Cationic Two-Photon Lanthanide Bioprobes Able to Accumulate in

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Cationic Two-Photon Lanthanide Bioprobes Able to Accumulate in Live Cells Anh Thy Bui,† Maryline Beyler,‡ Yuan-Yuan Liao,† Alexei Grichine,§ Alain Duperray,§ Jean-Christophe Mulatier,† Boris Le Guennic,∥ Chantal Andraud,† Olivier Maury,*,† and Raphael̈ Tripier*,‡ †

Univ Lyon, ENS Lyon, CNRS, Université Lyon 1, Laboratoire de Chimie, UMR 5182, 46 allée d’Italie, 69364 Lyon, France Université de Brest, UMR-CNRS 6521, UFR des Sciences et Techniques 6 avenue Victor le Gorgeu, C.S. 93837, 29238, Brest Cedex 3, France § INSERM, U1209, Université Grenoble Alpes, IAB, F-38000 Grenoble, France ∥ Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS, Université de Rennes 1, 263 Avenue du Général Leclerc, 35042 Rennes Cedex, France ‡

S Supporting Information *

ABSTRACT: An original cationic water-soluble cyclen-based Eu(III) complex [EuL1]+ featuring a chromophore-functionalized antenna to increase the two-photon (2P) absorption properties was synthesized. The photophysical properties were thoroughly studied in various solvents and rationalized with the help of theoretical calculations. The complex exhibits an optimized 2P absorption cross section. Finally, 2P microscopy imaging experiments on living T24 human cancer cells highlighted the spontaneous internalization and the biological stability of this 2P bioprobe in vitro. Macrocyclic-based antennas open new perspectives for future optimization of the photophysical properties and allows envisaging the design of Eu, Tb, Yb, and Sm bioprobes. This result also opens the way for the design of functional two-photon Ln complexes able to monitor intracellular physicochemical parameters.

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INTRODUCTION For about one decade, two-photon (2P) microscopy imaging using lanthanide complexes as probes has emerged as a valuable alternative to the classical one-photon confocal microscopy.1 The main achieved goal is to combine the intrinsic advantages of 2P excitation (three-dimensional resolution, NIR excitation localized in the biological transparency window) to the unique f-block element photophysical properties (sharp emission, long excited-state lifetime).2 Numerous examples of 2P cell imaging using Eu3+, Tb3+, Yb3+, and Sm3+ complexes have been reported in the literature3 including proofs of concept for 2Pmultiplexing,4 2P-time-gated (PSLIM), and 2P-lifetime (TSLIM) imaging.5 Optimization of the 2P absorption (2PA) cross-section requires the use of highly conjugated antennae,1,2 whereas the hydrosolubilization of the complex in biological media requires further functionalization with hydrophilic groups (PEG, sulfo-betaine, etc).6 The internalization of 2P lanthanide complexes into live cells remains a crucial point for the optimization of real bioprobes. The internalization mechanism has been thoroughly studied for one-photon lanthanide bioprobes, in particular, by the group of Parker. They demonstrated that cationic complexes based on cyclen or TACN chelates are easily internalized via macropinocytosis, a © XXXX American Chemical Society

form of nonspecific endocytosis involving the formation of vesicles internalized by invagination of the plasma membrane.6 In addition, Bünzli and co-workers described the rapid cell uptake of neutral binuclear europium(III) helicates via a similar mechanism,7a whereas the group of Miller reported the bioconjugation of the Lumi4(Tb) complex with cell-penetrating peptides to achieve internalization.7b In contrast, up to now the complexes featuring optimized 2P antennae, which were classically neutral or trianionic, were not spontaneously internalized in live cells, and most of the imaging experiments were performed on fixed cells or after membrane permeation using, for instance, dimethyl sulfoxide or saponine.3a,e−i,4,5 In the present paper, we describe the synthesis of an original cationic cyclen-based Eu(III) complex [EuL1]+ (Figure 1) inspired from the recently reported [EuMedo2pa]+ platform,8 further functionalized with conjugated antenna to increase the 2PA properties. The photophysical properties were thoroughly studied in various solvents and rationalized with the help of theoretical calculations. Finally 2P microscopy imaging experiReceived: April 11, 2016

