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Fluorescent BODIPY-anionic boron cluster conjugates as potential agents for cell tracking mahdi chaari, Nerea Gaztelumendi, Justo Cabrera-González, Paula Peixoto-Moledo, Clara Viñas, Elba Xochitiotzi-Flores, Norberto Farfan, Abdelhamid Ben Salah, Carme Nogues, and Rosario Nuñez Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00204 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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Bioconjugate Chemistry

Fluorescent BODIPY-anionic boron cluster conjugates as potential agents for cell tracking

Mahdi Chaari,1,4† Nerea Gaztelumendi,2† Justo Cabrera-González,1# Paula Peixoto-Moledo,2 Clara Viñas,1 Elba Xochitiotzi-Flores,3 Norberto Farfán,3 Abdelhamid Ben Salah,4 Carme Nogués,2* Rosario Núñez1*

1

Instituto de Ciencia de Materiales de Barcelona (ICMAB-CSIC), Campus de la UAB, E-

08193 Bellaterra (Barcelona), Spain. [email protected] 2

Departament de Biologia Cel·lular, Fisiologia i Immunologia. Universitat Autònoma de

Barcelona, E-08193 Bellaterra (Barcelona), Spain. [email protected] 3

Facultad de Química, Departamento de Química Orgánica, Universidad Nacional

Autónoma de México, 04510 México D. F., México. 4

Laboratoire des Sciences des Matériaux et de l’Environnement, Faculté des Sciences de

Sfax, Université de Sfax, B.P. 1171, 3000 Sfax, Tunisie.

†

M.C. and N.G. contributed equally to this work.

#

Current address: Department of Chemistry, Trinity College Dublin, Dublin 2, Ireland.

1 ACS Paragon Plus Environment

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Abstract A series of novel fluorescent BODIPY-anionic boron cluster conjugates bearing [B12H12]2- (5, 6), [3,3′-Co(1,2-C2B9H11)2]- (7, 8), and [3,3′-Fe(1,2-C2B9H11)2]- (9) anions have been readily synthesized

from

meso-(4-hydroxyphenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

(BODIPY 4), and their structure and photoluminescence properties have been assessed. Linking anionic boron clusters to the BODIPY (4) does not alter significantly the luminescent properties of the final fluorophores, showing all of them similar emission fluorescent quantum yields (3-6%). Moreover, the cytotoxicity and cellular uptake of compounds 5-9 have been analyzed in vitro at different concentrations of B (5, 50 and 100 µm B/ml) using HeLa cells. At the lowest concentration, none of the compounds shows cytotoxicity and they are successfully internalized by the cells, especially compounds 7 and 8, which exhibit a strong cytoplasmic stain indicating an excellent internalization efficiency. To the best of our knowledge, these are the first BODIPY-anionic boron cluster conjugates developed as fluorescent dyes aiming at prospective biomedical applications. Furthermore, the cellular permeability of the starting BODIPY (4) was improved after the functionalization with boron clusters. The exceptional cellular uptake and intracellular boron release, together with the fluorescent and biocompatibility properties make compounds 7 and 8 good candidates for in vitro cell tracking.


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Introduction The

purely

inorganic

metallacarborane

anions

[M(C2B9H11)2]-

(M

=

Co,

cobaltabisdicarbollide, commonly known as COSAN and M = Fe, ferrabisdicarbollide or FESAN) are boron cluster-based complexes in which two dicarbollide ligands, [C2B9H11]2-, are coordinated η5 to Co or Fe metals.1-2 These anions possess exceptional thermal and chemical stability.3-7 The protonated form and sodium salts of [3,3’-Co(C2B9H11)2]- anion (COSAN) have shown special physicochemical properties, such as amphiphilicity that makes COSAN being soluble in both water and organic solvents,8-14 but most remarkable is the property to self-assemble into micelles and monolayer vesicles in water.11, 15-16 It is noteworthy that COSAN is able to pass synthetic membranes with zero order kinetics,17 it can be accumulated in vitro within living cells,18 and it has shown antimicrobial activity.19 In this sense, the incorporation of COSAN-containing fluorescein probes inside phospholipid bilayers has allowed their visualization by fluorescence lifetime imaging.20 On the other hand, when radiolabelling COSAN with

125

I and

124

I, it can be visualized in vivo by Positron

Emission Tomography combined with X-ray Computed Tomography (PET-CT).21 Another well-known boron cluster anion is the closo-dodecaborate, [B12H12]2-, that has shown high solubility in water as sodium salt,22 chemical and hydrolytic stability,23 permeability across phospholipid membranes,24-25 and a low toxicity.26 Furthermore, the interactions of FESAN with DNA, and both COSAN and [B12H12]2- with proteins have been recently described.27-28 All these properties make these anions and their derivatives ideal candidates for potential medical applications as pharmacophores,23, 29-35 especially in boron neutron capture therapy (BNCT),36-41 but also when conjugated with fluorescent molecules they can act as excellent dyes.



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In this sense, the 4,4’-difluoro-4-bora-3a,4a,diaza-s-indacene or BODIPY derivatives are versatile dyes mainly known for their photoluminescence properties; BODIPY-based molecules usually exhibit chemical robustness, thermal and photochemical stability, synthetic versatility, good solubility in organic solvents and a few derivatives are also soluble in water.42-43 They possess a large absorption and fluorescence range in the green-yellow region of the visible spectrum, as well as high extinction coefficients, low molecular weights, high photochemical stabilities and cell membrane permeability.44-46 Due to the properties described above, BODIPY-based dyes have been mainly exploited in material science and biomedical applications47-48 i.e. as fluorescent probes,49-50 biological labeling,51 photodynamic therapy52 and theranostic agents.53 Examples of carboranyl BODIPYs bearing o- and p-carborane clusters have been reported;54-55 however, to our knowledge there are no examples in the literature of BODIPYs linked to anionic boron clusters (i.e COSAN, FESAN or [B12H12]2-) for biomedical applications. Moved by our interest in the development of new boron cluster-based derivatives with luminescent properties for biological applications, herein, we report the first set of conjugates based on BODIPY linked to [B12H12]2-, [3,3’-Co(C2B9H11)2]- and [3,3’-Fe(C2B9H11)2]- anions, through an ethylene glycol chain. The syntheses and complete characterization of these compounds are described along with the photophysical properties. Moreover, the cytotoxicity of the conjugates has been evaluated and their cellular uptake by HeLa cells have been compared by flow cytometry in order to assess their potential as fluorescent dyes for cell tracking. Bioimages of HeLa cells incubated with compounds have been also analyzed by confocal laser microscopy.


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Results and Discussion

Synthesis and structural characterization. Starting

from

boron

clusters

[Bu4N][B12H11(C4H8O2)]

(1),

[3,3’-Co(8-C4H8O2-1,2-

C2B9H10)(1’,2’-C2B9H11)] (2) and [3,3’-Fe(8-C4H8O2-1,2-C2B9H10)(1’,2’-C2B9H11)] (3), and the BODIPY derivative (4), a set of three new anionic BODIPY conjugates bearing closododecaborate (5), cobaltabisdicarbollie (7) and ferrabisdicarbollide (9) were synthesized by using similar conditions previously described for the nucleophilic oxonium ring-opening reaction (see Scheme 1).3 For all the reactions, in a first step the deprotonation of the phenol group in BODIPY (4) with K2CO3 in CH3CN is performed. Then, the nucleophilic attack of this deprotonated BODIPY to the oxonium ring in the anion [B12H11(C4H8O2)]-, and zwitterionic species 2 and 3 takes place to produce the ring opening reaction. The reactions were monitored by

11

B NMR spectroscopy by comparison with their parent species 1-3.56-57

The compounds 5, 7 and 9 were isolated from the reaction mixture as [NBu4]+ or [NMe4]+salts in good yields, ranged from 68 to 88%, and no further purification is required. With the aim to obtain more biocompatible compounds, the cation exchange to Na+ by using cation-exchange resin was possible for 5 and 7 to give 6 and 8, respectively.



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Scheme 1. Procedure to obtain compounds 5-9.

The structures of compounds 5-9 were confirmed by IR-ATR, NMR (1H,

13

C{1H} and

11

B{1H}), MALDI-TOF mass spectroscopies and elemental analysis.

