Introduction

Fluorescent organic materials have attracted increasing interest due to their potential applications in bioimaging and chemosensing (Zhang et al. 2017; Huang et al. 2016; Chang et al. 2016). However, a majority of fluorescence organic materials face the problem of aggregation-induced quenching (ACQ) because of strong π–π stacking interactions in the enlarged π-conjugated system (Thomas et al. 2007), resulting in limitation of their practical applications. Aggregation-induced emission (AIE), as a novel phenomenon, was first discovered by Tang’s group (Luo et al. 2001). The compound exhibits very weak fluorescence in solution but emits strong fluorescence in the aggregated state. Currently, AIE materials have gained widespread attentions for their potential application in various fields, for instance bioimaging, chemosensors and organic light-emitting diodes (Gui et al. 2017; Mei et al. 2015). However, the working mechanism of the AIE processes is very complicated, and the reason is still unclear. At present, some theories or hypothesises are proposed to explain the AIE phenomenon, e.g., intramolecular restrictions, J-type aggregates, restriction of intramolecular rotation, twisted intramolecular charge transfer (Li et al. 2017a ; Kwork et al. 2015; Ding et al. 2013). Therefore, a large number of compounds with AIE properties have been developed. To further enlarge the family of specific AIE-active compounds, it is necessary to carry out more extensive investigations in this field.

Herein, we developed a novel A–D–π–A type triphenylamine-based derivative (Fig. 1), which displays AIE behavior in water–ethanol system. First, triphenylamine moiety with a strong electron donor can help to delocalize the electron distribution and extend the conjugation system (Liu et al. 2014). Second, owing to the strong electron-withdrawing ability and large π-conjugation system, terpyridine ligand exhibits good optical properties (Wang et al. 2018a). Third, the pyridinium cation moiety with high membrane potential is able to target intracellular mitochondria (Tian et al. 2017). Compound L displays good AIE characteristic and emits red fluorescence. As expected, compound L is capable of targeting mitochondria.

Fig. 1
figure 1

Synthesis of the compound L

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Experimental section

Materials and measurements

All of the reagents were available commercially and used without further purification. 1H NMR and 13C NMR spectra were obtained on Bruker 400 Ultrashield spectrometer. Mass spectrum was measured on HRMS-LTQ Obritrap XL (ESI source). IR spectra (KBr pressed pellets) were obtained on Nicolet FT-IR-870SX spectrophotometer. UV–Vis absorption and one-photon fluorescence spectra were obtained on UV-1700 and Hitachi F-4600 fluorescence spectrophotometer, respectively.

Synthesis of L

M1 (Wang et al. 2016) (0.50 g, 1 mmol) were dissolved in ethanol (50 mL), M2 (Tian et al. 2017) (0.25 g, 1 mmol) and 3 drops piperidine were subsequently added. The reaction mixture was refluxed for 24 h. The solid was filtered off and washed with ethanol. The product was recrystallized from methanol to afford red solids. IR (cm−1) selected bands: 2918, 2135, 1796, 1646, 1585, 1510, 1468, 1439, 1418, 1389, 1323, 1292, 1175, 972, 875, 841, 793, 699, 660, 621, 557, 540. 1H NMR (d6-DMSO) δ: 8.77 (m, 4 H), 8.70 (m, 4 H), 8.16 (d, J = 6.9 Hz, 2 H), 8.05 (t, J = 7.8 Hz, 2 H), 7.97 (m, 3 H), 7.69 (d, J = 8.7 Hz, 2 H), 7.54 (m, 2 H), 7.44 (t, J = 7.9 Hz, 2 H), 7.36 (d, J = 16.2 Hz, 1 H), 7.23 (m, 5 H), 7.12 (d, J = 8.7 Hz, 2 H), 4.23 (s, 3 H). 13C NMR (d6-DMSO) δ: 156.1, 155.4, 149.7, 145.2, 140.7, 137.9, 130.4, 130.1, 128.6, 126.2, 125.4, 124.9, 124.8, 123.4, 122.7, 121.5, 121.3, 117.7, 56.4. MS: 594.26 ([M-PF6]+).

