Significant emission enhancement of a bola-amphiphile with salicylaldehyde azine moiety induced by the formation of [2]pseudorotaxane with γ-cyclodextrin

Abstract

A cationic bola-amphiphile with salicylaldehyde azine moiety exhibited weak emission in water, whereas around 30-fold enhancement of emission was observed upon addition of γ-cyclodextrin due to the formation of [2]pseudorotaxane inclusion complex, which can specifically localize in mitochondria of living cells for fluorescent imaging.

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As one of the famous macrocyclic host molecules in supramolecular chemistry, cyclodextrins (CDs) are a family of natural cyclic oligosaccharides formed by six or more α-1,4-linked D-glucopyranosyl residues, having a hydrophobic cavity and a hydrophilic outer surface.^1–4 The hydrophobic interior of CDs allows encapsulation of a suitable hydrophobic guest, while the hydrophilic exterior makes the host–guest complexes water-soluble. Their salient inclusion capabilities have resulted in a broad range of applications,^1–17 including drug delivery, catalysis, bioimaging, and environmental science, among others.

Salicylaldehyde azine and its derivatives, owing to their both of aggregation induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) characteristics,^18–23 have received a great deal of research interest over the past years and have exhibited highly promising applications,^18–33 such as optoelectronic materials and biomedical areas. In particular, Liu's group recently developed a series of light-up salicyladazine fluorogens by conjugating different functional groups, achieving the purposes of in situ monitoring vital biological processes and/or therapeutic effects.^18,27–33 Besides, Zhang and his co-workers recently reported a series of tetraphenylethylene and silole fluorophores with AIE properties for various applications.^34–37 Song's group extensively investigated self-assembly behaviours of bola-amphiphiles appending tetraphenylethene AIE moiety³⁸ and other functional groups.^39–41

As ESIPT process typically involves the enol (E)–keto (K) phototautomerization by the intramolecular proton transfer via hydrogen bonding, therefore, its emission is very sensitive to the environmental conditions.^18,19 Generally, the high polarity and hydrogen-bond donor ability of solvents, such as water, could inhibit the ESIPT emission from K*. In contrast, such emission with a large Stokes shift can be restored in the lower polar environment.^18,19 As a consequence, molecules with ESIPT properties often give rise to the enhanced emission, when they are incorporated into the nonpolar and hydrophobic microenvironment of proteins or lipid membranes in cells and organisms.^20,42,43 This makes them act as suitable light-up bioprobes for biosensing and imaging applications. However, it seems difficult to investigate the precise location and specific interaction of probes within bio-macromolecules. This should be ascribed to the complicated structures of these biological species.

In comparison with bio-macromolecules, the simpler inclusion complex of guest molecules by CDs can serve as a versatile model for helping to understand the supramolecular interactions or mechanisms of these small molecular bioprobes with biological species. Moreover, the supramolecular host–guest CDs inclusion complexes may exhibit more biocompatible and more appealing characteristics. Herein, we reported a [2]pseudorotaxane inclusion complex 1@γ-CD formed by a cationic salicylaldehyde azine bola-amphiphile 1 with γ-CD (Scheme 1), which exhibited a significant enhancement of emission in water. Besides, the [2]pseudorotaxane inclusion complex can act as an effective and specific probe for mitochondrial imaging in living cells even at a very low concentration.

Scheme 1 The chemical structure of 1 and schematic representation of [2]pseudorotaxane 1@γ-CD. The right photographs are their aqueous solution under 365 nm light.

The cationic salicylaldehyde azine bola-amphiphile 1 was synthesized according the literature,²⁸ and ¹H NMR confirmed its identity (Scheme S1 and Fig. S1†). The diluted water solution of 1 exhibited a very weak emission, while a bright green emission was observed upon addition of γ-CD (Scheme 1, right). We suggest that the enhanced emission should be attributed to the inclusion of salicylaldehyde azine part into the hydrophobic cavity of γ-CD, thus, restoring the ESIPT process of 1,^18,19 together with the shielding effect for the quenching ability of pyridinium at the ends.^44–47