The IR spectra for all compounds show the typical υ(B-H) strong bands between 2529 and 2537 cm-1 settling the presence of boron clusters. Other bands at 1482 (N-C), 1604 (C-O), 2867, 2925, 2953 cm-1 (C-H) were observed. Conversely to the 1H NMR spectrum of precursor 1 that shows two multiplets at 3.94 and 4.54 ppm attributed to the CH2 protons from the dioxane ring, the derivative 5 shows four resonances (3.63, 3.73, 3.91 and 4.32 ppm) for the CH2 protons after the ring opening has occurred.58 Similarly, compound 7 shows three resonances at 3.62, 3.90 and 4.30 ppm. Besides, five additional resonances corresponding to the BODIPY moiety are also observed in both compounds, 5 and 7. 1H resonances due to the cations [NBu4]+ and [NMe4]+ observed in 5 and 7, respectively, have disappeared in compounds 6 and 8 confirming the cation exchange. A very different 1H NMR spectrum was observed for compound 9 that, as expected, exhibits appreciable paramagnetic shifts of resonances in the range from δ -10 to +87 ppm, especially in those protons closer to the Fe(III) (see SI). The 13C{1H} NMR spectra of compounds 5-8 show resonances corresponding 6 ACS Paragon Plus Environment

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to Cc, -OCH2-, BODIPY and cations carbon atoms, complementing the 1H NMR spectra and corroborating the formation of the expected compounds. Compound 9 shows resonances for the ether groups protons (-OCH2-) between 0.40 and -10.40 ppm because of their nearness to Fe(III).3, 59 The 11B{1H} NMR spectrum for 5 shows four resonances corresponding to the B atoms from the closo-dodecaborate cluster, which has been slightly shifted with respect to 1, confirming the opening of the dioxane ring. An additional triplet at 1.57 ppm attributed to BF2 from the BODIPY is also observed. Likewise, for compound 7 the typical pattern 1:1:1:1:2:3:3:2:2:1:1 in the range from 24.37 to -27.21 ppm is observed, including an additional triplet at 1.57 ppm that indicates the presence of BODIPY. Compound 9 shows 11

B{1H} NMR patterns in the region between δ +118 and -468 ppm, in agreement with

reported studies of other oxonium FESAN derivatives.59 Elemental analyses also confirmed the stoichiometries of all these compounds, whereas MALDI-TOF was only possible for compounds 7, 8 and 9.

Photophysical Properties Compounds 5, 7, 8 and 9 are soluble in organic solvents such as acetone, THF, CH3CN and DMSO, whereas compound 6 is soluble in water. The photophysical properties of compounds 5-9 were investigated by UV-vis and fluorescence spectroscopy in THF solutions and for 6 also in water at room temperature (Figure 1). The UV-vis spectra are very similar for all of them, showing the absorption maxima around 497 nm (Table 1), which corresponds to the S0S1 (π-π*) transitions in the boron dipyrromethene BODIPY. A shoulder at higher energy attributed to vibrational transitions is also present. The maximum wavelength for compound 6 in water was slightly blue shifted (~3 nm) respect to THF, indicating a very little solvatochromic effect. The emission spectra for all compounds show maxima in the range from 504 to 520 nm due the BODIPY fluorophore. The Stokes shift values are very low, as



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usually occur with this kind of dyes, varying between 7 and 23 nm. The largest one was observed for the COSANE derivative 7. In general, the link of the different anionic boron clusters to the BODIPY 4 does not alter significantly the absorption and emission maxima probably due to the distance between the cluster and the dye. The quantum yields (φF) values for compounds 5-9 are in the range 3-6%, a little bit lower than the φF of the starting compound 4 (7%), which seems to indicate that linking these boron clusters to the BODIPY produces a slight decrease of the fluorescence. Nevertheless, the fluorescence intensities of 59 are adequate for their visualization in cells with the confocal microscope and to be used as cell tracking agents (vide infra).

Figure 1. Normalized absorption and emission spectra in THF of 5-9, using as λexc the respective maximum absorption peaks.


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Table 1. Photophysical data for compounds 5-9. Stokes shift Compound

λabs (nm)

ε/105 (M-1 cm-1)

λem(nm)

φFa (nm)

a

5

496

0.536

517

0.06

21

6

497

0.495

516

0.06

19

6b

494

0.350

513

0.03

19

7

497

0.582

520

0.04

23

8

497

0.433

519

0.04

22

9

497

0.560

504

0.03

7

Reference compound rhodamine 6G (EtOH, φF = 0.94); bin water

Cytotoxicity Considering the photophysical properties and the biological potential of these compounds for cell tracking, we aimed to assess their cytotoxicity and cellular uptake in HeLa cells. The three different “families” of BODIPY-anionic boron cluster conjugates were tested: two closo-dodecaborate derivatives (5-6), two derivatives of COSAN (7-8) and the FESAN derivative (9). In the present work, the cytotoxicity of these boron-clusters was analyzed by MTT assay at three different concentrations, based on the final concentration of boron in the cell culture (5, 50 and 100 µg B/ml) instead of using the molarity (Table S1), because even though the aim of this work was to evaluate the use of these compounds as imaging agents, we were interested in knowing if some of them could be also applied as potential anticancer agents, especially in BNCT.



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Noticeably, no cytotoxicity was observed in cells incubated with compounds 5-9 at the lowest concentration (5 µg B/ml). On the other hand, only compound 6, bearing [B12H11]2- and Na+ as cation, was non-cytotoxic at any concentration, while the rest of compounds were highly cytotoxic at 50 and 100 µg B/ml (Figure 2a). The percentage of living cells in cultures incubated with compound 5 progressively decreased with increasing the concentration (60 and 35% of cell viability at 50 and 100 µg B/ml, respectively). As the only difference between 5 and 6 is the cation, we could attribute the toxicity to the [NBu4]+ cation in compound 5. By contrast, the toxicity was higher for metallacarborane-containing dyes 7-9 at 50 µg B/ml and 100 µg B/ml, since the percentage of viable cells was less than 17% and 5%, respectively. In this case, although the Na+ is a more biocompatible cation than [NMe4]+, the toxicity cannot be attributed to the [NMe4]+, as no differences were found between compounds 7 (carrying [NMe4]+) and 8 (carrying Na+) in terms of viability (Figure. 2a). Moreover, the toxicity should not be attributed to the presence of impurities since compounds 5-9 are pure compounds according to their characterization. Surprisingly, using the MTT assay, cell viability in cultures incubated with products 7-9 at 5 µg B/ml was extremely high, more than two-fold the cell viability of control cultures (Figure 2a). However, when cells incubated with these compounds were visualized under an inverted microscope (Figure 2c), the number of cells was comparable to the control cultures. To find out if the values obtained using the MTT assay were reliable or were due to a technical artifact, two different methods to quantify again the cytotoxic effect of compounds 7-9 (5 µg B/ml) were used, the Trypan Blue and the Live/Dead assays. The results demonstrated that compounds 7-9 do not increase the cell viability since the cell number obtained, using these methods, was lower than those from control cultures (Figure 2b). A possible explanation of these contradictory results relies on the method used to assess the cell viability and the physicochemical properties of the COSAN and FESAN derivatives. 10 ACS Paragon Plus Environment

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MTT assay is a common approach to test the cytotoxicity (recommended by the ISO 10993-5) based on the metabolic redox activity of the cells; only intracellular NAD(P)H-dependent oxidoreductase enzymes of live cells can reduce the MTT dye to insoluble formazan salts. Therefore, the amount of formazan is directly proportional to the number of viable cells. However, when molecules to be tested: i) can induce changes in the cell metabolic activity, or ii) can directly reduce the MTT, the results obtained could be misinterpreted. In order to elucidate which of these two mechanisms takes place, and considering that metallacarboranes are redox-active molecules,3, 60 compounds 7-9 were incubated with MTT in absence of cells. The results showed that these compounds were not able to reduce the MTT dye (Figure S1a), or to contribute to increase the absorbance of the MTT, as the values obtained were similar among the compounds and the control medium. However, when formazan salts formation was monitored under an inverted microscope, many cells incubated with compounds 7-9 showed extremely high amounts of formazan (black precipitates) while control cells homogeneously produced less formazan salts (Figure S1b). Therefore, this result suggests that compounds 7-9 increase the cells metabolic activity, as has been reported with some iron-based biomaterials.61 Moreover, it has been recently demonstrated that ionizing radiation promotes mitochondrial biogenesis and enhanced metabolic activity in many different cell lines (included Hela cells) leading to an underestimation of growth inhibition when MTT assay is used.62 In fact, discrepancies between the values obtained using direct and indirect methods for cell viability determination have been described not only for the MTT assay but also for ATP content assays.63 Therefore, we would like to stress that cell viability assessment based on the intracellular metabolic activity should be validated with other methods, which directly determine the number of viable cells.



