Mitochondria-targeting fluorescent probe with a pH/viscosity response for assisted detection of non-alcoholic fatty liver in mice

Abstract

Cancer cells are characterized by high viscosity, low pH and high levels of reactive oxygen species. Both pH and viscosity are key indicators which can respond to the microenvironment of cancer cells and are also important indicators for monitoring fatty liver. Based on this, we have constructed a hemicarbocyanine fluorescent probe (HTC) possessing a D–D′(π)–π–A structure using phenol as an electron donor (D), a thiophene moiety as an electron donor and a partial π bridge. The fluorescent probe HTC exhibits pH-responsive properties, and the fluorescence intensity of the probe at 627 nm gradually decreases and the fluorescence at 720 nm gradually increases with the increase of pH for pH = 7 to 10.5. Meanwhile, with an increase of viscosity the fluorescent probe HTC exhibited gradually enhanced fluorescence at 627 nm, thus revealing a response to the change of viscosity. In addition, the fluorescent probe HTC can target mitochondria for cellular fluorescence imaging, and co-localized fluorescence imaging with the commercial mitochondrial probe Mito-Tracker Green with a Pearson's coefficient of 0.91. We have utilized the properties of the fluorescent probe HTC, which exhibits a pH and viscosity response, and have applied it to monitor the change of pH or viscosity in cells and differentiate between normal and cancerous cells, as well as for the identification of non-alcoholic fatty liver mice. In addition, the probe is easily metabolized in mice and has good biocompatibility.

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Introduction

In recent years, the incidence and mortality rates associated with cancer have been on the increase.¹ However, if it can be detected as early as possible and treated in time, then the survival rate of patients can be significantly improved.^2,3 Therefore, it is crucial for human health to identify tumor markers to enable early diagnosis and treatment of cancer. Viscosity is an important indicator of normal cell growth. It is one of the key factors for maintaining normal cell function, influences cell metabolism and differentiation⁴ and is of great importance for the physiological processes of cells. Abnormal cell viscosity is associated with a number of diseases, such as Parkinson's disease,⁵ diabetes⁶ and sickle cell disease.⁷ In addition, the normal functioning of cells is affected by their acid–base balance, and imbalances in pH homeostasis have been linked to the development of a number of diseases, including metabolic disorders,⁸ hepatic encephalopathy⁹ and hyperchloremia.¹⁰ Most importantly, cells are able to sense changes in the surrounding microenvironment, regulate cell morphology and influence physiological processes. Compared to normal cells, cancer cells have a unique microenvironment with low pH, high viscosity and high levels of reactive oxygen.¹¹ This unique microenvironment favours the survival and metastasis of cancer cells. Viscosity and pH can be used as important parameters to distinguish cancer cells from normal cells. Therefore, real-time monitoring of changes in cell viscosity and pH is important to understand the normal cellular mechanisms and the principles underpinning the development of associated diseases.

Mitochondria are the main sites of cellular energy production, controlling signaling pathways for longevity and health and determining the fate of the cell. Therefore, a balance in the mitochondrial microenvironment is very important as it influences the physiological activities of cells.^12–14 There are many factors that affect the mitochondrial microenvironment, such as protease content, oxidative factors, pH and viscosity. Among them, pH and viscosity are particularly important because viscosity can directly affect mitochondrial signal transduction and energy production^15–17 and pH affects the mitochondrial metabolism.

Non-alcoholic fatty liver disease (NAFLD) includes a range of liver lesions, usually due to the metabolic accumulation of fat in the liver.¹⁸ At the same time, the accumulation of lipid droplets in hepatocytes increases the viscosity of mitochondria, resulting in abnormal mitochondrial function. Therefore, monitoring changes in mitochondrial viscosity is important for the study of NAFLD.

There are many traditional methods for measuring viscosity and pH, such as the rotational viscometer method,¹⁹ the capillary viscometer method,²⁰ the acid–base indicator method, and via a pH meter. However, these methods are only suitable for solution assays and are not suitable for detecting the viscosity and pH of the intracellular environment in vitro as well as changes in viscosity and pH in organisms. It has been shown that fluorescent probes can be utilized for biomonitoring because of their ease of handling, high sensitivity and high resolution.^21,22 In recent years, a number of fluorescent probes have been used to monitor changes in intracellular viscosity and pH.^23–26 As mitochondria are the key sites of cellular aerobic respiration, the development of fluorescent probes that can efficiently target mitochondria as well as sensitively detect changes in cellular viscosity and pH is particularly important in understanding the effects of viscosity and pH on mitochondrial diseases.^27–30 Currently, most of the fluorescent probes for monitoring changes in mitochondrial viscosity are cationic fluorescent probes based on twisted intramolecular charge transfer (TICT) designs, which have the advantages of fast response and high spatial resolution, and whose non-radiative decay in the excited state is affected by the viscosity of the surrounding environment.³¹ These fluorescent molecular rotors generally have rotatable conjugated groups, and so the molecular rotor rotates rapidly in low-viscosity media, reducing the degree of conjugation and consuming large amounts of energy, which attenuates the fluorescence. However, under high viscosity conditions, rotation is hindered, and the fluorescence is enhanced. However, such molecules still have defects, such as poor water solubility, high background interference, complex synthesis, and a low rotor efficiency.

