Borondifluoride β-diketonate complex as fluorescent organic nanoparticles: aggregation-induced emission for cellular imaging

Abstract

A novel borondifluoride curcuminoid complex TB showing an AIE effect is designed and prepared. The photophysical properties were investigated by tuning the TICT and AIE state in various solutions. In particular, the TB loaded nanoparticles acted as an excellent reagent for cellular imaging.

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Fluorescent molecular aggregates with well-defined shapes have shown promising applications in biomimetic chemistry and materials sciences.^1,2 Compared to the inorganic fluorescent nanoparticles with possible cytotoxicity from heavy metal ions, the organic ones have shown their merits such as large Stokes shifts, high photostability, low cytotoxicity and size-tuneable emission.^3–6 Although great progress of small molecules-based fluorescent nanoparticles has been made, most of them have been suffering the aggregation-caused quenching (ACQ) in the particles and prohibit their further imaging applications.^7–9 To address this challenge, Tang and co-workers developed a series of luminogenic molecules showing aggregation-induced emission (AIE) effect.^10–15 These molecules display weak emission due to the intramolecular rotation in good solvents but become highly luminescent in the aggregated state.

Borondifluoride luminogens have attracted great interest due to their excellent photophysical properties.^16–18 The most widely used dye in this family is boron-dipyrromethene (BODIPY).^19,20 However, BODIPY dyes exhibit small Stokes shifts (typical less than 15 nm), which would increase the background interference in cellular imaging. Two strategies referring to ligand design have been developed to address this issue. First, high Stokes shifts would be realized by changing the symmetric N,N-bi-chelating ligand to unsymmetrical ones;²¹ second, O,O-bi-chelating ligand modified with electron donating groups have been used to prepare borondifluoride curcuminoids complexes which show large Stokes shifts.²² The later derivatives have been demonstrated two-photon absorption and versatile bioimaging.²³ Several O,O-bi-chelating borondifluoride complexes have shown the AIE effect, nevertheless, researches are focus on their mechanochromism;^24–26 applications of the chromophore to bioimaging have not been developed.

In this contribution, a novel borondifluoride curcuminoid complex TB is prepared and its AIE properties and fluorescent nanoparticle imaging were investigated. DFT calculation results indicate that triphenylamine (TPA) moiety behaves as an electron donor while the borondifluoride (BDF) curcuminoid fluorophore acts as an electron acceptor (Fig. 1). Therefore, TB would show large solvatochromism and exhibit excited twist intramolecular charge transfer (TICT) state.²⁷ In addition, AIE effect comes to play for restriction of the intramolecular rotation in the aggregated state. Furthermore, bovine serum albumin (BSA) was used as matrix to prepare TB nanoparticles due to the excellent biocompatible properties of albumin.²⁸ These BSA-based nanoparticles loaded TB could be accumulated by HeLa cells and show enhanced green fluorescence emission.

Fig. 1 (a) Compound TB studied in this work; DFT-calculated (b) HOMO and (c) LUMO orbits for TB using the B3LYP/6-31G(d) basis set.

TB was prepared according to the synthetic route shown in Scheme S1.† Detailed procedure for the synthesis and characterization data is presented in the ESI.† The photophysical properties of TB in various organic solvents were investigated (Fig. 2). The absorption maximum of TB red-shifted about 15 nm when the solvents changed from hexane to dichloromethane, meanwhile the colorless solution turned to a deep yellow one. The donor–acceptor dye TB shows a bathochromic shift with the solvent polarity because the polar solvents lower the energy gap by stabilization of the charge transfer exited state. Interestingly, the spectrum of TB in dichloromethane exhibited the largest shift from 450 to 582 nm. This observation is consistent with Tang's report.²⁹ However, in the more polar methanol and acetonitrile, the localized excited state (LE) change to TICT state, which quenches the fluorescent emission dramatically (Fig. 2b).

Fig. 2 Photographs of TB in various solvents under (a) visible light, (b) 365 nm UV illumination. Normalized (c) UV-vis and (d) fluorescent spectra of TB in various solvents. Concentration is 10 μM, excitation wavelength is 395 nm. HEX: hexane; TOL: toluene; DCM: dichloromethane; THF: tetrahydrofuran; EA: ethyl acetate; MeCN: acetonitrile; MeOH: methanol.

To further investigate the solvent effect, the emission measurements of TB in THF/hexane mixtures with varying fraction of hexane were performed. Both the emission maximum and wavelength changed gradually with the hexane addition (Fig. 3). No sudden change was observed when this solvent polarity varied. This result is different to the observation of BODIPY derivatives with same triphenylamine as the electron donor moiety.¹⁶ Compared to the intensity determined in pure THF, the emission maximum of TB in THF/hexane mixture with 90% of hexane increased by 8-fold. The emission band exhibits a hypsochromic shift more than 90 nm during this process. Furthermore, the favourable liner relation (R² = 0.992) between hexane fraction and peak intensity indicates the TB can be used as a probe to monitor the environment polarity.

Fig. 3 (a) Photographs of TB in THF/hexane mixtures with different fraction of hexane under 365 nm UV illumination. (b) Fluorescent emission spectra of TB in THF/hexane mixtures with different fraction of hexane. (c) The dependence of fluorescent emission maximum intensity and wavelength on hexane fraction. Concentration is 10 μM, excitation wavelength is 395 nm.

