Simultaneous two-color visualization of lipid droplets and lysosomes for cell homeostasis monitoring using a single fluorescent probe†

Mengxiao Liu‡ ^a, Yingcui Bu‡*^a, Dongxiao Wang^a, Lihua Tang^a, Didi Hu^a, Longchun Li^a and Xiaoping Gan*^ab
aSchool of Materials and Chemistry, Anhui Agricultural University, 230036 Hefei, P. R. China. E-mail: gan-xp@ahau.edu.cn; byc@ahau.edu.cn
^bKey Laboratory of Agricultural Sensors, Ministry of Agriculture Rural Affairs, 230036 Hefei, P. R. China

First published on 22nd August 2024

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Introduction

Organelles are relatively independent functional units separated by membrane systems in eukaryotic cells, which is also the key difference between eukaryotic and prokaryotic cells.^1,2 Different organelles possess diverse compositions and structures and play critical but distinct roles during various biological procedures. Meanwhile, some important physiological and pathological processes require the cooperation of several organelles.^3–5 In particular, LDs and lysosomes establish dynamic inter-organelle membrane contact sites to facilitate the exchange of metabolites, influencing the development of diseases associated with the dysfunction of both organelles.^6–8 Therefore, the design and synthesis of specific fluorescent probes for simultaneous dual-color labeling of LDs and lysosomes in live systems are of great significance for further cell homeostasis monitoring and early disease diagnosis.

As is well known, LDs are recognized as the most hydrophobic organelles, containing a lipophilic core composed of neutral lipids coated with a phospholipid monolayer,⁹ while lysosomes are intracellular acidic vesicles containing more than 60 kinds of hydrolases.¹⁰ Consequently, there exist notable distinctions between the microenvironments of lysosomes and LDs in terms of polarity, pH, and other factors.^11,12 For instance, Yu et al. constructed a polarity-sensitive fluorescent probe, in which morpholine was selected as a lysosome targeting group and N,N-dimethyl-4-vinylaniline connected naphthalimide acted as a LD targeting group, capable of simultaneously imaging LDs and lysosomes in cells and tissues.¹³ Zhao et al. developed a pH-sensitive ratiometric fluorescent probe by introducing a proton acceptor of the N,N′-diethylamino unit into an AIE-active pyrazoline-based skeleton, which can simultaneously dual-color stain LDs and lysosomes for quantitatively monitoring the lipophagy process.¹⁴ In light of the fact that the acidic lysosome internal environment was usually prone to quenching the fluorescence of probes that were equipped with a basic group (lysosome targeting group),^15,16 an ideal two-color probe for LDs and lysosomes should possess high stability, especially for pH, high selectivity and distinguishable optical properties.^17,18 To date, despite various efforts that have been devoted, the probes that simultaneously meet the above requirements have rarely been reported.^19,20

In this study, we selected TPE, a classical AIE rotor, electron-donating group (D), and lipid droplet-targeting group to link with a naphthalimide, NDI, a classic fluorophore and electron-accepting group (A) through a single bond, where the morpholine (Mor) moiety is selected as the lysosomal targeting moiety²¹ to construct the target molecule TPE-NDI-Mor (Scheme 1). Cell experiments demonstrated that TPE-NDI-Mor could label both LDs and lysosomes based on the specific response to polarity through dual-color fluorescence imaging, excited by the same excitation wavelength (λ_ex = 405 nm).²² Also, the as-prepared compound exhibited excellent pH stability, immunity to interference, and a large Stokes shift, exhibiting significant potential for further cellular tracking.^23,24 Moreover, TPE-NDI-Mor can efficiently monitor the physiological and pathological processes of cells by monitoring the dynamic changes of LDs and lysosomes.²⁵

Experimental section

General procedures

The chemicals and solvents were purchased from commercial companies and used directly without further purification. Human hepatocellular carcinoma cells (HepG2) were purchased from BeNa Culture Collection. ¹H NMR and ¹³C NMR spectra were recorded on an Agilent DD2 600 Ultrashield spectrometer using dimethylsulfoxide-d6 (DMSO-d6) or CDCl₃ as a solvent. Chemical shifts were reported in parts per million (ppm) relative to the internal standard TMS (0 ppm) and coupling constants were reported in Hz. Splitting patterns were described as singlet (s), doublet (d), triplet (t) or multiplet (m). The mass spectra were obtained on an LTQ-Orbitrap XL mass spectrometer. The raw materials used for the synthesis of M1 and M2 in this paper were all from Adamas.