It was found³² that fluorescent probes with an electron donor (D)–π–electron acceptor (A) backbone can improve the fluorescent molecule rotor efficiency and can enhance the fluorescence response to viscosity through TICT.³³ In addition, probes with this structure have good photostability and biocompatibility.^34,35 Based on this, we have constructed HTC, a fluorescent probe with a D–D′(π)–π–A conformation, by introducing phenol as an electron donor (D) and a thiophene moiety as an electron donor and partial π-bridge into a conventional hemicarbocyanine molecule. HTC carries a positive charge to target mitochondria, and the inclusion of phenolic hydroxyl groups increases the aqueous solubility and responsiveness to changes in pH. More importantly, HTC provides two viscosity recognition sites, a single bond between the benzene ring and the thiophene ring and a double bond between the thiophene ring and the indole ring, which can effectively improve the sensitivity. In low viscosity media, the free intramolecular rotation of HTC leads to an increase in the non-radiative attenuation rate and almost no fluorescence, whereas in high viscosity media, the intramolecular rotation of the probe is hindered and the fluorescence is enhanced. Since the probe contains the Ar–OH moiety, it is sensitive to pH changes in the environment. We have utilized the pH-responsive and viscosity-responsive properties of the fluorescent probe and applied them to cellular fluorescence imaging. It was found that the fluorescent probe could target mitochondria and differentiate between normal and cancerous cells. In addition, the fluorescent probe could be applied for the identification of non-alcoholic fatty liver mice. The probe is easily metabolized and has good biocompatibility as it is excreted through urine 8 h after entering into the mice. This work provides a visualization means for the identification of cancer cells and non-alcoholic fatty liver and provides a design strategy for the construction of fluorescent probes.

Results and discussion

Synthesis and crystal structure of HTC

Compound 1 was prepared using the literature method³⁶ using 5-bromothiophene-2-formaldehyde and p-hydroxyphenyl boronic acid via a Suzuki reaction. Compound 1 was then treated with 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium iodide in anhydrous ethanol to obtain the target compound HTC (Fig. S1, ESI†) via a Knoevenagel condensation reaction. The molecular structure of HTC was characterized using X-ray crystallography (Fig. S2–S4, ESI†). The crystal structure of HTC shows that the C=C double bond connects the indole ring and the thiophene ring, and the C=C is in an “E” configuration. The thiophene ring and the benzene ring are basically parallel, and the dihedral angle between them is 3.30°; the dihedral angle between the indole ring and the thiophene ring is 27.19°. The phenolic hydroxyl group of the probe HTC and the I⁻ ions form intermolecular O1–H1⋯I1 hydrogen bonds (see Table S2 for hydrogen bonding parameters, ESI†). Due to the presence of hydrogen bonding, it is difficult to dissociate the hydrogen from the phenolic hydroxyl group, so the pK_a of the probe is larger, and the probe HTC structure extends to form a one-dimensional helical structure through atypical intermolecular C12–H12⋯O1 and C23–H23A⋯O1 hydrogen bonding (Fig. 1). Selected crystallographic parameters are given in Table S1 (ESI†).

Fig. 1 (A) Crystal structure ellipsoid of probe HTC (ellipsoid chance is 30%); (B) HTC probe forms a one-dimensional helical structure through atypical C12–H12⋯O1 and C23–H23A⋯O1 hydrogen bonds.