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DOI: 10.1021/acs.inorgchem.6b00891 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

Figure 1. Structure of the complexes [EuMedo2pa]+ (left) and [EuL1]+ (middle) and optimized geometry of [YL1′]+ (right). Y, O, N, and C atoms are in blue, red, purple, and gray, respectively. H atoms are removed for clarity.

centered around 345 nm, signature of a intra-ligand charge transfer transition (ILCT). Upon excitation in this lowestenergy ILCT transition (320−360 nm), the characteristic Eu3+ emission profile is obtained (Figure 3). The relative intensity pattern of the 5D0 → 7FJ (J = 0−4) transitions are 0.1, 1, 5, 0.6, 2.5 with an intense J = 4 transition signature of a low-symmetry environment of Eu3+ (here c2 according to DFT calculations). It is worth noting that the quantum yields are high in nonprotic solvents with different polarity like dichloromethane and acetonitrile and dramatically drop in methanol and water (Table 1). The luminescence lifetimes follow exactly the same trend. Such observations generally suggest the coordination of a water or methanol molecule, resulting in a luminescence quenching due to the coordinated O−H vibrator. However, in the present case, the lifetime measured in deuterated methanol (τ(CD3OD) = 0.76 ms) is close to that in CH3OH (Figure S3) and in deuterated water (τ(D2O) = 0.75 ms) suggesting a partial hydration (q = 0.5).9 Furthermore, the comparison of the I(J=1)/Itot in the four different solvents gives exactly the same value (Figure S4). Since Eu3+ emission is very sensitive to any modification of its coordination polyhedron, this result demonstrates that the symmetry around the metal is identical in all solvents excluding any water or solvent coordination. Finally the optimized geometry of [YL1′(H2O)]+ never converges to a stable structure with the water molecule directly coordinated to yttrium independently of the starting position of the coordinated water (on the c2 axis (Figure 4, bottom) or on the side of the available cavity (Figure 4, top). The final optimized geometries consist in [YL1′]+ with

ments were performed highlighting the spontaneous internalization of this 2P bioprobe into living T24 human cancer cells.

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RESULTS AND DISCUSSION Synthesis. Synthesis of ligand L1 was achieved by a trans-Ndialkylation of dimethyl cyclen (1)8 with the mesylated derivative of the picolinate chromophore 2 followed by saponification of the methyl ester groups in a biphasic mixture of tetrahydrofuran (THF)/MeOH/KOH(aq) (Scheme 1). The europium complex [EuL1]Cl was prepared in water at pH = 6 by mixing L1 and an excess of europium salt (1.5 equiv). Its purity was ensured by analytic high-performance liquid chromatography (HPLC; see Supporting Materials, Figures S1 and S2) and characterized by 1H NMR and high-resolution mass spectrometry (HRMS). To circumvent the lack of X-ray structure for complex [EuL1]+, geometry optimization using density functional theory (DFT) was realized on the yttrium parent [YL1′]+, where the difference between L1′ and L1 consists in the replacement of the O(CH2CH2O)3-Me moieties by O(CH2)2OMe (see computational details in Supporting Information). The obtained geometry strongly resembles the one of the parent [EuMedo2pa]+ complex.8 Importantly, as shown in Figure 1, a c2 symmetry axis is found in the optimized structure. Photophysical Measurements. The photophysical properties were measured in water and organic solvents (methanol, dichloromethane, and acetonitrile), and the data are compiled in Table 1. As expected for this type of antenna, the absorption spectrum (Figure 2) shows a broad, structureless band, B


Article

Inorganic Chemistry Scheme 1. Synthesis of Ligand L1 and Its Europium Complex

Table 1. Photophysical Data of [EuL1]Cl in Different Solvents at Room Temperature solvent

λabs (nm)

ε (L·mol−1·cm−1)

Φ (%)

τ (ms)