Optical measurements

The UV-visible spectra were recorded on a TU-1901 Double Beam UV-visible spectrophotometer and the fluorescence emission spectra were recorded using a G9800A fluorescence spectrophotometer. In the measurements of absorption and emission spectra, the pass width was 5 nm for the compound. The spectra of the compounds were recorded in ten solvents with different polarities at a concentration of 1 × 10⁻⁵ mol L⁻¹. The quartz cuvettes used were of 1 cm path length. In addition, the fluorescence quantum yields of the two compounds were calculated using the formula for fluorescence quantum yields, which is given below:

	(1)

Cytotoxicity assays in cells

To ascertain the cytotoxic effect of the compound, the 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay was performed. HepG2 cells were trypsinized and plated to reach ∼70% confluence in 96-well plates for 24 h before treatment. Prior to the treatment of the compounds, Dulbecco's modified Eagle's medium (DMEM) was removed and replaced with fresh DMEM, and aliquots of the compound stock solutions (1 × 10⁻³ mol L⁻¹ DMSO) were added to obtain final concentrations of 5, 10, 20, 40 and 60 × 10⁻⁶ mol L⁻¹. The treated cells were incubated for 24 h at 37 °C and under 5% CO₂. Subsequently, the cells were treated with 5 mg mL⁻¹ MTT (40 μL per well) and incubated for an additional 4 h (37 °C, 5% CO₂). Then, DMEM was removed, the formazan crystals were dissolved in DMSO (150 μL per well), and the absorbance at 570 nm was recorded. The cell viability (%) was calculated according to the following equation:

Cell viability % = OD570 (sample)/OD570 (control) × 100

where OD₅₇₀ (sample) represents the optical density of the wells treated with various concentrations of the compounds and OD₅₇₀ (control) represents that of the wells treated with DMEM supplemented with 10% fetal calf serum (FCS, Gibco). Three independent trials were conducted, and the averages and standard deviations were reported. The reported percent cell survival values were relative to untreated control cells.

Confocal microscopy imaging of cells

For HepG2 cells, DMEM supplemented with 10% fetal calf serum, penicillin and streptomycin, L-glutamine, and fungizone was used. For the living cell confocal laser scanning microscopy experiment, HepG2 cells were seeded in 24-well glass plates at a density of 2 × 10⁵ cells per well, incubated for 96 h at 37 °C under a 95% air and 5% CO₂ atmosphere to allow the cells to reach ∼90% confluence, and the medium was changed every two days. Then DMEM was removed and replaced with target compounds in DMSO (5 × 10⁻⁶ mol L⁻¹) for 15 min at 37 °C under a 95% air and 5% CO₂ atmosphere. Then the cells were washed with phosphate buffered saline (PBS) (3 × 1 mL per well) and 1 mL of PBS was added to each cell. The cells were then imaged on a TCS SP8 DIVE/SP8 DIVE upright confocal laser scanning microscope using 60 × magnification oil-dipping lenses for monolayer cultures. Image data acquisition and processing were performed using Zeiss LSM Image Browser, Zeiss LSM Image Expert, and Image J.

Oleic acid treatment

Living HepG2 cells were initially cultured in a glass-bottom culture dish for 24 h to adhere. The cells were incubated with 1 mL of 5 × 10⁻⁴ mol L⁻¹ of OA for 2 h and then washed with PBS buffer twice. Subsequently, the cells were stained with 5 × 10⁻⁶ mol L⁻¹ of TPE-NDI-Mor for 15 min and then washed twice with PBS to wash away the excess probe. The cells were observed under a confocal microscope.

Chloroquine treatment

Living HepG2 cells were initially cultured in a glass-bottom culture dish for 24 h to adhere. Then 5 μL of 0.01 mol L⁻¹ chloroquine (CQ) was suspended in PBS buffer, which was used to incubate the cells for 1 h. The cells were then washed with fresh culture medium twice to remove the excess chloroquine, which was afterward stained with 5 × 10⁻⁶ mol L⁻¹ TPE-NDI-Mor for 15 min. The cells were then washed twice with PBS to wash away the excess probe and then observed under a confocal microscope.