Spectral response to viscosity

First, to verify the relationship between the fluorescence intensity of HTC and viscosity, we examined the absorbance of the probe in water and glycerol (Fig. 2A), using different ratios of glycerol and pure water as the viscosity detection system. As shown in Fig. 2B, by adjusting the glycerol volume ratio, the viscosity of the solution was gradually increased from 1.2 cp (pure water) to 499.5 cp (glycerol), and the fluorescence of the probe at 627 nm was enhanced with the increase of the solution viscosity, and the fluorescence intensity was increased 20-fold. In addition, as shown in Fig. 2C, there was a significant linear correlation (R² = 0.989) between log I_627nm and log(viscosity) according to the equation of Förster–Hoffmann with log I_627nm = 0.396 log η + 1.819. Meanwhile, we examined the fluorescence spectra of HTC in different polar solutions (Fig. 2D). It was evident that the positions of the maximum emission peak of HTC in different solvents was almost unaffected by the solvent, which were all around 627 nm. However, due to the restricted intramolecular rotation of the TICT process, the corresponding fluorescence spectra varied according to the change of polarity in different solvents. The probe's fluorescence was strongest in glycerol, and the fluorescence in the other solvents was weak. Subsequently, we examined the quantum yield of the probe, as shown in Fig. 2E. The quantum yield of the probe in water is only 1.5%, while it is 32.5% in glycerol, which is a 22-fold increase.

Fig. 2 (A) Absorbance of HTC (10 μM) in water and glycerol; (B) fluorescence spectra of HTC (10 μM) in different viscosities; (C) linear relationship between log I_627nm and log(viscosity); (D) fluorescence intensity of HTC in different polar solutions; (E) quantum yields of HTC in water and glycerol, respectively. λ_ex = 520 nm, λ_em = 610–630 nm; and (F) the fluorescence intensity of HTC (10 μM) in relation to various analytes (1 blank; 2 Cu²⁺; 3 Ca²⁺; 4 H₂O₂; 5 Ag⁺; 6 Na⁺; 7 SO₃²⁻; 8 Zn²⁺; 9 Br⁻; 10 Cl⁻; 11 F⁻; 12 I⁻; 13 CO₃²⁻; 14 SO₄²⁻; 15 GSH; 16 NO₃⁻; 17 ClO⁻; 18 Mg²⁺; 18 C₂O₄²⁻; 19 Cys; 20 TBHP; 21 SNP; 22 Gly); relative fluorescence intensity at 627 nm.

To investigate the specificity of the probe response to viscosity, we added analytes to the probe and detected the fluorescence intensity at 627 nm. The results are shown in Fig. 2F, where the probe still has a high selective response to viscosity. We then examined the stability of the probe in water and glycerol (Fig. S5, ESI†), and the results showed that the probe possesses good stability.

Spectral response of the probe to pH

Since the probe incorporates an Ar–OH moiety, which is sensitive to pH changes, we examined the effect of pH on the spectral properties of the probe. The UV-visible absorption and fluorescence spectra of the probe did not change much over pH = 2–7 (Fig. S6 and S7, ESI†). For pH values in the range of 7–10.5, there was a gradual decrease of the absorption peak at 520 nm and a gradual increase of the absorption peak at 600 nm with increasing pH, whilst equi-absorption points appeared at 424 nm and 535 nm (Fig. 3A). The UV-visible absorption spectra of HTC corresponded to the colour change of the solution we observed (Fig. S9, ESI†); the colour of the solution gradually changed from red to purple and then to yellow.

Fig. 3 (A) UV spectra of HTC at different pH values; (B) fluorescence spectra of HTC (10 μM) under λ_ex = 500 nm excitation at different pH values; (C) fluorescence spectra of HTC (10 μM) under λ_ex = 600 nm excitation at different pH values; (D) the emission intensity normalized ratio (I₆₂₇/I₇₂₀) of HTC and the Henderson–Hasselbalch equation fitting curves.

Similarly, the fluorescence spectra showed that under 520 nm excitation, the fluorescence intensity of the probe at 627 nm gradually decreased with increasing pH (Fig. 3B). Under 600 nm excitation, the fluorescence intensity of the probe at 720 nm gradually increased (Fig. 3C). The coincidence with the change of the UV-vis absorption spectrum of HTC further proved our speculation. Finally, we plotted the fluorescence intensity ratio of the probe at 621 nm and 720 nm (I_627nm/I_720nm) against pH, and obtained a good linear relationship as shown in Fig. 3D, with R² = 0.99. By means of the Henderson–Hasselbalch equation (pH = pK_a + lg[(I_max − I)/(I − I_min)]), the pK_a of the probe was obtained as 8.6. This is in agreement with the crystal structure we obtained, which shows that the phenolic hydroxyl group and the I⁻ ion form an intermolecular O1–H1⋯I1 hydrogen bond, making it is difficult to dissociate the hydrogen from the phenolic hydroxyl group, and so the pK_a is larger.³⁶

