ligand–metal complex in the presence of various metal ions. However, KCN
Figure 4. Effects of reaction times on the fluorescent intensity of ligand (3 lM) at maximum emission of 599 nm in the presence of Fe3+ (5–9 lM) in aqueous/ methanol (60:40, v/v) at pH 7.0.
Figure 5. The fluorescence intensity contrast bars in order to investigate the interference effect of other metal ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v) at ambient temperature on the detection ability of ligand for Fe3+, red bars represent the fluorescence response of ligand (3 lM) to Fe3+ (18 lM) in the presence of interfering metal ions (18 lM) and black bars represent the fluorescence intensity of ligand (3 lM) with competing ions (18 lM) in the absence of Fe3+.
reverse the ligand–metal complex to the backward direction and caused the complete quenching of fluorescence as shown in Figure 5.
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Figure 6. Fluorescence intensity variation at maximum emission of 599 nm, with alteration of pH from 2 to 11, in MeOH/Tris–HCl buffer solution.
by spontaneous measurement of UV-visible absorption spectra. The results clearly showed that organic solvent exert continuous increment on the absorption intensity of probe–metal complex formation up to 40%, with further increase in concentration of organic solvent; there was a negligible effect on the peak intensity. These results inspired us to use 40% aqueous organic media for further measurements. Conversely, acetonitrile exhibited reverse effect on the ligand–metal complexation reaction. In acetonitrile solvent system, there was no UV-visible absorption as shown in Figure 7. This may be due to strong affinity of ferric ion toward cyanide ion. To confirm this, we have further investigated the ligand–iron complexation in the presence of KCN, which cause complete quenching of UV-visible absorption and this experiment support our expectation of reversible nature of ligand in the presence of cyanide ion. While, there was no absorption signal for ligand in the given range. The effect of different organic solvent on the fluorescence emission and UV-visible absorption spectra and corresponding intensity values are tabulated in Table 1. The Stokes shift was calculated by Eq. 1.29
ðtA tFÞ ¼
1 1 kA kF
107
ð1Þ
3.4. Bioimaging applications of ligand 3 in L-929 and BHK-21 cell lines
Figure 7. Variation of absorption intensity of ligand 3 upon two equivalent addition of ferric ion at absorption maxima of 557 nm with different aqueous/organic solvent ratio.
3.3.2. pH effect The effect of pH was evaluated in MeOH/Tris–HCl buffer in the pH range of 2–11 as shown in Figure 6. The probe works well in neutral pH condition which is more suitable for ferric ion detection in the biological fluid and living cells under neutral pH range. However, the sensor molecules are sensitive to acid with pH less than 5 that bring spirolactam ring opening of the ligand by the activation of ligand spirolactam carbonyl while there was no acid induced ring opening in MeOH/Tris–HCl buffer under pH span of 5–11. This property of ligand makes it a reliable source for ferric ion detection in natural water system and in living cells. 3.3.3. Effect of media Solvent showed direct effect on the ligand–iron chelation. A suitable ratio of aqueous organic solvent was finding out by varying the concentration of methanol into aqueous solution followed
3.4.1. Bioimaging applications of ligand 3 in L-929 cells (mouse fibroblast cells) To explore the sensing performance of the ligand 3 toward Fe3+ in biological samples, fluorescence imaging experiments were performed using L-929 (mouse fibroblast cells) cells on confocal fluorescent microscope following the reported procedure30 with slight modification. Briefly, L-929 cells were incubated with ligand (5 lM) in complete MEM (minimum essential media) for 3 h at 30 °C, and very week fluorescence was observed. The samples were washed with PBS three times and then treated with various concentrations of Fe3+ ranging from 5 to 25 lM for 30 min at 37 °C, which displayed bright intracellular fluorescence depending upon the Fe3+ concentrations. The results of confocal fluorescence microscopic experiments against L-929 (mouse fibroblast cells) are given in Figure 8. 3.4.2. Bioimaging applications of ligand 3 in BHK-21 cells (hamster kidney fibroblast) To investigate the practical applicability of ligand 3 toward several cells of living system, the different cell lines were utilized including BHK-21 cells (hamster kidney fibroblast). The confocal fluorescent microscopic experimental results for BHK-21 (hamster kidney fibroblast) are shown in Figure 9; suggesting extensive applicability of synthesized ligand over a wide biological cell lines. 3.5. Cytotoxicity assay Furthermore, the ligand cytotoxicity was accessed after 24 h treatment to the cells by MTT assay, a frequently used method in
Table 1 Solvent effect on the spectroscopic properties of ligand upon treating with ferric ion
