Cell membrane permeable fluorescent perylene bisimide derivatives for cell lysosome imaging

Abstract

We have developed acid activated fluorescent probes based on amphiphilic perylene bisimide with morpholine groups on the bay (Lyso-APBI). Incorporating one morpholine group, Lyso-APBI-1 showed an acid activated fluorescence increase of 70-fold upon pH values lowering from 8.0 to 4.0. Lyso-APBI-2, with two morpholine groups on the bay of PBI, had a red-shifted emission and a 190-fold fluorescence enhancement within same pH variation range. These Lyso-APBI probes have excellent membrane permeability, low cytotoxicity, and can rapidly accumulate and specifically activate in cell lysosomes. The double morpholine moieties make Lyso-APBI probes have higher acid activation ratio and better cell lysosome specificity. Moreover, long-time cell imaging proved the relatively high photostability of Lyso-APBI probes in a harsh physiological environment.

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Introduction

Fluorescence molecular imaging plays a major role in living tissues and cells research since it is noninvasive, rapid, highly sensitive and inexpensive.^1–3 With appropriate fluorescent probes, many biological species in living cells can be targeted and visualized.^4,5 Several characteristics of a successful fluorescent probe in live-cell imaging include water solubility, brightness, photostability, low cytotoxicity and live-cell permeability.^6,7 Furthermore, the long-wavelength excitation/emission fluorophores are favorable for in vivo bioimaging because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems.^8,9 However, the long-wavelength excitation/emission fluorophores tend to be larger molecules with extended π-conjugation, and they often have poor water solubility and are impermeable to cells.^10,11 Therefore, a pressing need exists to develop fluorescent dyes satisfying the multiple criteria of long-wavelength excitation/emission, photo-chemistry stability, high selectivity, and live-cell permeability.

Perylene bisimide derivatives (PBI) are chromophores that display long-wavelength excitation/emission, high fluorescence quantum yields and photostability.^12,13 The high stability is a crucial advantage for long-term sensitive fluorescence applications.¹⁴ PBI probes for fluorescence lifetime imaging of intracellular pH have been designed by introducing amino groups. Our group is interested in the development of water soluble and biocompatible perylene dyes for live-cell imaging.¹⁵ Here, we report the synthesis of amphiphilic perylene bisimide probes with morpholine moieties and polyethylene glycol (PEG) groups (Lyso-APBI), and investigate the live cell lysosome imaging (Chart 1). The Lyso-APBI probes in this study are specifically designed to contain two pairs of three-unit PEG groups on the imides of perylene bisimides, making the probe water solubility and live-cell permeability.¹⁵ The designed probe also contain a typical lysosome-targeting moiety of morpholine on the bay.¹⁶ We anticipated that the photoinduced electron transfer (PET) from the morpholine units to perylene bisimide dyes would quench the fluorescence in neutral conditions. After the nitrogen is protonated in acidic solution, the probe would show enhanced fluorescence emission due to the blocked PET.¹⁷ The synthetic approach for Lyso-APBI is outlined in Scheme 1.

Chart 1 Chemical structures of Lyso-APBI probes.

Scheme 1 (i) MeCN, K₂CO₃, NaI, N₂, overnight; (ii) concentrated sulfuric acid, r.t., 12 h; I₂, 85 °C, Br₂, 85 °C, 10 h; (iii) compound 1, HAc, N-Methyl pyrrolidone, 85 °C, N₂, 1 h; (iv) compound 2, 2 M K₂CO₃, Pd(PPh₃)₄, toluene, ethanol, N₂, 80 °C, 15 h.