Mechanism of probe response to viscosity and pH

Fluorescence detection reveals that the probe fluoresces most strongly in glycerol and weakly in all other solvents. Meanwhile, the fluorescence of the probe increases gradually with viscosity enhancement, which is due to the formation of a push–pull electronic structure in low viscosity media. The Ar–OH and thiophene in HTC act as the electron donor and the C=N⁺ in the indole ring part as the electron acceptor. The single bond between the benzene and thiophene rings and the double bond between thiophene and indole rings can freely rotate due to the free rotation of the benzene and thiophene rings. This promotes the TICT and consumes the HTC in the nonradiative pathway of the excited state energy, so the fluorescence is very weak. By contrast, in high viscosity media, the free rotation of the molecules is blocked, which inhibits the non-radiative energy consumption and the fluorescence is enhanced.³⁷

During the detection of the pH changes, since the probe has an Ar–OH group, which is sensitive to pH changes, in alkaline solutions, the phenolic hydroxyl group gradually loses its protons, leading to an enhanced ICT effect. At pH 11 and 12, the probe C=C decomposes in strong alkaline solution to form aldehydes, and we found a molecular ion peak of 203.01742 (theoretical value of the molecular weight of aldehydes: 203.0172) at pH = 11 and 12 (Fig. S8, ESI†), which is consistent with our speculation.

Ability to target mitochondria

The biocompatibility of the probe, as an important indicator of potential biological application, is crucial for the probe in practical applications. Given that the probe has a pH and a viscosity response, we attempted to detect pH and fluorescence imaging in living cells. First, we examined the cytotoxicity of the probe, and the CCK8 method showed that the survival rate of all cells was more than 80% after co-incubation at different concentrations (0–10 μM) over 24 h (Fig. 4C). This indicates that the probe has low toxicity to the cells and is biocompatible. Next, the targeting of the probe toward organelles was investigated. Mitochondria are organelles with a double membrane and negative membrane potential.^38,39 Therefore, the positively charged probe HTC can easily penetrate the mitochondrial matrix and should therefore target mitochondria. The cellular co-localization experiments showed that the probe was able to target mitochondria for cytofluorescence imaging (with a Pearson coefficient of 0.91, Fig. 4A).

Fig. 4 Co-localization images of HTC (5 μM) in HepG2 cells. (A) Cells were pre-incubated with serum-free medium for 4 h and then co-stained with HTC and Mito-Tracker Green (200 μM) for fluorescence imaging. Green channel: fluorescence image of Mito-Tracker Green (200 μM) (λ_ex = 488 nm, λ_em = 500–550 nm). Red channel: fluorescence image of HTC (5 μM) (λ_ex = 520 nm, λ_em = 610–630 nm). Merge: fluorescence image of green and red channel combined; (B) map of fluorescence intensity distribution in the shaded region; (C) toxicity map of HTC in LO₂, HepG2 and 4T1. Scale bar: 20 μm.

Reaction to the pH value in the cell

Confocal laser microscopy was used to examine the fluorescence of living HepG2 cells in an environment with a pH of 6.0 to 8.0. The fluorescence intensity under the red channel was stronger at pH = 6 or 7, and the red light under the red channel was refracted at pH = 8. The fluorescence intensity at pH = 6 or 7 was four times higher than at pH = 8 (Fig. 5). This result is consistent with the fluorescence change at 627 nm in the solution state, indicating that the HTC probe can detect pH changes in cells.

Fig. 5 (A) Confocal imaging of PBS-induced HepG2 cells at different pH values. Scale bar: 20 μM; (B) quantitative fluorescence intensity of the red-light channel. Data are presented as mean ± SD (standard deviation, n = 3), ***p < 0.001.

Detection of changes in viscosity in NAFLD cell models

To determine whether the probe could detect changes in the viscosity of living cells, we chose HepG2 as a cell model. The mitochondrial viscosity of the cells was increased using mycobacterium and serum-free HBSS medium as external and internal factors,⁴⁰ and then fluorescence imaging of the cells was performed. The results (Fig. 6A) showed only faint red fluorescence when the cells were incubated with the probe HTC. When the mitochondrial viscosity of the cells increased, the red light was significantly enhanced. These results indicate that HTC can monitor changes in cell viscosity induced by endogenous and exogenous factors in living cells. It is known that cancer cells and normal cells differ in viscosity and pH, and cancer cells are characterized by low pH and high viscosity, which can be used to distinguish normal cells from cancer cells. Fluorescence imaging by confocal laser microscopy showed that the fluorescence of cancer cells HepG2 and 4T1 was significantly stronger than that of normal cells LO2, with the intensity of HepG2 and 4T1 being five times that of LO2 (Fig. S10, ESI†). This indicates that the HTC probe can be used for cell fluorescence imaging to distinguish cancer cells from normal cells.