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Figure 2. Cell viability of HeLa cells after 24 h of incubation with BODIPY-boron clusters. a) Cell viability after incubation with compounds 5-9 at three different concentrations of B, using MTT assay. Data were normalized with control cultures. Mean values and SD from three independent experiments. b) Comparison among MTT, Trypan blue and Live/Dead assays. Cell viability in cultures incubated with compounds 7-9 (5 µg B/ml) was twofold higher than in control cells using MTT, whereas the number of viable cells was lower than control cultures when Trypan blue and Live/Dead tests were performed. c) Bright field images showing no differences in the cell density between control cells and cultures incubated with the compounds 5-9 (5 µg B/ml). Cell density drastically diminished in all cultures incubated with compounds at ≥ 50 µg B/ml except in cultures incubated with compound 6. Scale bar: 100 µm.

Remarkably, these results agree with previous studies performed with pristine COSAN and I2COSAN, where a 100% and 0% of HeLa cells viability were obtained using 25 and 250 µM of COSAN, respectively (similar to the molarities of compounds 7-9 at 5 and 50 µg/ml of B),


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although the incubation time and the GI50 were higher than in the present work (Figure S2).64 On the other hand, it has been reported that carboranyl-BODIPYs have no cytotoxic effect up to 100 µM,54 and closo-dodecaborate-encapsulating liposomes have shown GI50 values ranging from 2 to 33 mM,32 which is in line with our results, since no cytotoxic effect was observed in cultures incubated with the closo-dodecaborate 6 at more than 700 µM.

Cellular uptake and intracellular localization Cellular uptake of compounds 5-9 was assessed by flow cytometry, a method that detects the fluorescence intensity emitted by single cells. Remarkably, the results showed that all cells analyzed were able to internalize the different compounds at the three concentrations tested (100 % of positive cells for all compounds and concentrations with the exception of compounds 5 and 6 at 5µg B/ml, in which positive cells were 99.2% and 96.1, respectively), demonstrating that all of them can penetrate into the cells regardless their chemical composition and structure (Figure 3a). When the internalization efficiency was compared in terms of fluorescence intensity, COSAN derivatives 7 and 8 were the most efficient for all concentrations (Figure 3b), whereas the closo-dodecaborates 5 and 6 were the least ones. In order to interpret these results and avoid any misunderstanding, we first measured the fluorescence emission of each compound at 5 µg B/ml in freshly cell culture medium (MEM) to analyze if there were intrinsic differences among the fluorescence intensity due to the compound nature (Figure 3c). The fluorescence intensities (FI) of compounds 7, 8 and 9 (57, 40, 13 arbitrary units, respectively) were lower compared to 5 and 6 (538, 293 respectively, Table S2), which fits with the quantum efficiencies obtained in THF solution. It is well known that fluorescence intensity is proportional to the fluorophore concentration. Accordingly, if all compounds were internalized with similar efficacy, the amount of compound inside the cells



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would be equivalent for all of them, and thus, the fluorescence detected by flow cytometry at 5 µg B/ml should be correlated with the fluorescence intensity of each compound in MEM (higher values for 5 and 6 and lower for 7-9). However, the results obtained were quite the opposite (high values for 7-9 and lower for 5 and 6), suggesting that the intracellular concentration of each compound was different and therefore, the internalization efficiency. Taking into account that cells have a small volume, compounds with a higher cellular uptake would become extremely concentrated compared to those presenting a poorer one; therefore, the higher the intracellular uptake, the higher the fluorescence intensity (FI) emitted. For instance, compound 9 exhibits a FI in MEM around 40-fold lower than compound 5 (Figure 3c), but almost 20-fold higher than the same compound after internalization (Figure 3b, Table S2). In fact, when the FI detected by flow cytometry at 5 µg B/ml was corrected considering the intrinsic FI of the compounds in MEM (Table S2), metallacarboranes 7-9 were 1000 fold better internalized than closo-dodecaborates 5 and 6 (Figure 3d). On the other hand, the cellular uptake of all compounds at 100 µg B/ml was equal or higher than the starting BODIPY 4 (Figure S3), indicating that the linking of the boron cluster anions to BODIPY improves its internalization. Therefore, the herein reported compounds could be considered good candidates for fluorescence cell imaging applications, especially compounds 7 and 8 that show extraordinary cellular uptake. In addition, due to the low toxicity and good cellular uptake of these two compounds at low concentration, they could be analyzed in the future to explore their suitability as BNCT agents.


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Figure 3. Cellular uptake of the BODIPY-boron clusters after 2 h of incubation by flow cytometry. a) Fluorescence intensity of cells incubated with BODIPY-boron clusters. Fluorescence threshold was established with the control culture (untreated cells). The plot corresponds to one replicate at 5 µg B/ml. b) Mean values and SD of the fluorescence intensity from three independent experiments showing the cellular internalization of each compound at 5, 50 and 100 µg B/ml. c) Emission spectra of compounds diluted in culture medium (MEM) at 5 µg B/ml. d) Comparison of cellular uptake at 5 µg B/ml before and after the correction of the fluorescence intensity (FI). Due to the differences in FI values among the compounds, data from products 5 and 6 are represented in an inset. FI expressed in arbitrary units (a.u.).

Moreover, a qualitative analysis of HeLa cells incubated with compounds at 5 µg B/ml concentration for 2 h was also assessed by confocal microscopy (Figure 4). This microscope discriminates the fluorescence emitted by compounds located inside the cells from those outside the cells because it allows scanning many thin sections (optical sections in the z15 ACS Paragon Plus Environment


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plane) through the sample, obtaining a three-dimensional image of the cell. Noticeably, all the compounds were internalized by cells in varying degrees, with the green fluorescence being much less intense for compounds 5 and 6 than for COSAN derivatives 7 and 8 (Figure 4a). The orthogonal projections (Figure 4b), optical sections in the XZ and YZ axis of the merged images from each sample, definitively demonstrated that all compounds were located inside the cells and not adhere to the cell surface. Compounds reported here are the first BODIPYanionic boron cluster conjugates that have been visualized in living cells using confocal microscopy, being compounds 7 and 8 those that show exceptional cytoplasmic staining. These results agree with those obtained by flow cytometry, and confirm that COSAN derivatives have an extraordinary cellular uptake.


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Figure 4. Cellular uptake of BODIPY-boron clusters after 2 h of incubation by confocal microscopy. a) Confocal images of the cellular uptake at 5 µg B/ml concentration (scale bar: 50 µm). Amplified images in the bottom row (scale bar: 25 µm). b) Orthogonal projections of compound 6 and 7 demonstrating the cytoplasmic localization of the compounds (Scale bar: 25 µm). Plasma membrane (red); BODIPY-boron clusters (green) and nucleus (blue). a.u.: arbitrary units.

Conclusions In summary, we have synthesized, in a straightforward and efficient way, a series of novel BODIPY-boron cluster-based anions (5-9) to further study their photophysical properties and biocompatibility. After functionalization, the compounds have preserved the luminescence properties of the starting BODIPY (4) with slight changes in the quantum yield values, which allows their use as fluorescent probes in different cellular methodologies such as flow 17 ACS Paragon Plus Environment


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cytometry and confocal microscopy. In general, compounds 5-9 are better internalized than the non-functionalized BODIPY (4), showing a clear cytoplasmic staining. The closododecaborate derivative 6 is extremely innocuous for HeLa cells in terms of cytotoxicity but its internalization efficiency is lower than COSAN derivatives 7 and 8. Our hypothesis is that probably the high cytotoxic effect observed for compounds 7 and 8 at concentrations of B ≥ 50 µg B/ml could be related to their high penetration efficiency. Remarkably, COSAN derivatives are exceptional candidates as fluorescent dyes for in vitro cell tracking due to their extraordinary cellular uptake, particularly at low concentrations where they are not cytotoxic.