Results and discussion

Lyso-APBI probes were synthesized according to Scheme 1. PEG 1,¹⁸ bromoperylene tetracarboxylic acid bisanhydride 2, 3 (ref. 19) were prepared according to published procedures. The morpholine reacted with 4-(bromomethyl)benzeneboronic acid pinacol ester to afford the morpholine benzeneboronic acid ester 4. The amphiphilic perylene bisimide compounds 5 and 6 was obtained via a coupling reaction between PEG 1 and bromoperylene tetracarboxylic acid bisanhydride 2 or 3. Lyso-APBI probes were synthesized by Suzuki coupling between compounds 5 (or 6) and morpholine compound 4. The Lyso-APBI probes were fully characterized by H-NMR spectroscopy, C-NMR spectroscopy and mass spectrometry. The details of the synthetic compounds can be found in the ESI.†

These Lyso-APBI probes show amphiphilic properties with high solubility in common organic solvents such as toluene, dichloromethane, tetrahydrofuran, and good water solubility. The solubility of Lyso-APBI-1 in water is 164 g L⁻¹. With two morpholine moieties on the bay of PBI, the water solubility of Lyso-APBI-2 was improved to 235 g L⁻¹. The optical properties of Lyso-APBI probes in aqueous solution were investigated ultraviolet-visible (UV-vis) absorption spectroscopy (Fig. 1) and fluorescence spectroscopy. In amphiphilic tetrahydrofuran solvent, the maximum absorption of Lyso-APBI-1 appeared at 532 nm along with higher vibronic transition at 488 nm. Lyso-APBI-2, two benzene rings on the bay, had a red-shifted maximum absorption to 551 nm, suggesting the Lyso-APBI with the introduction of benzene units have increased π-conjugation.

Fig. 1 Concentration-dependent UV-Vis absorption spectroscopy of Lyso-APBI in tetrahydrofuran and aqueous solution.

In aqueous solution, Lyso-APBI probes showed aggregation behaviour, which was investigated via concentration-dependent UV-Vis absorption spectroscopy. In a low-concentration aqueous solution (1.0 × 10⁻⁶ mol L⁻¹), the absorption bands of Lyso-APBI probes showed maximum absorption at 538 nm (for Lyso-APBI-1), and 555 nm (for Lyso-APBI-2). When the aqueous solution concentration increased from 1.0 × 10⁻⁶ mol L⁻¹ to 1.0 × 10⁻⁴ mol L⁻¹, a blue shift of the absorption maximum was observed (Lyso-APBI-1 from 538 nm to 515 nm; Lyso-APBI-2 from 555 nm to 536 nm), implying the formation of H-type cofacial π–π stacking in a high-concentration aqueous solution.²⁰

The pH-dependent fluorescence properties of Lyso-APBI probes in aqueous solution were shown in Fig. 2. The fluorescence intensity of Lyso-APBI probes was too small to be distinguished from the baseline. In contrast, these Lyso-APBI probes showed relatively high fluorescence increase as the pH range is lowered (Fig. 2A and B). Lyso-APBI-1, the maximum emission wavelength at 607 nm, showed a fluorescence increase of about 70-fold upon pH values lowering from 8.0 to 4.0. Lyso-APBI-2, two morpholine moieties on the bay of PBI, had a red-shifted emission maximum at 624 nm and a 190-fold fluorescence enhancement from pH 8.0 to 4.0. Compared with other acid activated probes, a noteworthy aspect of Lyso-APBI probes is that these probes have large Stokes shifts in aqueous solution (Lyso-APBI-1 for 97 nm, Lyso-APBI-2 for 73 nm. Table S1†). Large Stokes shifts can reduce self-absorption of the higher energy part of their emission spectra and eliminate measurement interference by excitation light and scattered light.²¹

Fig. 2 The optical features of Lyso-APBI (10 μM) at different pH values. Fluorescence spectra of Lyso-APBI-1 (A) and Lyso-APBI-2 (B) recorded at different pH values; plot of fluorescence intensity of Lyso-APBI-1 (C) and Lyso-APBI-2 (D)vs. pH; the fluorescence intensity of Lyso-APBI-1 (E) and Lyso-APBI-2 (F) between pH 4.0 and pH 8.0.