I(J=1)/Itot

H2O CH3OH CH2Cl2 CH3CN

341 345 349 339

40 500 47 000 43 000 45 000

1a 7a 35b 36b

0.47 0.67 1.03 1.16

0.11 0.10 0.11 0.11

coordination sphere (Figure 4, top), the latter being slightly energetically preferred (8.7 kcal·mol−1). As a consequence, the strong decrease of the quantum yield in protic solvents (H2O and to a less extent MeOH) compared to dichloromethane cannot be explained by water (respectively, methanol) coordination, but it can be ascribed to the increase of dynamic fluctuation of the complex induced by the formation of H-bonds with the carbonyl moieties. Similar behavior has already been observed for bipyridine azamacrocyclic Eu3+ complexes featuring identical antennae.11 This preliminary hypothesis will be further developed in a forthcoming study. The 2P absorption properties were measured in methanol using two-photon excited fluorescence (TPEF) method using a Ti/sapphire laser source (700−900 nm) and coumarin 307 as external standard. Upon excitation at 750 nm, the characteristic

Using quinine sulfate in H2SO4 1N as standard (Φ = 54.6%, λex = 345 nm). bRelative to the data measured in MeOH. Errors in quantum yield and lifetime are ±10%. a

the water molecule linked either (i) in the second coordination sphere by a strong hydrogen bond with the carbonyl group (Figure 4, bottom) or (ii) to an O atom of the first C


Article

Inorganic Chemistry

and at the plasmalemma in spotlike cytoplasmic structures. The long-term cell incubation (28 h) in the presence of the complex does not impair cell viability or proliferation (Figure S7), and the staining remains present in dividing mitotic cells (Figure S7). The unambiguous identification of [EuL1]Cl inside living cells was performed with the help of emission spectroscopy under confocal spectral microscope and the measurements of luminescence lifetime by TSLIM method (Figure S8). A lifetime of 0.51 ms is measured in the cells by TSLIM, almost identical to that measured in water using classical methods. To get better insight of cytosolic localization of [EuL1]Cl, the T24 cells, loaded with [EuL1]Cl, were costained with 10 μM of Nil Red, a common lipophilic membrane stain (Figure 5). After few minutes of incubation, the plasma membrane and intracellular vesicles were imaged in confocal mode under 561 nm excitation. Most of the vesicular structures, labeled with Nile red, corresponded well to the structures containing [EuL1]+ complex. Such localization may suggest the endocytic or pinocytic internalization pathway of the hydrophilic [EuL1]+ complex. Unfortunately, the strong spectral overlap between the two probes (550−630 nm) and the long luminescence lifetime of Eu3+ required a slow-scan sequential acquisition of the two channels. Thus, the small fast-moving cytosolic vesicles were imaged with a temporal delay and do not show the exact spatial colocalization.

Figure 2. Normalized absorption spectrum of [EuL1]Cl in MeOH at RT (red line, lower abscissa). Superimposed on this plot is the 2P absorption measured by TPEF method in methanol in wavelength doubled scale (■, upper abscissa).

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CONCLUSION The cationic cyclen-based Eu(III) complex [EuL1]+, soluble in water and stable in biological medium, exhibits optimized 2P antenna ligand, and for the first time is spontaneously internalized into live cells. This platform opens new perspectives for future optimization of the photophysical properties and allows envisaging the design of Eu, Tb, Yb, and Sm bioprobes. This result also opens the way for the design of functional two-photon Ln complexes able to monitor intracellular physicochemical parameters; these projects are currently developed in our group.

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Figure 3. Normalized emission spectrum of [EuL1]Cl in MeOH at RT (λex = 345 nm).