Experimental Instrumentation Elemental analyses were performed using a Carlo Erba EA1108 microanalyzer. ATR-IR spectra were recorded on a high-resolution spectrometer FT-IR Perkin Elmer Spectrum One. The 1H NMR (300.13 MHz), 11B {1H} (96.29 MHz) and 13C {1H} NMR (75.47 MHz) NMR spectra were recorded on a Bruker ARX 300 spectrometer. All NMR spectra were recorded in CD3COCD3 solutions at 25ºC. Chemical shift values for referenced to external BF3·OEt2, and those for 1H and

13

11

B {1H} NMR spectra were

C{1H} NMR were referenced to

SiMe4 (TMS). Chemical shifts are reported in units of parts per million downfield from the reference, and all coupling constants are reported in Hertz. MALDI-TOF-MS mass spectra were recorded in the negative ion mode using a Bruker Biflex MALDI-TOF [N2 laser; λexc = 337 nm (0.5 ns pulses); voltage ion source 20.00 kV (Uis1) and 17.50 kV (Uis2)]. UV-Vis spectra were recorded on Shimadzu UV-1700 Pharmaspec spectrophotometer, using 1 cm cuvettes. The fluorescence emission spectra were recorded in a Perkin-Elmer LS-45 (230V) Fluorescence spectrometer. Samples were prepared in spectroscopic grade solvents and adjusted to a response within the linear range. No fluorescent contaminants were detected on 18 ACS Paragon Plus Environment

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excitation in the wavelength region of experimental interest. The fluorescence quantum yields of all compounds were calculated by comparison using Rhodamine 6G in EtOH (ϕF = 0.94) as a standard, and corrected for the refractive index of the solvent. Samples were prepared in such a way as to obtain an absorbance around 0.1 at the excitation wavelength.

Materials All reactions were performed under atmosphere of dinitrogen employing standard Schlenk techniques. 1,4-Dioxane was purchased from Merck and distilled from sodium benzophenone previously to use. Acetonitrile was dried for 2 days over molecular sieves (3Å; Merck; activated for 16h at 300 ºC under vacuum). After drying, acetonitrile was degassed using the well stablished freeze-pump-thaw protocol. Commercial grade tetrahydrofuran, methanol, ethanol, hexane, dichloromethane and acetone were used without further purification. Compounds 1,58 2,57 359 and 465 (SI) were synthesized according to the literature. Compounds NaBF4, [Bu4N]Br, pyrrole, p-hydroxybenzaldehyde, CF3CO2H, DDQ, Et3N, and BF3.Et2O were purchased from Aldrich. Compounds [NMe4]Cl and HCl solution (4M in 1,4-dioxane) were purchased from Acros. K2CO3 was obtained from Labkem. Cation exchange resin strongly acidic PA was acquired from Panreac.

Biological assays Products 5-9, previously dissolved in 1% of DMSO (Sigma/Aldrich), were diluted in Minimum Essential Medium (MEM) supplemented with 10% of Fetal Bovine Serum (FBS) (both from Gibco) and 1% (200 mM) of L-glutamine (Biowest) at final concentrations of 5, 50 and 100 µg/ml of B. The molarity of these 5, 50 and 100 µg/ml of boron concentrations were 36, 356 and 712 µM for compounds 5 and 6 and 25, 243 and 487 µM for compounds 7-



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9, respectively (see Table S.1 for molarities). Compounds were then sterilized by filtration (0.2 µm of diameter. Fisher Scientific) and kept at 4 ºC until its use in cell cultures. The HeLa cell line, derived from a human cervical cancer, was used to perform the biological assays. Cells were routinely maintained with MEM + 10% FBS + 1% L-glutamine in T25 culture flasks at 37 ºC and a constant atmosphere of 5% CO2 (standard conditions).

Cytotoxicity The MTT (Thiazolyl Blue Tetrazolium Bromide, from Sigma/Aldrich) assay was used to assess the cell toxicity of the BODIPY-boron clusters. MTT compound can be reduced to formazan salts by living cells, being a common method to indirectly determine cell viability and proliferation. To perform the experiments, 500 µl (6x104 cell/ml) of HeLa cells were seeded in 24 well plates, kept in culture for 24 h to allow cell adhesion, and then, incubated 24 h with 500 µl of products 5-9 at the different concentrations. After that, the compounds were removed; the cultures were rinsed three times with a salt solution (HBSS from Biowest) and finally, incubated with 0.1 µg/ml of MTT in dark during 3 h at 37ºC. After removal the MTT, formazan salts were dissolved in 500 µl of pure DMSO and the absorbance measured at 540 nm using the VICTOR™ X3 Multilabel Plate Reader coupled to the PerkinElmer 2030 Manager software. Four replicates of each compound and concentration were analyzed for experiment. Experiments were performed in triplicate and the values obtained were normalized with control cultures. In cultures incubated with compounds 7-9 at 5 µg/ml of B, cell viability was also analyzed using the Trypan blue exclusion test (Sigma/Aldrich) and the Live/Dead viability/cyotoxicity kit (Thermo Fisher Scientific). Trypan blue dye can only stain non-viable cells with damaged plasma membrane, whereas Live/Dead kit is based on the cellular esterase activity and membrane integrity, differently staining live (green) and dead cells (red). Both methods were 20 ACS Paragon Plus Environment

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performed following the manufacturer's instructions and cells were counted using an Olympus IX71 inverted fluorescence microscope. Nonlinear regression curves for growth inhibition (GI50) were obtained by GraphPad Prism software, using the results obtained by MTT assay except for compounds 7-9 at 5 µg/ml, where Live/dead results were considered. To assess if there was an interference between the compounds and the MTT reagent, the direct reduction of MTT by the compounds in absence of cells was analyzed. To perform this study, 250 µl of compounds 7-9 at 5 µg/ml of B were mixed and incubated with 250 µl of MTT for 3 h at 37ºC. Absorbance was read at 540 nm and data were normalized with the control medium (MTT + culture medium).

Cellular uptake Cell internalization of the compounds was assessed by flow cytometry at all concentrations and by confocal microscopy at 5 µg/ml of B. For flow cytometry, HeLa cells were seeded into 6 well plates (2x105 cells/well), kept in standard conditions 24 h to allow cell attachment, and then incubated 2 h with the different concentrations of the products 5-9. Afterwards, the products were removed, the cells were rinsed twice with HBSS and detached by trypsinization. Cell suspensions were centrifuged 5 min at 300 g and rinsed again with HBSS. Finally, cells were resuspended in 1X PBS at a final concentration of 1x106 cell/ml with 1µl of propidium iodide (1mg/ml from Sigma/Aldrich) to discard false positive (cells with internalized product due to plasma membrane damage). Cellular uptake was determined using the laser 488 of the BD FACSCanto flow cytometer coupled to the FACSDiva software. A total number of 10.000 single cells were analyzed for sample and experiments were performed in triplicate for each product and concentration. Untreated cells and cultures incubated with BODIPY alone at 487 and 712 µM (molarities of the boron-clusters at 100 21 ACS Paragon Plus Environment


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µg/ml, see table S1) were included at each experiment as negative and positive controls, respectively. Fluorescent emission spectra of compounds in freshly culture medium (MEM + 10% FBS + 1% L-glutamine +1% DMSO) was also determined at 5 µg B/ml using the same excitation wavelength that those for flow cytometry and confocal microscopy experiments (488 nm). As the fluorescence intensity (FI) depends on the concentration and the chemical nature of the compounds, flow cytometry results at the lowest concentration were corrected, taking into account the intrinsic FI of the compounds in culture medium (MEM) which was previously measured. The FI correction factor for each compound was calculated considering as one unit, the FI of the compound with the highest value in MEM (Table S2). For the confocal microscopy analysis, HeLa cells were seeded into glass bottom culture dishes (MatTek) at a density of 1x105 cell/ml and after their attachment to the plate surface (24 h) were incubated with the products at 5 µg/ml. Afterwards, cells were rinsed three times with 1X PBS and counterstained with 1 µl/ml of Hoescht (10 mg/ml. Thermo Fisher) and Cell Mask (5 mg/ml. Thermo Fisher) for nuclear and plasma membrane staining, respectively. Serial images (25 optical sections of 1µm each one) were captured with the Leica TCS SP5 confocal microscope coupled to a Leica LAS AF software using 10 % or 70 % of laser power for capturing the signal from compounds 7-9 and 5-6, respectively. Images were analyzed with ImageJ and orthogonal projections were obtained using the Imaris software.