In pH 8.0 condition, the fluorescence quantum yield of Lyso-APBI-2 is 0.0015, which lower than that of Lyso-APBI-1 (0.0025). It is attributed to the double quench from two morpholine moieties of Lyso-APBI-2 in basic condition. However, the double protonation of nitrogen of Lyso-APBI-2 in acid condition not only blocked PET, but the dication was also able to suppress the aggregation of the PBI more effectively.²² Accordingly, Lyso-APBI-2 had higher fluorescence quantum yield (0.28) than Lyso-APBI-1 (0.18) in acid condition. On the basis of the above, the acid activation ratio of Lyso-APBI-2 is 2.7-fold larger than that of Lyso-APBI-1. Furthermore, the Lyso-APBI probes showed also good reversibility between pH 4.0 and pH 8.0 (Fig. 2E and F), which is attributed to the protonation/deprotonation of the morpholine group. The pK_a value of Lyso-APBI-1 and Lyso-APBI-2 was calculated to be 5.7 ± 0.2 and 5.4 ± 0.2, respectively. This indicates that the fluorescence response of Lyso-APBI matches well with the physiological pH range (pH 3.8–5.0) of lysosomes,²³ making it promise as red fluorescent probe for lysosome imaging.

To demonstrate the potential utility of Lyso-APBI probes for cellular imaging, their cytotoxicity was assessed using MTT cell-viability assay. We have reported that the perylene bisimide probes with PEG groups show very low cytotoxicity.¹⁵ Incorporating morpholine moieties on the bay of PBI, Lyso-APBI probes have slight increase in cytotoxicity but still remain low cytotoxicity (Fig. S2†). The cell viability was over 85% even though 20 μM Lyso-APBI was added for 48 h (Fig. 3), indicating that Lyso-APBI probes have a low cytotoxicity. We ascribed the exceptionally low cytotoxicity to PEG chains which protect the dyes from interacting nonspecifically with the extracellular proteins and triggering immunogenicity and antigenicity inside the cells.²⁴

Fig. 3 In vitro viability of HeLa cells treated with Lyso-APBI-1 (A) and Lyso-APBI-2 (B) for 24 h and 48 h.

To evaluate the feasibility of imaging in living cells using Lyso-APBI probes, they were employed to stain HeLa cells for the CLSM images (Fig. 4). A few fluorescent dots with low brightness are observed in the cells stained by Lyso-APBI-1(Fig. 4B), while, there were highly fluorescent dots in cells stained by Lyso-APBI-2 (Fig. 4E). These results indicate that Lyso-APBI probes have good cell-membrane permeability. The quantified fluorescence intensities of multi-points in cells stained with Lyso-APBI-1 and Lyso-APBI-2 were shown in Fig. 4C and F. The Lyso-APBI-2 showed two-times higher mean fluorescence intensity than Lyso-APBI-1 in cells.

Fig. 4 Confocal microscopy images of HeLa cells treated with Lyso-APBI: the bright-field (A) and confocal fluorescence (B) images of HeLa cells co-incubated with Lyso-APBI-1 and fluorescence intensities of multi-points in cells (C) the bright-field (D) and confocal fluorescence (E) images of HeLa cells co-incubated with Lyso-APBI-2 and fluorescence intensities of multi-points in cells (F).

In order to examine the lysosomes imaging ability of Lyso-APBI probes, HeLa cells were co-stained with commercially available lysosome-special LysoTracker green probe and Lyso-APBI probes. As shown in Fig. 5, both Lyso-APBI probes and LysoTracker green display strong localized fluorescence within lysosomes. The fluorescence images of Lyso-APBI (red) and LysoTracker green (green) can be merged rather well (yellow), and the intensity profiles of the linear regions of interest across HeLa cells stained with Lyso-APBI and LysoTracker display good overlap. These results indicate that Lyso-APBI probes can specifically target the lysosomes of living cells. The co-localization of Lyso-APBI with the LysoTracker green was quantified by correlation analysis. The Pearson coefficient of co-localization for Lyso-APBI-1 with LysoTracker green is 0.76, while that for Lyso-APBI-2 is 0.90, indicating that the lysosome specificity of Lyso-APBI-2 is better than that of Lyso-APBI-1. We attribute the high lysosome specificity to the two acid activated morpholine moieties in Lyso-APBI-2. Furthermore, the accumulation rate of Lyso-APBI-2 in cell lysosome was investigated by in situ real-time CLSM imaging and compared with commercially available lysosome probes such as LysoTracker green and LysoTracker red. After 10 min of cells incubation with 1.0 μM of Lyso-APBI-2, the fluorescence signal from cells clearly appeared (Fig. 6). In contrast, cell incubation with 1.0 μM of LysoTracker green or LysoTracker red, fluorescent dots in cells gradually formed with a lag time of ∼10 min. It clearly indicates that the accumulation rate of Lyso-APBI-2 in cell lysosome is faster than that of LysoTracker green and LysoTracker red.