Computational details

The ground state geometries of the molecules are optimized at the time-dependent density functional theory (TD-DFT)/CAM-B3LYP (Yannai et al. 2004) functional level without any symmetry restraint (Wang et al. 2012) using Gaussian 09 program (Frisch et al. 2009). The UV–Vis absorption properties are obtained based on the optimized structures with CAM-B3LYP functional utilizing Gaussian 09 package. The basis set of 6-31G* is chosen for all atoms. An analytical frequency confirms evidence that the calculated species represents a true minimum without imaginary frequencies on the respective potential energy surface. The lowest 25 spin-allowed singlet–singlet transitions, up to energy of about 5 eV, were taken into account in the calculation of the absorption spectra. The molecular orbitals were visualized using GaussView 5.0.9 (Dennington et al. 2009).

Results and discussion

Photophysical properties

The UV–Vis absorption and one-photon fluorescence spectra of L in different polar solvents were initially investigated. The corresponding photophysical data were summarized in Table 1. As depicted in Fig. 2, compound L exhibited two strong absorption bands at about 460 nm and 275 nm in different solvents, which can be assigned to the π–π* transition mixed with intramolecular charge transfer process (ICT) and πterpyridine–πterpyridine* transition, respectively. Weak solvatochromism was observed in the maximum absorption band, indicative of the little difference in dipoles between the ground and the excited states of the compound. Increasing the solvent polarity from benzene to DMF (Fig. 2 and Table 1), the fluorescence maxima of L shifted from 593 to 608 nm and the fluorescence was quenched at the same time, which might be assigned to ICT. Moreover, the Stokes’ shifts also exhibited an increasing tendency with the increase of the solvent polarity. To better understand the ICT process, we performed theoretical calculations with the density functional of CAM-B3lyp/6-31 g(d) (Fig. 3 and Table 2). From Fig. 3, the electron cloud of the highest occupied molecular orbital (HOMO) was dominated by the triphenylamine unit, –C=C– moiety and pyridine unit. Meanwhile, the lowest unoccupied molecular orbital (LUMO) was localized on the pyridine group due to the strong electron-withdrawing ability. Due to the HOMO → LUMO transition, the absorption band at 484 nm was assigned as the ICT transition mixed with π–π* transition, keeping consistent with the experimental ones.

Table 1 The photophysical data of L in different solvents
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Fig. 2
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UV-Vis absorption (left) and one-photon fluorescence (right) spectra of L in different solvents

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Fig. 3
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Electron density distributions of frontier molecular orbitals of L

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Table 2 Calculated linear absorption properties (nm), excitation energy (eV), oscillator strengths and major contribution for L using CAM-B3LYP/6-31G*
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Aggregation-induced emission (AIE) properties

Compound L could be dissolved in many organic solvents, such as THF, ethanol, acetonitrile and DMSO, but has poor solubility in water. To determine whether L is AIE active and considering the biological application, the UV–Vis absorption and fluorescence spectra of L in water–ethanol mixtures with different fraction of water (fw) were investigated. From Fig. 4, we can see that compound L displayed three absorption bands in dilute ethanol solution. As the water fraction increased to 90%, the band at about 460 nm gradually decreased, accompanying with slightly blue shift. Moreover, the absorption curves decayed further from zero in the long wavelength region with high water fraction, indicating the existence of aggregative species in these solutions.

Fig. 4
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UV-Vis absorption spectra of L in H2O/ethanol mixtures with different fractions of water

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Figure 5 exhibited the changes of fluorescence intensity of L in aqueous mixtures. In dilute ethanol solution, compound L was nearly non-emissive. The fluorescence intensity of L obviously decreased with the gradual addition of water amounts into ethanol solution. This phenomenon could be reasonably explained by a twisted intramolecular charge transfer (TICT) mechanism. The fluorescence intensity was invigorated at fw = 80% and reached the maximum fluorescence intensity with λ = 618 nm at fw = 90%, which was a threefold enhancement to that of L in absolute ethanol solution, demonstrating the AIE characteristic of compound L. In this stage, compound L begins to aggregate because the solvating power of the aqueous mixture decrease, making the emission enhanced due to the restriction of intramolecular rotation effect. Meanwhile, the ICT effect is efficiently weakened. As depicted in Fig. 5b, when fw reached 95%, the fluorescence intensity of L in the water–ethanol mixture decreased. This phenomenon has often been detected in some dyes possessing AIE properties, but the reason is still unclear. Up to now, this phenomenon could be explained by two possible factors: (1) in the extensive aggregation state, the molecules on the nanoparticle surface contributes mainly to the emission, while the molecules in interior contributes faintly, resulting in a decrease of fluorescence intensity. (2) When adding water to the solution, the molecules could form amorphous particles and crystal particles. The former one could decrease the fluorescence intensity, while the latter will enhance the intensity of fluorescence (Zheng et al. 2013).