Synthesis of 5 A dry round-bottomed flask equipped with a condenser and a magnetic stirring bar was charged under N2 with 1 (47 mg, 0.1 mmol), 4 (28.37 mg, 0.1 mmol), K2CO3 (55 mg, 0.4 mmol), [NBu4]Br (34.49 mg, 0.1mmol) and 3 mL of a hydrous acetonitrile. The mixture was stirred under reflux overnight. After, the reaction mixture was filtered off and the solvent was 22 ACS Paragon Plus Environment

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removed under vacuum. The residue was washed with THF and filtered to give 5 as a brown sticky solid. Yield: 80.6mg, 81 %. 1

H NMR, δ(ppm): 7.98 (s, 2H; NCH), 7.66 (d, 3J(H,H) = 6 Hz, 2H; C6H4), 7.26 (d, 3J(H,H) =

9 Hz, 2H; C6H4), 7.13 (d, 2H; 3J(H,H) = 3 Hz, C-CH), 6.68 (m, 2H; CHCHCH), 4.32 (t, 3

J(H,H) = 4.5 Hz, 2H; OCH2), 3.91 (t, 3J(H,H) = 6 Hz, 2H; OCH2), 3.73 (t, 3J(H,H) = 6 Hz,

2H; CH2OCH2), 3.63 (t, 3J(H,H) = 6 Hz, 2H; CH2OCH2), 3.44 (t, 3J(H,H) = 9 Hz, 16H; N– CH2), 1.86–1.75 (m, 16H; CH2–CH2), 1.51–1.41 (m, 16H; CH2–CH3), 0.98 (t, 3J(H,H) = 7.5 Hz, 24H; CH2–CH3); 11B{1H} NMR, δ(ppm): 8.11 (s, 1B, B–O), 1.57 (t, 1J(B,F) = 28.32 Hz, 1B, BF), –15.06 (s, 5B), –16.44 (s, 5B), –21.20 (s, 1B); 13C{1H} NMR, δ(ppm): 13.14 (s, CH3), 19.51 (s, CH3CH2CH2), 23.75 (s, CH3CH2CH2), 58.62 (s, NCH2), 68.03 (s; O–CH2), 68.40 (s; O–CH2), 69.10 (s; O–CH2), 73.19 (s; O–CH2), 115.15 (s; C6H4), 118.54 (s; CHCHCH), 125.14 (s, CC), 131.53 (s, CCH), 132.67(s; C6H4), 134.62 (s; C6H4–C), 143.28 (s, N-CH), 147.81 (s; C6H4), 162.14 (s; C6H4); ATR-IR (cm−1): ν = 1044,1256,1297 (C-O), 1178,1223 (C-H), 1481 (N-C), 1574,1604 (C=C), 2529 (B-H), 2865, 2924, 2960 (Car-H); elemental analysis calcd. (%) for C51H101B13F2N4O3: C 61.44, H 10.21, N 5.62; found: C 62.44, H 10.32, N 5.02.

Preparation of 6 Compound 5 was dissolved in a minimum volume of acetonitrile/water (50:50). The solution was passed repeatedly through a cation exchanging resin, previously loaded with NaCl. The solvent mixture was finally evaporated to give compound 6. The total solubility of the new salt in distilled water was indicative of the full cation exchange, which was also confirmed by 1

H and 13C{1H} NMR.



1

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9 Hz, 2H; C6H4), 7.08 (d, 2H; 3J(H,H) = 4 Hz, CCH), 6.67–6.68 (m, 2H; CHCHCH), 4.36 (t, 3

J(H,H) = 4.65 Hz, 2H; OCH2), 3.94 (t, 3J(H,H) = 4.5 Hz, 2H; OCH2), 3.70 (s, 4H;

CH2OCH2); 11B{1H} NMR, δ (ppm): 7.26 (s, 1B, B–O), 1.55 (t, 1J(B,F) = 28.22 Hz, 1B, BF), –15.79 (s, 10B), –20.96 (s, 1B); 13C{1H} NMR, δ(ppm): 66.81 (s; O–CH2), 67.78 (s; O–CH2), 68.89 (s; O–CH2), 72.48 (s; O–CH2), 115.10 (s; C6H4), 118.56 (s; CHCHCH), 126.18 (s, CC), 131.43 (s, CCH), 132.55 (s; C6H4), 134.63 (s; C6H4–C), 143.48 (s, NCH), 147.61 (s; C6H4), 161.68 (s; C6H4); ATR-IR (cm−1): ν = 1045,1259,1295 (C-O), 1180, 1225 (C-H), 1576, 1603 (C=C), 2476 (B-H), 2873, 2927, 2959 (CarH); elemental analysis calcd. (%) for C19H29B13FeN2Na2O3: C 40.90, H 5.24, N 5.02; found: C 39.89, H 5.34, N 4.00; MALDITOF-MS: m/z calcd for [6]-: 256 and 535; found: 566.

Synthesis of 7 A dry round-bottomed flask equipped with a condenser and a magnetic stirring bar was charged under nitrogen with 2 (100 mg, 0.24mmol), 4 (66 mg, 0.23mmol), K2CO3 (128 mg, 0.93 mmol) and 8 mL of CH3CN. The mixture was stirred under reflux overnight. After the reaction mixture was washed with THF, filtered off and the solvent was removed under vacuum. Then, the orange oil was dissolved in 1 mL of MeOH and a saturated solution of [NMe4]Cl in water was added to obtain 7 as an orange solid. Yield: 164.8 mg, 88 %. 1


6 Hz, 2H; C6H4), 7.12 (s, 2H; CCH), 6.68 (s, 2H; CHCHCH), 4.30 (s, 6H; Cc–H and OCH2), 3.90 (s, 2H; OCH2), 3.62 (s, 4H; CH2OCH2), 3.44 (s, 12H; N–CH3); 11B{1H} NMR, δ(ppm): 24.37 (s, 1B, B–O), 5.25 (s, 1B, B–H), 1.57 (t, 1J(B,F) = 28.32 Hz, 2B, B–H and BF2), –1.07 (s, 1B, B–H), –2.79 (s, 2B, B–H), –6.13 (s, 3B, B–H), –6.84 (s, 3B, B–H), –15.95 (s, 2B, B– H), –19.09 (s, 2B, B–H), –20.43 (s, 1B, B–H), –27.71 (s, 1B, B–H); 13C{1H} NMR, δ(ppm): 24 ACS Paragon Plus Environment

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46.42 (s; Cc–H), 54.55 (s; Cc–H), 55.28 (s; N–CH3), 68.07 (s, O–CH2), 68.51 (s; O–CH2), 69.27 (s, O–CH2), 72.14 (s; O–CH2), 114.92 (s,C6H4), 118.43 (s, CHCHCH), 126.00 (s, CC), 131.52 (s, CCH), 132.61 (s; C6H4), 134.69 (s, C6H4–C), 143.37 (s, NCH3), 147.79 (s,C6H4), 161.95 (s,C6H4); ATR-IR (cm−1): ν = 1019, 1045, 1260, 1296 (C-O), 1178, 1223 (C-H), 1482 (N-C), 1574, 1604 (C=C), 2537 (B-H), 2867, 2925, 2953 (CarH), 3049 (Cc-H); elemental analysis calcd. (%) for C27H51B19Co1F2N3O3·(CH3)2CO: C 43.62, H 6.95, N 5.09; found: C 43.63, H 6.92, N 5.06; MALDI-TOF-MS: m/z calcd for [7]-: 693.9; found: 694.4.