The emission spectra of TB in THF/water mixtures with varying water fraction were collected. In polar solvents, TB presents a bathochromic shift in emission with decreased intensity. As a contrast, TB shows good solubility in THF but forms aggregates in water, which might restrict the intramolecular rotation and restore fluorescent emission. Therefore, it is anticipated that the photophysical properties of TB can be well tuned by changing the solvent polarity and its aggregation in THF/water mixtures. As shown in Fig. 4, the solution of TB became turbid when the water fraction reached 70% in the mixture solution, indicating that the aggregates were formed. Spherical shaped aggregates with uniform size were observed in THF/water mixture with a low fraction of water in SEM images (Fig. S1†). The pure THF solution of TB shows strong yellow-green emission with the maximum at 540 nm. After addition of a low fraction of water, the emission intensity was dramatically quenched and reached to its minimum at 60% water, which was evidenced by both photograph and spectra. At the same time, the emission maximum was red shifted about 20 nm to 560 nm. Such a TICT effect is consistent with the results in Tang's report.

Fig. 4 Photographs of TB in THF/water mixtures with different fraction of water under (a) visible light, (b) 365 nm UV illumination. (c) Fluorescent spectra of TB in THF/water mixtures with different fraction of water. (d) The dependence of intensity on water fraction. Concentration is 10 μM, excitation wavelength is 395 nm.

Considering the single bond rotates between the donor triphenylamine and acceptor borondifluoride luminogen, we anticipate that TB is an AIE molecule according to Tang's well-developed model. The photographs and fluorescent spectra collected in high fraction of water (≥70%) proved our hypothesis (Fig. 4). The quenched emission of TICT state in THF/water mixture with 60% of water was enhanced after further addition of water. The intensity of TB in 90% of water was even higher than that in pure THF solution. Interestingly, the emission maximum exhibits a hypsochromic shift of 25 nm during this enhancement, because the polarity in the interior aggregates is lower than that in the external environment. The solvent polarity and AIE effects on the TB photoluminescent properties were further tested in two kinds of viscous mixtures: polar methanol/glycerol and non-polar DCM/silicon oil. The emission intensities increased with viscosity and showed polarity dependence (Fig. S2†).

The TB nanoparticles (NPs) were prepared by using polymer bovine serum albumin (BSA) as matrix. Compare to the aggregates of AIE molecules formed in THF/water mixture, these kinds of nanoparticles have been proved to be superior in emissive brightness and photostability.³⁰ In fact, Wei and Zhang also developed several strategies to prepare AIE molecules loaded particles showing great advantages in bioimaging.^31–36 Three different concentrations of TB solutions were slowly added to a BSA aqueous solution under stirring, separately. The TB aggregates would form in the nonpolar solution and further entangled with the hydrophobic moieties of BSA. The TB nanoparticles were obtained after sonication for 90 seconds, evaporation THF and filtration through the syringe filter (ESI†). The average size of the particles is dependent on the molecular concentration (Fig. S4†). Take one sample as an example, dynamic light scattering (DLS) result and transmission electron microscopy (TEM) image of the TB nanoparticles (10 μM) were shown in Fig. 5. TEM image shows that the particles size is ranged in 20–80 nm. The average size of the NPs is 141.8 nm with a little broad size distribution (PDI = 0.248).

Fig. 5 (a) DLS diagram and (b) TEM image of BSA nanoparticle loaded with TB (10 μM).

The superiority of TB NPs over the pure TB aggregates was further evaluated in vitro cellular imaging using confocal laser scanning microscopy (CLSM). Weak fluorescent emission could be detected for the pure TB due to its bad cellular uptake ability (Fig. S3†). In contrast, after incubation of the TB NPs in HeLa cells for 3 h, the CLSM image shows strong green emission with 405 nm laser excitation (Fig. 6). This result suggests that BSA encapsulated TB nanoparticles could be effectively internalized via endocytosis pathway into the cells. Photostability of TB-loaded BSA particles was investigated by laser scanning. The fluorescence of these particles showed about 60% intensity after 1 hour continuous exposure. In addition, the cells showed high viability even the incubation was longer than 48 h. The cell toxicity tests results demonstrate that the cellular viabilities were higher than 90% in the determined low concentration (10 μM) (Fig. S6†). These results indicate that that the particle is a good imaging reagent with high biocompatibility.

Fig. 6 (a) Confocal images of HeLa cells after incubation with TB loaded BSA nanoparticles for 3 h at 37 °C. (b) The phase contrast image of nanoparticles at the same condition. Concentration is 10 μM.

In conclusion, we have developed a novel donor–acceptor borondifluoride derivative which shows interesting solvent polarity dependence and AIE effects. LE and TICT were the crucial mechanism to tune its photoluminescence spectra by changing the solvents or protonation of the molecule. AIE effect was utilized to prepare TB-containing BAS nanoparticles, which exhibited increased HeLa cell uptake ability and excellent bioimaging ability.³⁷ Further work along this way to prepare near-infrared AIE fluorescent nanoparticles for imaging is ongoing in our laboratory.

Acknowledgements

This work was supported by State Key Laboratory of Powder Metallurgy, Central South University, the Natural Science Foundation of China (21473257, 91127024, 21501031 and 51574267) and Natural Science Foundation of Jiangxi, China (20151BAB213001). The authors also thank the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) and the National High Technology Research and Development Program of China (2015AA020502).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Synthetic route and characterization of TB, NMR spectra of TB, DLS and SEM images of aggregates, cell imaging test. See DOI: 10.1039/c6ra23719c

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