EXPERIMENTAL SECTION

Synthesis. Reagents were purchased from ACROS Organics and from Aldrich Chemical Co. Cyclen was purchased from Chematech (Dijon, France). Dimethyl-cyclen (1) 8 and pegylated 6(methylmethanesulfonate)pyridine-2-carboxylic acid methyl ester (2)11 were synthesized as previously described. The solvents were freshly distilled prior to use and according to the standard methods. The analytic HPLC was performed on a Prominence Shimadzu HPLC/LCMS-2020 equipped with a UV SPD-20 A detector. The chromatographic system employs HPLC (VisionHT C18 HL 5 μ 250 × 4.6 mm) with 0.1% aqueous trifluoroacetic acid−MeCN (v/v) as eluents [isocratic 10% MeCN (4 min), linear gradient from 10 to 90% MeCN (6 min), isocratic 90% MeCN (4 min)] at a flow rate of 1 mL/ min and UV detection at 254 and 350 nm. NMR spectra (1H and 13C) were recorded at the core facilities of the University of Brest, with Bruker Avance 500 (500 MHz) or Bruker AMX 300 (300 MHz) spectrometers. The HRMS analyses were performed at the Institute of Analytic and Organic Chemistry, ICOA in Orléans. Compound 3. A solution of compound 2 (415 g, 0.82 mmol) in dry CH3CN (15 mL) was added dropwise to a solution of 1 (81 g, 0.40 mmol) and K2CO3 (226 mg, 4 equiv) in the same solvent (10 mL). The mixture was stirred at reflux for 3 d. The solution was cooled to room temperature (RT), and K2CO3 was filtrated off and washed with CH3CN (3 × 5 mL). The filtrate was evaporated and taken up in CH2Cl2 and washed with water (2 × 20 mL). The organic layer was dried under MgSO4, filtrated, and concentrated under vacuum. The mixture was purified by flash chromatography using C18 reverse phase

europium(III) emission is obtained and displays a quadratic variation of intensity with respect to the laser intensity, signature of a 2P antenna effect (Figures S5 and S6). As expected for a non-centrosymmetric compound, the 2P absorption spectrum (Figure 2) matches very well the wavelength-doubled one-photon absorption, indicating that the low energy ILCT transition responsible for the europium(III) sensitization is one- and two-photon allowed. Because of the spectral restriction of the laser source the maximum of 2P cross section is not achieved, and only the red tail of 2P spectrum can be measured. At 700 nm, the 2P cross section is ∼100 GM, a value in the same range as that of other complexes featuring similar antenna chromophores.3a,10,11 Confocal and Two-Photon Microscopy. Biphotonic imaging of live T24 cells was performed. A solution of [EuL1]Cl in phosphate buffer was added to the culture medium, and strongly red emissive microaggregates are immediately observed outside of the cells under near UV mercury lamp excitation. After 4 h of incubation, [EuL1]Cl is internalized in cells and accumulated in the perinuclear zone D


Article

Inorganic Chemistry

Figure 4. Initial structure with a water molecule directly coordinated to the Y center (a, b) and final optimized geometries (c, d) of [YL1′(H2O)]+. Only the central coordination sphere is represented for clarity, the rest of the antenna being suppressed.

Figure 5. Confocal imaging of live T24 cells costained for 4 h with [EuL1]Cl (C = 10−5 mol·L−1, 2P excitation at λex = 780 nm) (left) and Nile red (C = 10 μM, 1P excitation at λex = 561 nm) (middle). Colocalization of both labels and transmitted light DIC image (right). Compound L1. Compound 3 (150 mg, 0.15 mmol) was dissolved in 10 mL of a mixture of THF/MeOH (8/2), and an aqueous solution of KOH 6 M (2 mL) was added. The mixture was vigorously stirred at 80 °C for 24 h. The mixture was cooled to RT and poured into a separating funnel, and the organic layer was recovered and concentrated under vacuum; L1 was used without further purification. HRMS: m/z: 498.2600 [MH2+2H]+ calcd. 498.2599 for C54H70N6O12 + 2H+. Compound [EuL1]Cl. The previously hydrolyzed ligand was dissolved in water, and the pH of the solution was adjusted to 6 by addition of 1 M HCl. Then EuCl3·6H2O was added (82 mg, 1.5 equiv). The solution was stirred under argon for 1 h. The pH of the solution was measured and increased to 5.5 by addition of solid K2CO3. The mixture was further heated to reflux overnight. A cloudy solution was obtained and was then centrifuged. The filtrate was recovered, and the solvent was evaporated to dryness. The solid was taken up in acetonitrile and was only partially soluble. The solid was filtrated off. The filtrate was evaporated to give [EuL1]Cl as a yellow