a b c
S. No.
Solvent
F/Foa
Emission maxima (nm)
Ab
Absorption maxima (nm)
Stokesshift (cm1)
1 2 3
MeCN: H2O (40:60, v/v) MeOH: H2O (40:60, v/v) EtOH: H2O (40:60, v/v)
3.27 181 174
599 599 599
0.001 0.475 0.471
557 557 557
—c 1258 1258
F/Fo = Fluorescence intensity of 3 lM ligand at emission maxima 599 nm, in the presence and absence of 6 lM Fe3+. A = Absorption signal intensity of 6 lM ligand at absorption maxima 557 nm, in the presence of 12 lM Fe3+. — = Absorption and emission intensity in case of MeCN solvent was quenched, so, Stokes shift was not calculated in aqueous MeCN media.
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Figure 8. Confocal fluorescence microscopic images for L-929 cells; (A1-A3) L-929 cells incubated with probe (5 lM); (B1-B3) L-929 cells incubated with probe (5 lM) in the presence of (5 lM) ferric ion; (C1-C3) L-929 cells incubated with probe (5 lM) in the presence of (10 lM) ferric ion; (D1-D3) L-929 cells incubated with probe (5 lM) in the presence of (25 lM) ferric ion. A1-D1: Bright field images; A2-D2: fluorescence images: A3-D3: merged images.
biology for cells toxicity. The results showed no toxicity for both BHK-2 (hamster kidney fibroblast) and L-929 (mouse fibroblast cells) cells as shown in Figure 10. The non-toxic behavior of ligand
3 at low doses and its ability to track the change in Fe3+ level in living cells suggest its possibility to use in biological system for Fe3+ detection.
Figure 9. Confocal fluorescence microscopic images for BHK-21 cells; (A1-A3) BHK-21 cells incubated with probe (5 lM); (B1-B3) BHK-21 cells incubated with probe (5 lM) in the presence of (5 lM) ferric ion; (C1-C3) BHK-21 cells incubated with probe (5 lM) in the presence of (10 lM) ferric ion; (D1-D3) BHK-21 cells incubated with probe (5 lM) in the presence of (25 lM) ferric ion. A1-D1: Bright field images; A2-D2: fluorescence images: A3-D3: merged images.
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Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2010-0027963). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.045. References
Figure 10. Cell viability of BHK-21 and L-929 cells cultured in complete media with 5 lM of ligand 3 and the control cells were cultured in the medium without ligand.
4. Conclusion In summary, alanine substituted rhodamine B derivative 3 was successfully prepared and its sensing capability for various metal ions were examined. The ligand exhibited high selectivity for Fe3+ over various metal ions including Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+ in aqueous/methanol (60:40, v/v) at neutral pH. The ligand was sensitive enough to detect the micro molar concentration of ferric ion with turn-on colorimetric as well as fluorescence change that was unaffected by the presence of other common coexisting metal ions, however, the recognition process was reversible confirmed by addition of KCN. Furthermore, confocal fluorescence microscopic experiments utilizing L-929 cells (mouse fibroblast cells) as well as BHK-21 (hamster kidney fibroblast), showed that the ligand exhibited good biocompatibility for cell imaging study with precise cell permeability and low toxicity against the tested cell lines. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0015056) and the National Research Foundation of
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