Synthesis and characterization

All compounds were prepared according to the synthetic routes shown in Scheme S1.† Experimental details are described in the Experimental section and the structural characterization data are shown in the ESI.†

Results and discussion

Design strategy and synthesis

TPE-NDI-Mor was obtained by the Suzuki reaction with a highly active borate ester to improve the reaction yield. The specific synthetic route is shown in Scheme S1,† which was further confirmed by ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (Fig. S1–S7†). Structurally, the introduction of a TPE scaffold enables the probe to possess an AIE effect that can be luminescent in aqueous environments, making the probe more suitable for application in the imaging of biological systems. Besides, the multi-aromatic ring possessed by TPE could enhance the lipid solubility of the probe and also endow the probe with the ability to target LDs. Meanwhile, the TPE and NDI groups are connected by a single bond to form a D–A molecular structure, which gives the probe a polarity response due to its TICT effect; then a morpholine group is introduced at the other end of the TPE group, which gives the probe the ability to target lysosomes at the same time.

Specific response to solvent polarity

At first, we tested the UV absorption and fluorescence emission spectra of TPE-NDI-Mor in six different polar solvents, including toluene, 1,4-dioxane (DIO), ethyl acetate, dichloromethane (DCM), dimethyl sulfoxide (DMSO), and acetonitrile (ACN). The results shown in Fig. 1a illustrate the fluorescence spectrum of TPE-NDI-Mor in different solvents, with an emission peak distributed between 500 nm and 620 nm. For instance, the maximum emission wavelength of TPE-NDI-Mor in nonpolar solvents, such as toluene, was 498 nm, while it redshifted gradually to 614 nm in DMSO as solvent polarity increased. To examine the regularity of TPE-NDI-Mor's response to solvent polarity, we normalized its fluorescence emission spectra in six different solvents. As depicted in Fig. 1b, the maximum fluorescence emission wavelength of TPE-NDI-Mor redshifted notably with increasing solvent polarity. To further explore the relationship between the probe and solvent polarity, we conducted a linear fit between the empirical polarity parameter E_T (30) of various solvents and the maximum fluorescence emission wavelength of TPE-NDI-Mor (Fig. 1c). The results demonstrated a strong linear relationship (linearity coefficient of 0.98) between the maximum fluorescence emission wavelength of TPE-NDI-Mor and the E_T (30) of the solvents. Additionally, we measured the fluorescence quantum yield of TPE-NDI-Mor in each solvent, with results presented in Table S1.† The fluorescence quantum yield of TPE-NDI-Mor in various solvents remained relatively stable, hovering around 4%. However, it is worth noting that the fluorescence quantum yield of TPE-NDI-Mor in dichloromethane and chloroform was notably high, above 20%, possibly due to the probe's better solubility in halogenated hydrocarbon solvents.

Many substances in organisms, including reactive small molecules and biomolecules, may interfere with fluorescent probes, so it is necessary to further explore the immunity of probes to these interferences. We selected some common biomolecules (2-GSH, 3-Cys, 4-Arg, 5-Glu), cations (6-K⁺, 7-Na⁺, 8-Cu²⁺, 9-Mg²⁺, 10-Fe³⁺, 11-Co²⁺, 12-Ag⁺), and anions (13-CO₃²⁻, 14-SO₄²⁻, 15-SO₃²⁻, 16-Cl⁻) to conduct anti-interference experiments. The results are illustrated in Fig. 1d, showing minimal fluctuation in fluorescence intensity compared to the control group, indicating that these interfering substances had no significant effect on the probe. Additionally, in Fig. 1e, the fluorescence emission intensity of TPE-NDI-Mor presented negligible changes compared to the polar response, indicating that the probe is not affected by viscosity changes. Also, complex microenvironments such as pH in organisms may affect the luminescence ability of fluorescent probes, thus highlighting the importance of quality fluorescent probes for bioimaging with good stability. As expected, the changes in the fluorescence intensity of TPE-NDI-Mor were minimal at pH values ranging from 4 to 12, as shown in Fig. 1f. Taken together, TPE-NDI-Mor possessed good anti-interference ability, suitable for subsequent biological application.