Fig. 5 Lysosome-targeting properties of Lyso-APBI in HeLa cells. (A) Colocalization images of HeLa cells stained with Hoechst 33342 (blue channel), LysoTracker green (green channel), Lyso-APBI-1 (red channel), overlay of three channels and the correlation of intensities. (B) Colocalization images of HeLa cells stained with Hoechst 33342 (blue channel), LysoTracker green (green channel), Lyso-APBI-2 (red channel), overlay of three channels and the intensity profile of region of interest (ROI) cross cell.

Fig. 6 Real-time confocal microscopy images of HeLa cells incubated with Lyso-APBI-2, LysoTracker green, and LysoTracker red.

Furthermore, we examined the feasibility of applying Lyso-APBI for tracking the pH changes in lysosomes. Treating HeLa cells with chloroquine to induce an increase in the lysosomal pH,²⁵ the fluorescence signal in the area of the lysosomes became weak (Fig. S3†). The results suggest that probe Lyso-APBI could be used for tracking pH changes in lysosomes. Furthermore, changes in the fluorescence images of HeLa cells stained by Lyso-APBI under continuous 559 nm laser scanning were monitored to evaluate the photostability of Lyso-APBI in cells. After scanning for 3 min, the fluorescence signal from cells remained almost unchanged. In contrast, the fluorescence signal from cells stained with the commercially available LysoTracker probes decreased significantly within same scanning time (Fig. S4†). These results prove the relatively high photostability of Lyso-APBI in harsh physiological environment.

Conclusions

In summary, we incorporated the morpholine and PEG moieties into perylene bisimide dyes and successfully synthesized Lyso-APBI probes. PEG chains imparted these probes amphiphilicity, excellent membrane permeability, low cytotoxicity. With morpholine moieties on the bay of perylene bisimide, Lyso-APBI probes can specifically activate in cell lysosomes, and track the pH changes. It is worthy of note that the double morpholine moieties make Lyso-APBI probes have faster accumulation rate, higher acid activation ratio and better lysosome specificity.

Experimental

Materials and instrumentation

All the chemicals used in synthesis are analytical pure and were used as received. UV/Vis spectra were recorded on a Shimadzu WV-2550 spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-5301 fluorescence spectrophotometer. The H-NMR spectra and C-NMR spectra were recorded at 20 °C on 600 MHz or 150 MHz NMR spectrometer (Bruker). Mass spectra were carried out with Agilent LC/MSD XCT Trap. CLSM images were obtained using Olympus confocal laser scanning microscopy (Olympus Fluoview FV1000).

Cell culture and imaging

HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (high glucose), supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units per mL) and streptomycin (100 μg mL⁻¹) at 37 °C in a 5% CO₂/95% air incubator in a humidified atmosphere, and culture media were replaced with fresh media two to three days. For fluorescence imaging, HeLa cells were grown in DMEM on a 35 mm glass bottom culture dishes for at least 24 h to enable adherence to the bottom.

The cells were loaded with Lyso-APBI according to a following procedure. Briefly, the 50 μL of 100 μM PBS solution of Lyso-APBI was added to the dish (final concentration of Lyso-APBI is 5 μM), and then the cells were incubated for 30 min at 37 °C. Afterward, the cells were washed several times with PBS for removing Lyso-APBI.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21274036), the Program for New Century Excellent Talents in University (NCET-12-0684), Training Program for Innovative Research Team and Leading talent in Hebei Province University (LJRC024). The Program for Innovative talent in Hebei Province University China (GCC2014054).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra20444a

‡ These authors contributed equally to this work and should be considered co-first authors.

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