Preparation of 8 Compound 7 was dissolved in a minimum volume of acetonitrile/water (50:50). Then, the solution was passed repeatedly through a cation exchanging resin, previously loaded with NaCl. The solvent mixture was finally evaporated to give compound 8. The partial solubilisation of the new salt in distilled water was indicative of the full cation exchange. Besides, the disappearance of the [NMe4]+ peaks in the 1H and 13C{1H} NMR was diagnostic of complete exchange to the Na+. 1

H NMR, δ(ppm): 7.98 (s, 2H; NCH), 7.67 (d, 3J(H,H)=9 Hz, 2H; C6H4), 7.22 (d, 3J(H,H) = 9

Hz, 2H; C6H4), 7.12 (d, 3J(H,H) = 3 Hz, 2H; CCH), 6.67 (d, 3J(H,H) = 3 Hz, 2H; CHCHCH), 4.30 (m, 6H; Cc–H and OCH2), 3.90 (t, 3J(H,H) = 4.5 Hz, 2H; OCH2), 3.64 (s, 4H; CH2OCH2); 11B{1H} NMR, δ(ppm): 24.37 (s, 1B, B–O), 5.35 (s, 1B, B–H), 1.57 (t, 1J(B,F) = 28.32 Hz, 2B, B–H and BF2), –1.09 (s, 1B, B–H), –2.83 (s, 2B, B–H), –6.09 (s, 3B, B–H), – 6.81 (s, 3B, B–H), –15.91 (s, 2B, B–H), –19.07 (s, 2B, B–H), –20.44 (s, 1B, B–H), –27.04 (s, 1B, B–H); 13C{1H} NMR, δ(ppm): 46.45 (s; Cc–H), 54.47 (s; Cc–H), 68.04 (s; O–CH2), 68.52 (s; O–CH2), 69.27 (s; O–CH2), 72.12 (s; O–CH2), 114.92 (s; C6H4), 118.43 (s; CHCHCH), 126.01 (s, CC), 131.52 (s, CCH), 132.60 (s; C6H4), 134.68 (s; C6H4–C), 143.38 (s, NCH), 147.77 (s; C6H4), 161.93 (s; C6H4); ATR-IR (cm−1): ν = 1012,1043,1261, 1294 (C-O), 1180, 25 ACS Paragon Plus Environment


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1226 (C-H), 1575,1604 (C=C), 2540 (B-H), 2872, 2924 (CarH), 3049 (Cc-H); elemental analysis calcd. (%) for C23H39B19CoF2N2NaO3·H2O: C 37.59, H 5.62, N 3.81; found: C 37.24, H 5.42, N 3.01; MALDI-TOF-MS: m/z calcd for [8]-: 693.9; found: 694.4.

Synthesis of 9 The procedure was the same as for compound 7 by using 3 (45 mg, 0.11mmol), 4 (30 mg, 0.11mmol), K2CO3 (58.5 mg, 0.42mmol) and 3 mL of CH3CN. After the workup compound 9 was obtain as a brown solid. Yield: 57 mg, 68 %. 1

H NMR, δ(ppm): 86.92 (br, B–H), 78.13 (br, B–H), 59.49 (br, B–H), 45.33 (br, Cc–H), 43.42

(br, Cc–H), 28.00 (br, B–H), 7.99(s, 2H; NCH), 7.03 (d, 3J(H,H)=6 Hz, 2H; C6H4), 6.96 (d, 3

J(H,H) = 6 Hz, 2H; C6H4), 6.77 (s, 2H; CCH), 5.33 (s, 2H; CHCHCH), 3.29 (s, 12H; N–

CH3), 0.40 (s, 2H; OCH2), -4.10 (s, 2H; OCH2), -10.40 (s, 2H; OCH2);

11

B{1H} NMR ,

δ(ppm): 119.35 (s, 1B, B–H), 102.56 (s, 1B, B–H), 42.59, 31.59, –0.77, –5.41, –40.94, – 357.69 (s, 14B, B–H), 0.95 (t, J(B,F) = 28.32 Hz, 1B, BF2), –431.31 (s, 1B, B–H), –467.74 (s, 1B, B–H); 13C{1H} NMR, δ (ppm): 55.03 (s; N–CH3), 60.74 (s; O–CH2), 63.22 (s; O–CH2), 113.23 (s; C6H4), 118.42 (s; CHCHCH), 125.15 (s, CC), 131.33 (s, CCH), 131.67 (s; C6H4), 134.50 (s; C6H4–C), 143.36 (s, NCH), 147.46 (s; C6H4), 159.60 (s; C6H4); ATR-IR (cm−1): ν = 1044,1259,1294 (C-O),1178,1224 (C-H),1482 (N-C), 1574,1604 (C=C), 2529 (B-H), 2863, 2924,

2953

(CarH),

3038

(Cc-H);

elemental

analysis

calcd.

(%)

for

C27H51B19F2Fe1N3O3·(CH3)2CO: C 43.74, H 6.98, N 5.11; found: C 43.55, H 7.12, N 4.82; MALDI-TOF-MS: m/z calcd for [9]-: 290.8; found: 291.4.

Acknowledgments The work was supported by Spanish Ministerio de Economía y Competitividad, MINECO, (CTQ2016-75150-R, MAT2017-86357-C3-3-R and “Severo Ochoa” Program for Centers of 26 ACS Paragon Plus Environment

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Excellence in R&D (SEV- 2015-0496)) and Generalitat de Catalunya (2017-SGR-1720, 2017SGR-503). J. C.-G. thanks to CSIC for an Intramural Grant, J. C.-G. was enrolled in PhD Program of the UAB. The authors wish to thank the SCAC for flow cytometry assistance and the Servei de Microscopia at the UAB.

ASSOCIATED CONTENT Supporting Information Electronic Supporting Information (ESI) including 1H, 13C{1H}, 11B{1H} NMR (2D experiments), as well as supplementary data regarding the cellular uptake and cytotoxicity, is available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION Corresponding authors *[email protected], *[email protected]

ORCID Rosario Núñez: 0000-0003-4582-5148 Carme Nogués: 0000-0002-6361-8559 Mahdi Chaari: 0000-0002-1888-2545 Nerea Gaztelumendi: 0000-0002-3837-2956 Justo Cabrera-González: 0000-0002-7733-9681 Paula Peixoto-Moledo: 0000-0003-3901-1922 Clara Viñas: 0000-0001-5000-0277 Elba Xochitiotzi-Flores: 0000-0002-3193-1911 Norberto Farfán: 0000-0003-4320-2975

Notes The authors declare not competing financial interest 27 ACS Paragon Plus Environment