column (40 g) eluted with water/acetonitrile (20:80 to 0:100). The compound stocked on the column and was finally recovered by elution with CH2Cl2 to give compound 3 as yellow oil. (245 mg; r = 60%). 1H NMR (300 MHz; CDCl3; 298 K) δ (ppm): 7.98 (s, 2 H), 7.56 (s, 2 H), 7.48 (d, 4 H, 3J = 6.6 Hz), 6.92 (d, 4 H, 3J = 6.3 Hz), 4.16 (t, 4 H, 4 J = 3 Hz), 3.92 (s, 2 H), 3.86 (t, 4 H, 4J = 3.6 Hz), 3.73−3.63 (m, 12 H), 3.57 (s, 6 H), 3.55−3.54 (m, 4 H), 3.46 (s, 2 H), 3.36 (s, 6 H), 3.07 (bs, 2 H), 2.86 (bs, 2 H), 2.48 (vbs, 12 H), 1.85 (s, 6 H). 13C NMR (Jmod; 100 MHz; CDCl3; 298 K) δ (ppm): 165.1 (CO), 159.9 (Cquat Pico), 158.9 (Cquat Pico), 158.5 (Cquat Pico), 147.6 (Cquat Ph), 133.7 (CH Ph), 127.6 (CH Pico), 125.7 (CH Pico), 114.9 (CH Ph), 113.5 (Cquat Ph), 96.2 (Cquat acetylene), 84.8 (Cquat acetylene), 71.8 (CH2−Peg), 71.8 (CH2−Peg), 70.8 (CH2−Peg), 70.5 (CH2−Peg), 70.4 (CH2−Peg), 69.5 (CH2−Peg), 67.46 (CH2−Peg), 62.0 (CH2− Pico), 58.6 (O−CH3 Pico), 55.0 (CH2 cyclen), 52.8 (O−CH3 Peg), 48.3 (CH2 cyclen), 42.36 (CH3 cyclen). HRMS: m/z: 1023.5430 [M + H]+ calcd. 1023.5437 for C56H74N6O12 + H+. E


Article

Inorganic Chemistry oil. (88 mg; r = 50% calculated from compound 3). 1H NMR (500 MHz; CDCl3; 298 K) δ (ppm): 37.59, 26.24, 6.98, 6.74, 4.03, 3.76, 3.64, 3.60−3.56, 3.47, 3.30, 2.37, 1.65, −0.05, −2.53, −6.65, −9.04, −11.29, −12.99. HRMS: m/z: 1145.4118 [M]+ calcd. 1145.4010 for C54H68EuN6O12.

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(6) (a) New, E. J.; Parker, D.; Smith, D. G.; Walton, J. W. Curr. Opin. Chem. Biol. 2010, 14, 238−246. (b) Butler, S. J.; McMahon, B. K.; Pal, R.; Parker, D.; Walton, J. W. Chem. - Eur. J. 2013, 19, 9511−9517. (7) (a) Deiters, E.; Song, B.; Chauvin, A.-S.; Vandevyver, C. D. B.; Gumy, F.; Bünzli, J.-C. G. Chem. - Eur. J. 2009, 15, 885−900. (b) Mohandessi, S.; Rajendran, M.; Magda, D.; Miller, L. W. Chem. Eur. J. 2012, 18, 10825−10829. (8) Rodríguez-Rodríguez, A.; Esteban-Gomez, D.; de Blas, A.; Rodríguez-Blas, T.; Fekete, M.; Botta, M.; Tripier, R.; Platas-Iglesias, C. Inorg. Chem. 2012, 51, 2509−2521. (9) The hydration number q is determined according to the equation q = 1.11 (k(H2O) − k(D2O) −0.31; see Supkowski, R. M.; Horrocks, W. D., Jr. Inorg. Chim. Acta 2002, 340, 44−48. (10) Soulié, M.; Latzko, F.; Bourrier, E.; Placide, V.; Butler, S. J.; Pal, R.; Walton, J. W.; Baldeck, P. L.; Le Guennic, B.; Andraud, C.; Zwier, J. M.; Lamarque, L.; Parker, D.; Maury, O. Chem. - Eur. J. 2014, 20, 8636−8646. (11) Bourdolle, A.; Allali, M.; Mulatier, J.-C.; Le Guennic, B.; Zwier, J.; Baldeck, P. L.; Bünzli, J.-C. G.; Andraud, C.; Lamarque, L.; Maury, O. Inorg. Chem. 2011, 50, 4987−4999.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b00891. General details of photophysical measurements, computational calculations and cell culture, semipreparative chromatograms, emission and luminescence spectra, 2P microscopy (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (O.M.) *E-mail: [email protected]. (R.T.) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors acknowledge the support of the Ministère de l’Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique, and the Institut National de la Santé et de la Recherche Médicale. R.T. thanks the “Service Commun de RMN” of the Univ. of Brest. Confocal and 2P facility of the IAB platform was cofunded thanks to grants of “Association pour la Recherche sur le Cancer” (ARC, Villejuif, France), “Ligue Contre le Cancer” (LCC Isère/ Ardèche), and the CPER program.