Fig. 6 Fluorogram of viscosity change of HTC (5 μM) in HepG2. (A) Control: cells were co-incubated with HTC and cells for 2 h; HBSS group: the cells were pre-incubated with serum-free HBSS medium for 2 h and then incubated with HTC for 2 h. Nys group: first incubated with HTC and HepG2 for 2 h, then incubated with Nys (10 μM) for 30 min. OA group: HepG2 was pre-incubated with HTC for 2 h, then OA (10 μM) was added and incubated together for 30 min. OA + EtOH group: HepG2 was pre-incubated with for 2 h, followed by the addition of OA (10 μM) and EtOH for 30 min. (B) Fluorescence relative pixel intensities. λ_ex = 520 nm, λ_em = 610–630 nm; scale bar: 20 μM. Data are presented as mean ± SD (standard deviation, n = 3), ***p < 0.001.

Since the HTC probe fluoresces more strongly in cancer cells and responds to changes in intracellular pH and viscosity, affecting the metabolic function of HepG2,⁴¹ HepG2 was induced with oleic acid, oleic acid and ethanol, which increase the viscosity of HepG2. Thus, a fatty liver cell model was established for use with the HTC probe to detect fatty hepatocytes, cell fluorescence imaging was performed using the HTC probe. Cells were incubated with HTC after induction with oleic acid, oleic acid and ethanol and showed a strong fluorescence – 4.2 times stronger than that of cells incubated with HTC alone (Fig. 6B). This indicates that the HTC probe can monitor fatty liver formation at the cellular level.

Detection of NAFLD mouse models

It is well known that fatty liver tissue has high viscosity.^42,43 Therefore, we attempted to identify hyperviscous organs in mice. We established a mouse model of nonalcoholic fatty liver by feeding mice with high-fat chow and intraperitoneally injecting olive oil containing CCl₄. Blood was taken from the eyes of the mice and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in the serum of the mice, and the results, as shown in Fig. 7A, showed that ALT and AST were higher in the mice with fatty liver than in the normal mice, which indicated the successful establishment of the nonalcoholic fatty liver model. First, we determined the optimal concentration of the probe for in vivo imaging in mice (Fig. S11, ESI†). Then, HTC was injected intraperitoneally in normal mice and fatty liver mice, respectively, followed by fluorescence imaging with a small animal imager, and the results, as shown in Fig. 7D, showed that the fluorescence was weaker in normal mice and stronger in fatty liver mice. Quantifying the fluorescence in the abdomen of the mice (Fig. 7B), the fluorescence intensity of mice with fatty liver was about 2 times that of normal mice. This indicates that HTC can distinguish between normal mice and mice with fatty liver. Next, we executed the mice, dissected and removed the vital organs, and performed fluorescence imaging of the organs, and the results are shown in Fig. 7E. The fluorescence of the liver of the normal mice was weaker, the fluorescence of the fatty liver model mice was stronger; the fluorescence of the rest of the organs was weaker. The fluorescence intensity was quantified in the mice (Fig. S12, ESI†), and the fluorescence intensity of the liver of the fatty liver mice was 2.4 times higher than that of normal mice, which indicates that the probe was mainly enriched in the liver. Subsequently, HE staining was performed on liver sections of mice in the normal and model groups. As shown in Fig. 7C, there were no obvious morphological changes in the liver tissues of normal mice, and the hepatocytes of mice with fatty liver showed vacuolization. From the tissue sections and serum measurements, it was clear that fatty liver would be accompanied by liver injury. In order to observe the metabolic pathway of the probe in the model mice, we monitored the fluorescence changes of the probe from 0 to 8 h in the mice (Fig. 7G), and found that the fluorescence of the probe was strongest at 4 h, and the fluorescence gradually weakened at 4–8 h. The urine of 0–8 h mice was collected (Fig. 7F) and fluorescence imaging was performed with a small animal imager; it was found that the urine glowed red, and the fluorescence intensity of the urine of mice with fatty liver was 3.2 times higher than that of normal mice (Fig. S12B, ESI†), which indicated that the probe was metabolized faster after entering into mice and being discharged out of the body along with the urine. The above experimental results demonstrated that the probe HTC can assist in the detection of NAFLD in mice and provide a visualization means for the diagnosis of NAFLD.

Fig. 7 (A) Serum levels of ALT and AST in mice with normal and fatty liver; (B) quantification of relative fluorescence intensity in normal and fatty liver mice; (C) HE staining of mouse liver tissue sections. Scale bar: 50 μM. (D) Fluorescence imaging of normal mice and mice with fatty liver after injection of HTC (10 μM); (E) fluorescence imaging of the major organs of mice with normal and fatty liver: (1) heart, (2) lung, (3) kidney, (4) spleen, and (5) liver. λ_ex = 520 nm, λ_em = 610–630 nm; (F) urine imaging of mice with normal and fatty liver after 8 h. (G) Metabolic fluorescence maps of the probe in mice with fatty liver 0–8 h after administration of HTC (10 μM). Data are presented as mean ± SD (standard deviation, n = 3), ***p < 0.001.