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References (1) Grimes, R. N. (2016) Carboranes (3rd Edition). Academic Press, US. (2) Housecroft, C. E. (2011) Boron: Metallacarbaboranes. Encyclopedia of Inorganic and Bioinorganic Chemistry. (King, R. B., Crabtree, R. H., Lukehart, C. M., Atwood, D. A., Scott, R. A., Eds.). John Wiley & Sons. (3) Cabrera-González, J., Sánchez-Arderiu, V., Viñas, C., Parella, T., Teixidor, F., and Núñez, R. (2016) Redox-Active Metallacarborane-Decorated Octasilsesquioxanes. Electrochemical and Thermal Properties. Inorg. Chem. 55, 11630–11634. (4) Cabrera-González, J., Cabana, L., Ballesteros, B., Tobias, G., and Núñez, R. (2016) Highly Dispersible and Stable Anionic Boron Cluster–Graphene Oxide Nanohybrids. Chem. Eur. J. 22, 5096-5101. (5) Grüner, B., Rais, J., Selucký, P., and Lucaníková, M. (2012) Recent Progress in Extraction Agents Based on Cobalt Bis(Dicarbollides) for Partitioning of Radionuclides from High-Level Nuclear Waste. Boron Science: New Technologies and Applications (Hosmane, N. S., Eds.) pp. 463-490, Ch. 19, Taylor & Francis, Bosa Roca. (6) Dash, B. P., Satapathy, R., Maguire, J. A., and Hosmane, N. S. (2011) Polyhedral boron clusters in materials science. New J. Chem. 35, 1955-1972. (7) Sivaev, I. B., and Bregadze, V. I. (1999) Chemistry of cobalt bis(dicarbollides). A review. Collect. Czech. Chem. Commun. 64, 783-805. (8) Uchman, M., Ďorďovič, V., Tošner, Z., and Matějíček, P. (2015) Classical Amphiphilic Behavior of Nonclassical Amphiphiles: A Comparison of Metallacarborane Self-Assembly with SDS Micellization. Angew. Chem. Int. Ed. 54, 14113-14117. (9) Gassin, P.-M., Girard, L., Martin-Gassin, G., Brusselle, D., Jonchère, A., Diat, O., Viñas, C., Teixidor, F., and Bauduin, P. (2015) Surface Activity and Molecular Organization of Metallacarboranes at the Air–Water Interface Revealed by Nonlinear Optics. Langmuir 31, 2297-2303. (10) Viñas, C., Tarrès, M., González-Cardoso, P., Farràs, P., Bauduin, P., and Teixidor, F. (2014) Surfactant behaviour of metallacarboranes. A study based on the electrolysis of water. Dalton Trans. 43, 5062-5068. (11) Bauduin, P., Prevost, S., Farràs, P., Teixidor, F., Diat, O., and Zemb, T. (2011) A Theta-Shaped Amphiphilic Cobaltabisdicarbollide Anion: Transition From Monolayer Vesicles to Micelles. Angew. Chem. Int. Ed. 50, 5298-5300. (12) Fernandez-Alvarez, R., Ďorďovič, V., Uchman, M., and Matějíček, P. (2017) Amphiphiles without Head-and-Tail Design: Nanostructures Based on the Self-Assembly of Anionic Boron Cluster Compounds. Langmuir 34, 3541–3554. (13) Rak, J., Dejlová, B., Lampová, H., Kaplánek, R., Matějíček, P., Cígler, P., and Král, V. (2013) On the Solubility and Lipophilicity of Metallacarborane Pharmacophores. Mol. Pharm. 10, 1751-1759. (14) Dąbrowska, A., Matuszewski, M., Zwoliński, K., Ignaczak, A., and Olejniczak, A. B. (2018) Insight into lipophilicity of deoxyribonucleoside‑boron cluster conjugates. Eur. J. Pharm. Sci. 111, 226-237. (15) Brusselle, D., Bauduin, P., Girard, L., Zaulet, A., Viñas, C., Teixidor, F., Ly, I., and Diat, O. (2013) Lyotropic Lamellar Phase Formed from Monolayered θ-Shaped CarboraneCage Amphiphiles. Angew. Chem. Int. Ed. 52, 12114-12118. (16) Farràs, P., Juárez-Pérez, E. J., Lepsik, M., Luque, R., Núñez, R., and Teixidor, F. (2012) Metallacarboranes and their interactions: theoretical insights and their applicability. Chem. Soc. Rev. 41, 3445-3463. (17) Verdiá-Báguena, C., Alcaraz, A., Aguilella, V. M., Cioran, A. M., Tachikawa, S., Nakamura, H., Teixidor, F., and Viñas, C. (2014) Amphiphilic COSAN and I2-COSAN 28 ACS Paragon Plus Environment

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crossing synthetic lipid membranes: planar bilayers and liposomes. Chem. Commun. 50, 6700-6703. (18) Tarrés, M., Canetta, E., Viñas, C., Teixidor, F., and Harwood, A. J. (2014) Imaging in living cells using [small nu]B-H Raman spectroscopy: monitoring COSAN uptake. Chem. Commun. 50, 3370-3372. (19) Zheng, Y., Liu, W., Chen, Y., Jiang, H., Yan, H., Kosenko, I., Chekulaeva, L., Sivaev, I., Bregadze, V., and Wang, X. (2017) A Highly Potent Antibacterial Agent Targeting Methicillin-Resistant Staphylococcus aureus Based on Cobalt Bis(1,2-Dicarbollide) Alkoxy Derivative. Organometallics 36, 3484-3490. (20) Uchman, M., Jurkiewicz, P., Cígler, P., Grüner, B., Hof, M., Procházka, K., and Matějíček, P. (2010) Interaction of Fluorescently Substituted Metallacarboranes with Cyclodextrins and Phospholipid Bilayers: Fluorescence and Light Scattering Study. Langmuir 26, 6268-6275. (21) Gona, K. B., Zaulet, A., Gómez-Vallejo, V., Teixidor, F., Llop, J., and Viñas, C. (2014) COSAN as a molecular imaging platform: synthesis and "in vivo" imaging. Chem. Commun. 50, 11415-11417. (22) Assaf, K. I., Ural, M. S., Pan, F., Georgiev, T., Simova, S., Rissanen, K., Gabel, D., and Nau, W. M. (2015) Water Structure Recovery in Chaotropic Anion Recognition: HighAffinity Binding of Dodecaborate Clusters to γ-Cyclodextrin. Angew. Chem. Int. Ed. 54, 6852-6856. (23) Karki, K., Gabel, D., and Roccatano, D. (2012) Structure and Dynamics of Dodecaborate Clusters in Water. Inorg. Chem. 51, 4894-4896. (24) Awad, D., Bartok, M., Mostaghimi, F., Schrader, I., Sudumbrekar, N., Schaffran, T., Jenne, C., Eriksson, J., Winterhalter, M., Fritz, J., et al. (2015) Halogenated Dodecaborate Clusters as Agents to Trigger Release of Liposomal Contents. ChemPlusChem 80, 656-664. (25) Awad, D., Tabod, I., Lutz, S., Wessolowski, H., and Gabel, D. (2005) Interaction of Na2B12H11SH with liposomes: Influence on zeta potential and particle size. J. Organomet. Chem. 690, 2732-2735. (26) Gabel, D., Awad, D., Schaffran, T., Radovan, D., Dărăban, D., Damian, L., Winterhalter, M., Karlsson, G., and Edwards, K. (2007) The Anionic Boron Cluster (B12H11SH)2− as a Means To Trigger Release of Liposome Contents. ChemMedChem 2, 5153. (27) García-Mendiola, T., Bayon-Pizarro, V., Zaulet, A., Fuentes, I., Pariente, F., Teixidor, F., Viñas, C., and Lorenzo, E. (2016) Metallacarboranes as tunable redox potential electrochemical indicators for screening of gene mutation. Chem. Sci. 7, 5786-5797. (28) Goszczyński, T. M., Fink, K., Kowalski, K., Leśnikowski, Z. J., and Boratyński, J. (2017) Interactions of Boron Clusters and their Derivatives with Serum Albumin. Sci. Rep. 7, 9800. (29) Leśnikowski, Z. J. (2016) Challenges and Opportunities for the Application of Boron Clusters in Drug Design. J. Med. Chem. 59, 7738-7758. (30) Plešek, J. (1992) Potential applications of the boron cluster compounds. Chem. Rev. 92, 269-278. (31) Viñas, C. (2013) The uniqueness of boron as a novel challenging element for drugs in pharmacology, medicine and for smart biomaterials. Future Med. Chem. 5, 617-619. (32) Tachikawa, S., Miyoshi, T., Koganei, H., El-Zaria, M. E., Viñas, C., Suzuki, M., Ono, K., and Nakamura, H. (2014) Spermidinium closo-dodecaborate-encapsulating liposomes as efficient boron delivery vehicles for neutron capture therapy. Chem. Commun. 50, 1232512328.