Subsequently, such inclusion event was thoroughly investigated by absorption and emission spectra. Fig. S2† shows the UV-vis absorption spectral changes of 1 as a function of added γ-CD. An isosbestic point at 393 nm was observed throughout the titration, indicating the presence of only two absorption species in solution: the free 1 and the inclusion complex 1@γ-CD. In sharp contrast to the little changes in absorption spectra, a remarkable fluorescence enhancement with a max λ_em at 523 nm was detected upon increasing addition of γ-CD, as shown in Fig. 1. About 30 fold enhancement was calculated after addition of one equivalent γ-CD. While little changes in emission spectra were observed after addition of α-CD or β-CD (Fig. S3†), which should be attributed their smaller size of hydrophobic cavity. This phenomenon also has been observed by Green⁴⁸ and Sukwattanasinitt.⁴⁹

Fig. 1 Fluorescence spectra (λ_ex = 400 nm) of 50 μM 1 upon the titration of γ-CD (0–3.0 equiv.) in PB buffer solution (10 mM, pH 7.0). Inset: fluorescence intensity at 523 nm as a function of γ-CD concentration.

On the other hand, the increase of emission intensity with increasing addition of γ-CD becomes much slower when one equiv. of γ-CD was introduced, suggesting their strong binding affinity and the formation of a 1 : 1 inclusion complex (Fig. 1 inset), which was further confirmed by Jobs's plot (Fig. S4†). Then, the binding constant (K) between 1 and γ-CD was determined by employing Benesi–Hilderbrand's double reciprocal plot for a 1 : 1 complex according to the following equation:

where F_i − F₀ refers to the difference in fluorescence intensity of 1 at 523 nm in the presence and absence of γ-CD; [1] and [γ-CD] are the concentrations of 1 and γ-CD, respectively. A straight line of 1/(F_i − F₀) vs. 1/[γ-CD] confirms that the interaction ratio of γ-CD and 1 is 1 : 1, and gives K value 7.2 × 10³, indicating the high binding affinity between them.

In order to prove our speculation that the salicylaldehyde azine part in 1 was threaded into the hydrophobic cavity of γ-CD, we performed the 1D and 2D ¹H NMR experiments. As shown in Fig. 2, a mixture of 1 and γ-CD (1 : 1) exhibited a quite different spectrum from that of 1 itself in D₂O. Upon addition of γ-CD, ¹H NMR spectra of 1 showed significant chemical shift changes. Specifically, the protons H(a,b,c,d) resonance of salicylaldehyde azine part shifted to upfield position (Δδ = 0.20, 0.21, 0.32, and 0.34 ppm, for Ha,b,c,d, respectively). In the ¹H–¹H NOESY spectra of 1 + γ-CD (Fig. 3), NOE crosspeaks (A–K) between the protons (Ha,b,c,d,e) of 1 and the protons (H3,5,6) of γ-CD are observed. These observations indicate that the salicylaldehyde azine part of 1 is encircled by the γ-CD ring, generating [2]pseudorotaxane inclusion complex 1@γ-CD.

Fig. 2 Partial ¹H NMR spectra (400 MHz, D₂O) of 1 (1 mM, bottom) and 1 + 1.2 equiv. of γ-CD (top) at ambient temperature.

Fig. 3 Partial ¹H–¹H NOESY spectra (500 MHz, D₂O) of 1 + 1.2 equiv. of γ-CD at ambient temperature.

Due to the presence of two cationic pyridinium groups in [2]pseudorotaxane 1@γ-CD together with its enhanced emission, we investigated its ability for specifically subcellular mitochondria fluorescent imaging by confocal laser scanning microscopy (CLSM), as exemplified in HeLa cells. A commercial mitochondrial dye, MitoTracker Deep Red FM, was used as a contrast agent to stain the mitochondria. After incubation with HeLa cells for 20 min, as shown in Fig. 4a, the complex 1@γ-CD can be readily taken up by the cells. The co-localization experiment with MitoTracker Deep Red FM exhibited the excellent superposition pattern between the green fluorescence emission from 1@γ-CD and the red fluorescence emission from MitoTracker, indicating that the [2]pseudorotaxane 1@γ-CD can be used as a promising biomarker for staining of the cell's mitochondria.