Conclusions

In summary, we constructed HTC, a hemicarbocyanine fluorescent probe with a D–D′(π)–π–A configuration, using phenol as an electron donor (D), thiophene moiety as an electron donor and a partial π-bridge. The fluorescent HTC was positively charged and could be targeted to mitochondria in the cell, with a Pearson's coefficient of 0.91 for cellular colocalized fluorescence imaging with commercial mitochondrial probes. In solution, the probe has pH-responsive and viscosity-responsive properties. In cells, the probe is able to monitor changes in pH and viscosity and can distinguish between normal and cancerous cells. Taking advantage of the fact that the probe has viscosity-responsive properties, we used the probe to identify the formation of non-alcoholic fatty liver in mice. In addition, the probe is easily metabolized in mice and has good biocompatibility. These experiments provide a visual means for the identification of cancer cells and non-alcoholic fatty liver and provide new ideas for the design of the fluorescent probe.

Experimental

Materials and characterization

Unless otherwise stated, all of the starting materials were commercially available and were used without further purification. The solutions of metal ions and anions were prepared from their nitrate and sodium salts, respectively. High resolution mass spectrometry (HRMS) data were obtained using an Agilent HPLC-6545. ¹H/¹³C NMR spectra were recorded on an Inova-400 Bruker AV 400 spectrometer using DMSO-d₆ as solvent and tetramethylsilane as the internal reference. UV-vis absorption spectra were obtained on a UV-2600 Milton Roy spectrofluorometer. PL spectra were recorded on a Cary eclipse Hitachi 4500 spectrofluorometer. All animal experiments were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Ethical Scientific Committee of Guizhou Medical University.⁴³

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This word was supported by the National Natural Science Foundation of China (22367004 and 22466012), the Guizhou Provincial Key Laboratory Platform Project (ZSYS[2025]008), and the Guizhou Provincial Natural Science Foundation (grant numbers ZK[2023]291 and ZK[2022]395). C. R. thanks the University of Hull for support.