Page 30 of 32

(33) Neumann, M., Kunz, U., Lehmann, H., and Gabel, D. (2002) Determination of the Subcellular Distribution of Mercaptoundecahydro-closo-dodecaborate (BSH) in Human Glioblastoma Multiforme by Electron Microscopy. J. Neuro-Oncol. 57, 97-104. (34) Cabrera-González, J., Muñoz Flores, B. M., Viñas, C., Chávez-Reyes, A., Dias, H. V. R., Jiménez Pérez, V. M., and Nuñez, R. (2018) Organotin dyes bearing anionic boron clusters as cell-staining fluorescent probes. Chem. Eur. J. 10.1002/chem.201705804 (35) Wu, C., Shah, A., Ye, H., Chen, X., Ye, J., Jiang, H., Chen, B., Wang, X., and Yan, H. (2015) Droplet electrochemical study of the pH dependent redox behavior of novel ferrocenyl-carborane derivatives and its application in specific cancer cell recognition. Anal. Chim. Acta 857, 39-45. (36) Hosmane, N. S. (2012) Boron Science: New Technologies and Applications. Taylor & Francis, Bosa Roca. (37) Sibrian-Vazquez, M., and Vicente, M. G. H. (2012) Boron Tumor-Delivery for BNCT: Recent Developments and Perspectives. Boron Science: New Technologies and Applications (Hosmane, N. S., Eds.) pp. 209-242, Ch. 10, Taylor & Francis, Bosa Roca. (38) Barth, R. F., Vicente, M. G. H., Harling, O. K., Kiger, W., Riley, K. J., Binns, P. J., Wagner, F. M., Suzuki, M., Aihara, T., Kato, I., et al. (2012) Current status of boron neutron capture therapy of high grade gliomas and recurrent head and neck cancer. Radiat. Oncol. 7, 146. (39) Scholz, M., and Hey-Hawkins, E. (2011) Carbaboranes as Pharmacophores: Properties, Synthesis, and Application Strategies. Chem. Rev. 111, 7035-7062. (40) Sivaev, I. B., and Bregadze, V. V. (2009) Polyhedral Boranes for Medical Applications: Current Status and Perspectives. Eur. J. Inorg. Chem. 2009, 1433-1450. (41) Barth, R. F., Coderre, J. A., Vicente, M. G. H., and Blue, T. E. (2005) Boron Neutron Capture Therapy of Cancer: Current Status and Future Prospects. Clin. Cancer Res. 11, 39874002. (42) Ulrich, G., Ziessel, R., and Harriman, A. (2008) The Chemistry of Fluorescent Bodipy Dyes: Versatility Unsurpassed. Angew. Chem. Int. Ed. 47, 1184-1201. (43) Loudet, A., and Burgess, K. (2007) BODIPY Dyes and Their Derivatives: Syntheses and Spectroscopic Properties. Chem. Rev. 107, 4891-4932. (44) Bañuelos, J. (2016) BODIPY Dye, the Most Versatile Fluorophore Ever? Chem. Rec. 16, 335-348. (45) Bessette, A., and Hanan, G. S. (2014) Design, synthesis and photophysical studies of dipyrromethene-based materials: insights into their applications in organic photovoltaic devices. Chem. Soc. Rev. 43, 3342-3405. (46) Vendrell, M., Zhai, D., Er, J. C., and Chang, Y.-T. (2012) Combinatorial Strategies in Fluorescent Probe Development. Chem. Rev. 112, 4391-4420. (47) Sozmen, F., Kolemen, S., Kumada, H.-O., Ono, M., Saji, H., and Akkaya, E. U. (2014) Designing BODIPY-based probes for fluorescence imaging of b-amyloid plaques. RSC Adv. 4, 51032-51037. (48) Kamkaew, A., Lim, S. H., Lee, H. B., Kiew, L. V., Chung, L. Y., and Burgess, K. (2013) BODIPY dyes in photodynamic therapy. Chem. Soc. Rev. 42, 77-88. (49) Xochitiotzi-Flores, E., Jimenez-Sanchez, A., Garcia-Ortega, H., Sanchez-Puig, N., Romero-Avila, M., Santillan, R., and Farfan, N. (2016) Optical properties of two fluorene derived BODIPY molecular rotors as fluorescent ratiometric viscosity probes. New J. Chem. 40, 4500-4512. (50) Kowada, T., Maeda, H., and Kikuchi, K. (2015) BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chem. Soc. Rev. 44, 4953-4972. (51) Boens, N., Leen, V., and Dehaen, W. (2012) Fluorescent indicators based on BODIPY. Chem. Soc. Rev. 41, 1130-1172. 30 ACS Paragon Plus Environment

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(52) Wang, J., Lu, Y., McGoldrick, N., Zhang, C., Yang, W., Zhao, J., and Draper, S. M. (2016) Dual phosphorescent dinuclear transition metal complexes, and their application as triplet photosensitizers for TTA upconversion and photodynamic therapy. J. Mater. Chem. C 4, 6131-6139. (53) Lu, H., Mack, J., Yang, Y., and Shen, Z. (2014) Structural modification strategies for the rational design of red/NIR region BODIPYs. Chem. Soc. Rev. 43, 4778-4823. (54) Xuan, S., Zhao, N., Zhou, Z., Fronczek, F. R., and Vicente, M. G. H. (2016) Synthesis and in Vitro Studies of a Series of Carborane-Containing Boron Dipyrromethenes (BODIPYs). J. Med. Chem. 59, 2109-2117. (55) Gibbs, J. H., Wang, H., Bhupathiraju, N. V. S. D. K., Fronczek, F. R., Smith, K. M., and Vicente, M. G. H. (2015) Synthesis and properties of a series of carboranyl-BODIPYs. J. Organomet. Chem. 798, Part 1, 209-213. (56) Plešek, J., Grüner, B., Macháček, J., Císařová, I., and Čáslavský, J. (2007) 8-Dioxane ferra(III) bis(dicarbollide): A paramagnetic functional molecule as versatile building block for introduction of a Fe(III) centre into organic molecules. J. Organomet. Chem. 692, 4801-4804. (57) Teixidor, F., Pedrajas, J., Rojo, I., Viñas, C., Kivekäs, R., Sillanpää, R., Sivaev, I., Bregadze, V., and Sjöberg, S. (2003) Chameleonic Capacity of [3,3‘-Co(1,2-C2B9H11)2]- in Coordination. Generation of the Highly Uncommon S(thioether)−Na Bond. Organometallics 22, 3414-3423. (58) Sivaev, I. B., Kulikova, N. Y., Nizhnik, E. A., Vichuzhanin, M. V., Starikova, Z. A., Semioshkin, A. A., and Bregadze, V. I. (2008) Practical synthesis of 1,4-dioxane derivative of the closo-dodecaborate anion and its ring opening with acetylenic alkoxides. J. Organomet. Chem. 693, 519-525. (59) Cioran, A. M., Teixidor, F., and Viñas, C. (2015) The effect of a paramagnetic metal ion within a molecule: comparison of the structurally identical paramagnetic [3,3-Fe(1,2C2B9H11)2]- with the diamagnetic [3,3-Co(1,2-C2B9H11)2]- sandwich complexes. Dalton Trans. 44, 2809-2818. (60) Hawthorne, M. F., Young, D. C., Andrews, T. D., Howe, D. V., Pilling, R. L., Pitts, A. D., Reintjes, M., Warren, L. F., and Wegner, P. A. (1968) p-Dicarbollyl derivatives of the transition metals. Metallocene analogs. J. Am. Chem. Soc. 90, 879-896. (61) Moravej, M., Purnama, A., Fiset, M., Couet, J., and Mantovani, D. (2010) Electroformed pure iron as a new biomaterial for degradable stents: In vitro degradation and preliminary cell viability studies. Acta. Biomater. 6, 1843-1851. (62) Rai, Y., Pathak, R., Kumari, N., Sah, D. K., Pandey, S., Kalra, N., Soni, R., Dwarakanath, B. S., and Bhatt, A. N. (2018) Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition. Sci. Rep. 8, 1531. (63) Chan, G. K. Y., Kleinheinz, T. L., Peterson, D., and Moffat, J. G. (2013) A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays. PLoS One 8, e63583. (64) Tarrés, M., Canetta, E., Paul, E., Forbes, J., Azzouni, K., Viñas, C., Teixidor, F., and Harwood, A. J. (2015) Biological interaction of living cells with COSAN-based synthetic vesicles. Sci. Rep. 5, 7804. (65) Baruah, M., Qin, W., Basarić, N., De Borggraeve, W. M., and Boens, N. (2005) BODIPY-Based Hydroxyaryl Derivatives as Fluorescent pH Probes. J. Org. Chem. 70, 41524157.



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Table of Contents Graphic

Fluorescent BODIPY-anionic boron cluster conjugates as potential agents for cell tracking

Mahdi Chaari,1,4† Nerea Gaztelumendi,2† Justo Cabrera-González,1# Paula Peixoto-Moledo,2 Clara Viñas,1 Elba Xochitiotzi-Flores,3 Norberto Farfán,3 Abdelhamid Ben Salah,4 Carme Nogués,2* Rosario Núñez1*


Fluorescent BODIPY-anionic boron cluster ... - ACS Publications

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