Fig. 5
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a Emission spectra of L in H2O/ethanol mixtures with different fractions of water, inset: photographs of L in H2O/ethanol taken under 365 nm UV illumination. b The changes of fluorescence intensity with different water fractions

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To further explore the enhanced emission of L in water–ethanol system, the aggregate process of L with different water fractions was investigated by scanning electron microscopy (SEM). As shown in Fig. 6a, a spherical particle of L formed immediately in the mixture of water–ethanol with fw of 40%. However, when fw = 90%, the AIE dots dispersed regularly and taken a block-like arrangement. These data demonstrated that the enhanced emission of L relies on the formation of nanoparticles. The comparative analysis of L with the previously reported AIE molecule was summarized in Table 3. Compared with the other reported AIE molecule, compound L exhibited long emission wavelength (> 600 nm), which encouraged us to explore its potential application in biological imaging.

Fig. 6
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SEM images of L in H2O/ethanol mixtures with different water fractions: a H2O/ethanol (40:60, v/v); b H2O/ethanol (90:10, v/v)

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Table 3 Comparison of the properties of L with the previously reported AIE molecule
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Biological application

High cell viability is essential for a fluorescent probe in its biological application. To evaluate the cytotoxicity of L, HepG2 cells were treated with L, and its viability was subsequently determined by MTT assay. Figure 7 revealed that HepG2 cells incubated with 15 μM of L remained 90% viable after 24 h of feeding time, suggesting the low toxicity and good biocompatibility of L. To assess the cellular uptake properties, HepG2 cell was used as a model, whereby 10 μM of L was treated for 30 min followed by confocal microscopy imaging after washing with PBS. As shown in Fig. 8a, L could penetrate into the cell cytosol within the short incubation period and exhibited intensive red fluorescence. It is well-known that fluorophores possessing a pyridinium moiety can function as mitochondria probes. A co-staining experiment with Mitotracker deep red was performed to establish whether L could be internalized with membrane-rich mitochondria. As displayed in Fig. 8a, a clear mitochondria profile with strong green fluorescence was observed from the green channel, which was ascribed to Mitotracker deep red. A similar mitochondria profile with red fluorescence was obtained after treating with L. The signal of L overlaid very well with the fluorescence of Mitotracker deep red (Fig. 8c). Moreover, Pearson’s co-localization coefficient, which describes the correlation of the intensity distribution between these two channels, was calculated to be 0.92 (Fig. 8b), indicating compound L could selectively accumulate into the mitochondria. The above results revealed that compound L is a promising fluorescent probe for targeting mitochondria.

Fig. 7
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Cytotoxicity data results obtained from the MTT assay at different concentrations for 24 h

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Fig. 8
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Colocalization fluorescence microscopy images of HepG2 cells treated with 10 μM of L and Mitotracker deep Red (MTR): a Fluorescence imaging of L and MTR in HepG2 cells. b Correlation plot of L and MitoTracker intensities. c Intensity profile of ROIs across HepG2 cells. Scale bar 10 μm

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Conclusion

In summary, an A–D–π–A type red fluorescent compound was designed and synthesized. Compound L was almost non-emissive when dissolved in ethanol, but became strongly emissive when aggregated in water solution, demonstrating a typical AIE phenomenon. Confocal imaging experiment indicated that compound L was capable of targeting the mitochondrial in living HepG2 cells. Current results offer a platform for the development of new AIE materials with red emission for targeting mitochondria.