Notes and references

1. J. Zheng, Y. Wang, N. Kawazoe, Y. Yang and G. Chen, Influences of viscosity on the osteogenic and adipogenic differentiation of mesenchymal stem cells with controlled morphology, J. Mater. Chem. B, 2022, 10, 3989–4001.
2. D. M. Hausman, What Is Cancer?, Perspect. Biol. Med., 2019, 62, 778–784.
3. J. D. Schiffman, P. G. Fisher and P. Gibbs, Early detection of cancer: past, present, and future, Am. Soc. Clin. Oncol., Educ. Book., 2015, 57–65.
4. M. Akbari, T. B. L. Kirkwood and V. A. Bohr, Mitochondria in the signaling pathways that control longevity and health span, Ageing Res. Rev., 2019, 54, 100940.
5. W. Shen, P. Wang, Z. Xie, H. Zhou, Y. Hu, M. Fu and Q. Zhu, A bifunctional probe reveals increased viscosity and hydrogen sulfide in zebra fish model of Parkinson's disease, Talanta, 2021, 234, 122621.
6. M. M. Sreejaya, V. M. Pillai, A. Ayesha, M. Baby, M. Bera and M. Gangopadhyay, Mechanistic analysis of viscosity-sensitive fluorescent probes for applications in diabetes detection, J. Mater. Chem. B, 2024, 20, 2917–2937.
7. E. Kucukal, Y. Man, A. Hill, S. Liu, A. Bode, R. An, J. Kadambi, A. J. Little and A. U. Gurkan, Whole blood viscosity and red blood cell adhesion: Potential biomarkers for targeted and curative therapies in sickle cell disease, Am. J. Hematol., 2020, 95, 1246–1256.
8. J. A. Kraut and N. E. Madias, Metabolic acidosis: pathophysiology, diagnosis and management, Nat. Rev. Nephrol., 2010, 6, 274–285.
9. E. Needham and G. Webb, Hepatic encephalopathy: a neurologist's perspective, Pract. Neurol., 2024, 24, 200–206.
10. T. G. Nagami, Hyperchloremia - Why and how, Nefrologia, 2016, 36, 347–353.
11. N. M. Anderson and M. C. Simon, The tumor microenvironment, Curr. Biol., 2020, 30, R921–R925.
12. U. Martinez-Outschoorn, F. Sotgia and M. P. Lisanti, Tumor microenvironment and metabolic synergy in breast cancers: critical importance of mitochondrial fuels and function, Semin. Oncol., 2014, 41, 195–216.
13. N. Li, Y. Li, J. Hu, Y. Wu, J. Yang, H. Fan, L. Li, D. Luo, Y. Ye, Y. Gao, H. Xu, W. Hai and L. Jiang, A Link Between Mitochondrial Dysfunction and the Immune Microenvironment of Salivary Glands in Primary Sjogren's Syndrome, Front. Immunol., 2022, 13, 845209.
14. Y. Zong, Y. Yang, J. Zhao, L. Li, D. Luo, J. Hu, Y. Gao, X. Xie, L. Shen, S. Chen, L. Ning and L. Jiang, Identification of key mitochondria-related genes and their relevance to the immune system linking Parkinson's disease and primary Sjögren's syndrome through integrated bioinformatics analyses, Comput. Biol. Med., 2024, 175, 108511.
15. A. P. Sommer, M. Kh Haddad and H. J. Fecht, Light Effect on Water Viscosity: Implication for ATP Biosynthesis, Sci. Rep., 2015, 5, 12029.
16. Q. Zhang, W. Liu, L. Jiang, Y. J. He, C. J. Wu, S. Z. Ren, B. Z. Wang, L. Liu, H. L. Zhu and Z. C. Wang, Real-time monitoring of abnormal mitochondrial viscosity in glioblastoma with a novel mitochondria-targeting fluorescent probe, Analyst, 2024, 149, 2956–2965.
17. M. Cieśluk, E. Piktel, U. Wnorowska, K. Skłodowski, J. Kochanowicz, A. Kułakowska, R. Bucki and K. Pogoda, Substrate viscosity impairs temozolomide-mediated inhibition of glioblastoma cells’ growth, Biochim. Biophys. Acta, Mol. Basis Dis., 2022, 1868, 166513.
18. K. K. Bence and M. J. Birnbaum, Metabolic drivers of non-alcoholic fatty liver disease, Mol. Metab., 2021, 50, 101143.
19. T. Seçkin and M. S. Kormaly, An easy-to-build rotational viscometer with digital readout, J. Chem. Educ., 1996, 72, 193–194.
20. Á. D. Rosa, E. Poveda, G. Ruiz, R. Moreno, H. Cifuentes and L. Garijo, Determination of the plastic viscosity of superplasticized cement pastes through capillary viscometers, Constr. Build. Mater., 2020, 206, 119715.
21. G. A. Kubi and D. Pei, Cell-penetrating and mitochondrion-targeting molecules, Methods Enzymol., 2020, 641, 311–328.
22. Y. Wei, Y. Liu, Y. He and Y. Wang, Mitochondria and lysosome-targetable fluorescent probes for hydrogen peroxide, J. Mater. Chem. B, 2021, 28, 908–920.
23. Y. Li, Y. Wang, Y. Li, W. Shi and J. Yan, Construction and evaluation of near-infrared fluorescent probes for imaging lipid droplet and lysosomal viscosity, Spectrochim. Acta, Part A, 2024, 316, 124356.
24. H. Xiao, P. Li and B. Tang, Small Molecular Fluorescent Probes for Imaging of Viscosity in Living Biosystems, Chemistry, 2021, 27, 6880–6898.
25. X. Zhang, F. Huo, Y. Zhang, Y. Yue and C. Yin, Dual-channel detection of viscosity and pH with a near-infrared fluorescent probe for cancer visualization, Analyst, 2022, 147, 2470–2476.