AIE properties of TPE-NDI-Mor

Then, to investigate the AIE effect of TPE-NDI-Mor, we tested the fluorescence emission spectra of TPE-NDI-Mor in different mixed solutions. Taking the THF/H₂O mixed solution as an example (Fig. 2a and b), there was no significant change in the fluorescence emission intensity when the water fraction (f_w) was less than 80%, while the fluorescence emission intensity quickly increased when f_w increased from 80% to 90% and reached the maximum value when f_w was 90%, finally decreasing with the increase of f_w. All the results showed that TPE-NDI-Mor possessed the AIE effect. In addition, similar experimental phenomena were observed in ACN/H₂O and DMSO/H₂O mixed solutions (Fig. S8 and Fig. S9†). In addition, a scanning electron microscopy (SEM) image of TPE-NDI-Mor in the THF/H₂O (f_w = 90%) mixed solvent was obtained, as shown in Fig. 2c. The SEM images showed that its particle size was about 100 nm, and this particle size could be suitable for entering the cells. To visually demonstrate the AIE performance of the probe, we wrote “AIE” using the THF solution of TPE-NDI-Mor.

Colocalization imaging of LDs and lysosomes

To further demonstrate the potential application of TPE-NDI-Mor, fluorescence imaging experiments were performed in living HepG2 cells. Before the bioimaging experiments, we evaluated the cytotoxicity of TPE-NDI-Mor using the MTT assay. The results, as quantified in Fig. S10,† showed that the cell viability was more than 80% at all concentrations, while the cell viability was up to more than 95% at a concentration of 5 × 10⁻⁶ mol L⁻¹. The above results indicated that TPE-NDI-Mor possessed much lower cell cytotoxicity, which allowed for next-cell staining. Later, we carried out the anti-photobleaching ability tests of TPE-NDI-Mor by continuous laser irradiation of the stained cells using CLSM. It could be seen that when the cell stained with TPE-NDI-Mor was scanned every 22 s, it still emitted light stably inside the cell within 440 s, and the fluorescence emission intensity did not decrease, which indicated that TPE-NDI-Mor had good photostability and was suitable for long-time dynamic tracking at the cellular level (Fig. S11†).

Furthermore, we performed fluorescence imaging experiments using TPE-NDI-Mor in living HepG2 cells, in which HepG2 cells were first pretreated with 5 × 10⁻⁶ mol L⁻¹ TPE-NDI-Mor for 15 min, and then the culture medium was washed away and the cells were observed by CLSM in the green and red channels at 405 nm excitation wavelengths simultaneously. The results are shown in Fig. 3a. It could be seen that HepG2 cells exhibited strong fluorescence in both green and red channels. The green emission formed dotted morphologies, which showed high overlapping with the dots in DIC images, indicating that the green emission mapped the distribution of LDs. Meanwhile, the red emission also appeared as dotted shapes, which were in accordance with the lysosomes in DIC images. To confirm the organelle localization ability of TPE-NDI-Mor, we performed co-localization experiments using the probe with Nile red and Lyso-tracker green (LTG), two commercial dyes targeting LDs and lysosomes, respectively. As shown in Fig. 3b, the green emission emitted from TPE-NDI-Mor was co-localized with Nile red, which is a commercial dye used for targeting LDs, and illustrated high overlapping with it. Pearson's co-localization coefficient was about 0.95, demonstrating that the green fluorescence of TPE-NDI-Mor was distributed in LDs. Subsequently, we found that the red fluorescence of TPE-NDI-Mor was colocalized with the green emission of LTG, a green emissive commercial dye used for staining lysosomes in living cells. As shown in Fig. 3c, the red emission of TPE-NDI-Mor showed high overlapping with LTG, and Pearson's coefficient was about 0.95, which indicated that TPE-NDI-Mor was also distributed in lysosomes. The co-localization results confirmed that the green and red emissions of TPE-NDI-Mor were localized in LDs and lysosomes, respectively. All the results indicated that TPE-NDI-Mor could be used as a dual-targeted multifunctional fluorescent probe with the potential to study the dynamics of LDs and lysosomes.