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REFERENCES

(1) (a) D’Aléo, A.; Andraud, C.; Maury, O. Luminescence of Lanthanide Ions in Coordination Compounds and Nanomaterials; De Bettencourt-Diaz, A., Ed.; Wiley, 2014; pp 197−226. (b) Bünzli, J.-C. G. ibid., pp 125−196. (2) (a) Andraud, C.; Maury, O. Eur. J. Inorg. Chem. 2009, 4357− 4371. (b) Ma, Y.; Wang, Y. Coord. Chem. Rev. 2010, 254, 972−990. (3) (a) Picot, A.; D’Aléo, A.; Baldeck, P. L.; Grichine, A.; Duperray, A.; Andraud, C.; Maury, O. J. Am. Chem. Soc. 2008, 130, 1532−1533. (b) Law, G.-L.; Wong, K.-L.; Man, C. W.-Y.; Wong, W.-T.; Tsao, S.W.; Lam, M. H.-W.; Lam, P. K.-S. J. Am. Chem. Soc. 2008, 130, 3714− 3715. (c) Kielar, F.; Congreve, A.; Law, G.-L.; New, E. J.; Parker, D.; Wong, K.-L.; Castreno, P.; De Mendoza, J. Chem. Commun. 2008, 2435−2437. (d) Eliseeva, S. V.; Auböck, G.; van Mourik, F.; Cannizzo, A.; Song, B.; Deiters, E.; Chauvin, A.-S.; Chergui, M.; Bünzli, J.-C. G. J. Phys. Chem. B 2010, 114, 2932−2937. (e) Hu, Z.-J.; Tian, X.-H.; Zhao, X.-H.; Wang, P.; Zhang, Q.; Sun, P.-P.; Wu, J.-Y.; Yang, J.-X.; Tian, Y.P. Chem. Commun. 2011, 47, 12467−12469. (f) Lo, W.-S.; Kwok, W.M.; Law, G.-L.; Yeung, C.-T.; Chan, C. T.-L.; Yeung, H.-L.; Kong, H.K.; Chen, C.-H.; Murphy, M. B.; Wong, K.-L.; Wong, W.-T. Inorg. Chem. 2011, 50, 5309−5311. (g) Baggaley, E.; Cao, D.-K.; Sykes, D.; Botchway, S. W.; Weinstein, J. A.; Ward, M. D. Chem. - Eur. J. 2014, 20, 8898−8903. (h) D’Aléo, A.; Bourdolle, A.; Brustlein, S.; Fauquier, T.; Grichine, A.; Duperray, A.; Baldeck, P. L.; Andraud, C.; Brasselet, S.; Maury, O. Angew. Chem., Int. Ed. 2012, 51, 6622−6625. (i) Bui, A.T.; Grichine, A.; Brasselet, S.; Duperray, A.; Andraud, C.; Maury, O. Chem. - Eur. J. 2015, 21, 17757−17761. (4) Placide, V.; Bui, A.-T.; Grichine, A.; Duperray, A.; Pitrat, D.; Andraud, C.; Maury, O. Dalton Trans. 2015, 44, 4918−4924. (5) Grichine, A.; Haefele, A.; Pascal, S.; Duperray, A.; Michel, R.; Andraud, C.; Maury, O. Chem. Science 2014, 5, 3475−3485. F


Cationic Two-Photon Lanthanide Bioprobes Able to Accumulate in

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