26. L. Yang, P. Gu, A. Fu, Y. Xi, S. Cui, L. Ji, L. Li, N. Ma, Q. Wang and G. He, TPE-based fluorescent probe for dual channel imaging of pH/viscosity and selective visualization of cancer cells and tissues, Talanta, 2023, 265, 124862.
27. E. G. Skurikhin, S. A. Afanas’ev, M. A. Zhukova, T. Y. Rebrova, E. F. Muslimova, E. S. Pan, N. N. Ermakova, O. V. Pershina, A. V. Pakhomova, O. D. Putrova, L. A. Sandrikina, L. V. Kogai and A. M. Dygai, Age-Related Features of the Viscosity of Plasma and Mitochondrial Membranes of Hepatocytes in Liver Cirrhosis, Bull. Exp. Biol. Med., 2021, 171, 707–712.
28. S. Chen, Y. Hong, Y. Liu, J. Liu, C. W. Leung, M. Li, R. T. Kwok, E. Zhao, J. W. Lam, Y. Yu and B. Z. Tang, Full-range intracellular pH sensing by an aggregation-induced emission-active two-channel ratiometric fluorogen, J. Am. Chem. Soc., 2013, 135, 4926–4929.
29. K. Kuter, M. Kratochwil, K. Berghauzen-Maciejewska, U. Głowacka, M. D. Sugawa, K. Ossowska and N. A. Dencher, Adaptation within mitochondrial oxidative pHospHorylation supercomplexes and membrane viscosity during degeneration of dopaminergic neurons in an animal model of early Parkinson's disease, Biochim. Biophys. Acta, 2016, 1862, 741–753.
30. M. Fu, W. Shen, Y. Chen, W. Yi, C. Cai, L. Zhu and Q. Zhu, A highly sensitive red-emitting probe for the detection of viscosity changes in living cells, zebrafish, and human blood samples, J. Mater. Chem. B, 2020, 8, 1310–1315.
31. K. Wong, H. Wang and G. Lanzani, Ultrafast excited-state planarization of the hexamethylsexithiopHene oligomer studied by femtosecond time-resolved pHotoluminescence, Chem. Phys. Lett., 1998, 288, 59–64.
32. C. Liu, D. Zhang, S. Ye, T. Chen and R. Liu, D-π-A structure fluoropHore: NIR emission, response to viscosity, detection cyanide and bioimaging of lipid droplets, Spectrochim. Acta, Part A, 2022, 267, 120593.
33. Y. Chen, W. Shi, Y. Xu and P. Wang, Real-time visualization of sulfatase in living cells and in vivo with a ratiometric AIE fluorescent probe, Chem. Commun., 2023, 59, 9754–9757.
34. Y. Wan, Z. Guo, Z. Wu, T. Liang and Z. Li, Visualization of Diabetes Progression by an Activatable NIR-IIb Luminescent Probe, Anal. Chem., 2024, 96, 14843–14852.
35. Q. Bai, C. Yang, M. Yang, Z. Pei, X. Zhou, J. Liu, H. Ji, G. Li, M. Wu, Y. Qin, Q. Wang and L. Wu, pH-Dominated Selective Imaging of Lipid Droplets and Mitochondria via a Polarity-Reversible Ratiometric Fluorescent Probe, Anal. Chem., 2022, 94, 2901–2911.
36. J. Liu, X. Ma, Q. Song, J. Zang, J. Hao, W. Liu and J. Jiang, Ratiometric fluorescent and colorimetric dual-modal sensing strategy for discrimination and detection of D₂O from H₂O, Chem. Commun., 2022, 58, 9262–9265.
37. Z. Peng, D. Zhang, H. Yang, Z. Zhou, F. Wang, Z. Wang, J. Ren and E. Wang, Mitochondria-targeted fluorescent probe for simultaneously imaging viscosity and sulfite in inflammation models, Analyst, 2024, 149, 3356–3362.
38. X. Zhang, Q. Sun, Z. Huang, L. Huang and Y. Xiao, Immobilizable fluorescent probes for monitoring the mitochondria microenvironment: a next step from the classic, J. Mater. Chem. B, 2019, 7, 2749–2758.
39. S. Sakamuru, M. S. Attene-Ramos and M. Xia, Mitochondrial Membrane Potential Assay, Methods Mol. Biol., 2016, 1473, 17–22.
40. M. Zhao, Y. Zhu, J. Su, Q. Geng, X. Tian, J. Zhang, H. Zhou, S. Zhang, J. Wu and Y. Tian, A water-soluble two-photon fluorescence chemosensor for ratiometric imaging of mitochondrial viscosity in living cells, J. Mater. Chem. B, 2016, 4, 5907–5912.
41. L. C. Lagrutta, S. Montero-Villegas, J. P. Layerenza, M. S. Sisti, M. M. García de Bravo and A. Ves-Losada, Reversible Nuclear-Lipid-Droplet MorpHology Induced by Oleic Acid: A Link to Cellular-Lipid Metabolism, PLoS One, 2017, 26, e0170608.
42. A. Popa, F. Bende, R. Şirli, A. Popescu, V. Bâldea, R. Lupuşoru, R. Cotrău, R. Fofiu, C. Foncea and I. Sporea, Quantification of Liver Fibrosis, Steatosis, and Viscosity Using Multiparametric Ultrasound in Patients with Non-Alcoholic Liver Disease: A “Real-Life” Cohort Study, Diagnostics, 2021, 26, 783.
43. Y. Chen, P. Zong, Q. Chen, X. Wang, J. Luo, K. Liu and R. Zhang, Construction of a pH- and viscosity-switchable near-infrared fluorescent probe and its imaging application, Spectrochim. Acta, Part A, 2024, 319, 124527.

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR spectra and crystal data and analysed data. CCDC 2385840. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4tb02711f

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