Fig. 4 CLSM images of live HeLa cells incubated with (a) 1@γ-CD and (b) 1 for 20 min at the concentrations of 5.0 μM. MitoTracker Deep Red FM (0.05 μM) was used to stain the mitochondria of the cell. Scale bar: 20 μm. Excitation and emission wavelength: 408 nm and 500–550 nm for 1@γ-CD and 1; 561 nm and 570–1000 nm for MitoTracker Deep Red FM.

Not surprisingly, 1 can also stain the cell's mitochondria (Fig. 4b). And no obvious fluorescent difference was observed when HeLa cells were incubated with 1@γ-CD or 1 at the concentration of 5.0 μM (Fig. 5), which should be mainly attributed to AIE characteristics of 1 at a high concentration for staining cells.^18,28 However, a significant fluorescent difference was observed when the concentration of 1@γ-CD or 1 decreased to 0.5 μM. A strong fluorescence was observed from HeLa cells incubated with 1@γ-CD at the concentration of 0.5 μM. In contrast, there is almost no fluorescence observed in HeLa cells incubated with 0.5 μM of 1 (Fig. 5b and Fig. S6†). The observations were further confirmed by Image J software analysis which showed a statistically significant decrease in green fluorescent staining from HeLa cells treated with 1 as compared with that treated with 1@γ-CD (Fig. 5c). When the concentration of 1@γ-CD was even reduced to be as low as 0.05 μM, fluorescent labelling efficacy of 1@γ-CD was still comparable to MitoTracker Deep Red FM (Fig. S7†). These results further confirm that the [2]pseudorotaxane 1@γ-CD can act as an effective mitochondrial probe, mainly due to its enhanced emission.

Fig. 5 CLSM images of live HeLa cells incubated with (a) 1@γ-CD and (b) 1 for 20 min at different concentrations (5.0 μM, 0.5 μM, and 0.05 μM). Scale bar: 20 μm. (c) Quantitative analysis of the mitochondria green fluorescent signal in cells was determined by Image J software.

The cytotoxicity of 1@γ-CD was evaluated against HeLa cells (Fig. S8†). Cells were respectively treated with 1@γ-CD, 1, and MitoTracker Deep Red FM for 12 h, and the percentage of viable cells was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide) assay.⁵⁰ The viability of untreated HeLa cells is assumed to be 100%. The results reveal that 1@γ-CD is not cytotoxic to the cells up to 5.0 μM. However, only less than 40% of the cells were viable after 12 h of incubation with 5.0 μM of MitoTracker Deep Red FM. Compared to MitoTracker Deep Red FM, significantly lower cytotoxicity was observed for both 1@γ-CD and 1. Also, it was found that the [2]pseudorotaxane 1@γ-CD exhibited a slightly lower cytotoxicity in comparison to 1, confirming the good biocompatibility of inclusion complex. All these results indicate that 1@γ-CD is a highly suitable fluorescent probe for cellular mitochondria imaging.

Conclusions

In summary, we reported that a cationic bola-amphiphilic salicylaldehyde azine derivative with ESIPT and AIE characteristics exhibited a weak emission in diluted water solution, while a significant emission enhancement was observed upon the formation of [2]pseudorotaxane inclusion complex with γ-CD. Such enhancement is attributed to the inclusion of salicylaldehyde azine part into the hydrophobic cavity of γ-CD, restoring the ESIPT process and shielding the quenching ability of pyridinium at the ends. The simpler host–guest inclusion complex enables us to employ a series of techniques to investigate the supramolecular interactions between them. Besides, the [2]pseudorotaxane 1@γ-CD exhibited low toxicity, good cell permeability, and high specificity for mitochondrial imaging, which is even comparable to the commercial mitochondrial dye. Also, it should be helpful in designing more complicated molecular machine for advanced applications and CDs-based controlled release systems.

Acknowledgements

We are grateful for financial support from National Natural Science Foundation of China (21302072 and 51403081), Natural Science Foundation of Jiangsu Province (BK20130226 and BK20140137), Natural Science Foundation of Jiangsu Higher Education Committee (13KJB150014), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of China, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra17346a

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