Self-reporting fusion and separation processes of LDs

LDs are dynamic organelles closely intertwined with cell metabolism and homeostasis. Herein, TPE-NDI-Mor was co-incubated with HepG2 cells to monitor the dynamic changes of LDs over different time intervals. Fig. 4 illustrates that four pseudocolored images were obtained using Image J software (0 min, 1 min, 2 min, and 3 min). Imaging analysis revealed significant movement of lipid droplets within the selected region. For example, time-lapse imaging from 0 to 3 minutes displayed the dynamic “run-kiss-run” events in each enlarged area. Details revealed that from 1 to 2 minutes, an obvious fusion process, viz the “kiss” event was clearly observed, while an obvious migration process (“run” event) occurred upon extending the observation time from 2 to 3 minutes. The above observation indicated that lipid droplet remodeling in the cells is primarily driven by lipid droplet movement rather than the probe's influence. In addition, the zoom-in of selected overlapped images (e, f, g, h) among a, b, c, and d collected by four different times also confirmed the existence of significant kinetic motion of the LDs.

Dynamic changes of LDs and lysosomes during cell homeostasis imbalance

Based on the above subcellular localization experiments, it can be seen that TPE-NDI-Mor can accurately localize both LDs and lysosomes through different emission wavelengths. Thus, we investigated the dynamic behaviors of LDs and lysosomes separately using TPE-NDI-Mor under OA and CQ conditions. Since OA could induce the formation of intracellular LDs, we incubated HepG2 cells with 0 and 5 × 10⁻⁴ mol L⁻¹ OA for 30 min, respectively, and then stained them with 5 × 10⁻⁶ mol L⁻¹ TPE-NDI-Mor for another 15 minutes. After that, the cell imaging was recorded by CLSM. As presented in Fig. 5a and b, in comparison with the control group (only dealt with TPE-NDI-Mor), a large amount of green fluorescence appeared in the cells treated with the OA group, which indicates that the number of LDs increased in the cells after incubation with oleic acid, but the fluorescence of the red channel was not significantly enhanced or weakened because OA did not have a direct effect on lysosomes in a short period. The dynamic changes in lysosomes were monitored by treating the cells with CQ, a lysosomal inhibitor, for half an hour and then staining them with 5 × 10⁻⁶ mol L⁻¹ TPE-NDI-Mor for 15 minutes to obtain the CLSM image shown in Fig. 5c. It could be seen that the green fluorescence localized in the LDs did not change significantly after CQ treatment, but the fluorescence of the lysosomal red channel disappeared compared with Fig. 5a, which was attributed to the inhibited binding of autophagosomes and lysosomes by CQ, resulting in a dramatic decrease in the number of lysosomes in the CQ-treated cells. In summary, TPE-NDI-Mor could not only monitor the increase in the number of LDs induced by oleic acid, but also record the changes in the number of lysosomes under the influence of CQ, which is a multifunctional fluorescent probe for monitoring the dynamic changes of cell homeostasis.

Conclusions

In summary, we have developed a superior fluorescent probe named TPE-NDI-Mor that exhibited a specific fluorescence response to polarity, which was the key difference between the microenvironments of LDs and lysosomes. Interestingly, cell imaging experiments indicated that TPE-NDI-Mor can effectively stain the dual organelles LDs and lysosomes with high specificity and monitor their dynamic changes for further cell metabolism and homeostasis imbalance. Furthermore, TPE-NDI-Mor exhibited excellent pH stability, low cytotoxicity, and good biocompatibility, making it suitable for further disease diagnosis.

Author contributions

Mengxiao Liu: data curation and writing – original draft. Dongxiao Wang: methodology and investigation. Lihua Tang: formal analysis and characterization analysis. Didi Hu: validation. Yingcui Bu: writing – review & editing and supervision. Longchun Li: software. Xiaoping Gan: conceptualization, writing – review & editing, funding acquisition, and supervision. All authors have read and agreed to the published version of the manuscript.

Data availability

The data that support the findings of this study are available in the ESI† of this article.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Natural Science Foundation of Education Committee of Anhui Province (2023AH050980), the Anhui Agricultural University Scientific Research Start-up Fund (rc382306), the Anhui Provincial Natural Science Foundation (2008085MB41) and the Young Backbone Teacher Project of CSC (202308770005).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4an00912f

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

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