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Synlett 2024; 35(01): 140-144
DOI: 10.1055/a-2012-0232
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Functional Dyes

A Novel Heterocyclic Xanthene-Analogous pH Probe for ­Quantitative Monitoring of Cell Surface pH by Fluorescence ­Lifetime Imaging

Shuyao Wu 
a   State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology, Dalian, 116024, P. R. of China
,
Xinfu Zhang  ∗
a   State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology, Dalian, 116024, P. R. of China
,
Xiaoxi Li∗
b   Central Laboratory, Liaoning Cancer Hospital & Institute, Cancer Hospital of Dalian University of Technology, Shenyang, 110042, P. R. of China
,
Shulan Sun
b   Central Laboratory, Liaoning Cancer Hospital & Institute, Cancer Hospital of Dalian University of Technology, Shenyang, 110042, P. R. of China
,
Ze Hui Wang
a   State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology, Dalian, 116024, P. R. of China
,
Dingxuan Li
a   State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology, Dalian, 116024, P. R. of China
,
Yi Xiao∗
a   State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology, Dalian, 116024, P. R. of China
› Author Affiliations

This work was supported by the National Natural Science Foundation of China (Nos. 21901031, 22078047, 22174009, 22278059, and 82202861), the Dalian Science and Technology Innovation Fund (No. 2020JJ25CY014), Science and Technology Foundation of Liaoning Province (2020-YQ-08 and 2022-YQ-08), and the Fundamental Research Funds for the Central Universities (Nos. DUT21YG131, DUT21YG126, DUT18RC(3)027, DUT20RC(5)024, LD202115, and DUT22LAB601).
› Further Information
 
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Abstract

Rapidly capturing slight changes in cell surface pH is extremely important to evaluate the rapid diffusion of acidic metabolites into the extracellular environment caused by disease and physiological pH fluctuations of cells. In this work, we designed a membrane-targeted pH probe, Mem-COC18 , based on a novel heterocyclic xanthene-analogous backbone. Mem-COC18 shows specific and stable staining ability towards membrane. Importantly, the fluorescence lifetime of Mem-COC18 is highly sensitive against acidity within membrane, which is in favor of quantifying pH through fluorescence lifetime imaging. Using Mem-COC18 , we recorded pH changes of 0.61 units on the surface of human cervical cancer cells (Hela) during glycolysis. Further on, we observed a robust pH-regulating mechanism of the plasma membrane that the pH fluctuation range within membrane (5.32–6.85) is much smaller than the change in extracellular environment (4.00–8.00). Consequently, we demonstrate a pH probe for quantifying small pH fluctuations within cell membrane that merits further evaluation for biology applications.


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Key words

FLIM - heterocyclic xanthene-analogous - plasma membrane targeted - cell surface pH - glycolysis

Cell surface pH is closely related to various pathological features, such as cellular inflammation and body pain in cancer.[1] In general, the surface pH of tumor cells is lower than that of normal cells, which is attributed to the production of more acidic metabolites, including carbonic acid from the pentose phosphate pathway and lactic acid from glycolysis.[2] In order to maintain intracellular homeostasis, tumor cells pump intracellular acid out of the cell through a series of regulatory mechanisms, such as Na–H+ exchangers and various transport enzymes.[3] In addition, metastatic cancer cells, which expend more energy, have higher surface acidity than nonmetastatic cancer cells.[4] Thus, cell surface pH levels can be used as a marker of disease remission and progression. Accurate measurement of pH changes on the surface of tumor cells is a prerequisite for in-depth understanding of tumor cell pathology and establishment of appropriate treatment strategies.

Due to the important role of the cell membrane in sensing microenvironmental fluctuations and making timely adjustments, monitoring of cell surface pH is of great physiological significance. At present, many reports of pH-sensitive small-molecule fluorescent probes are more inclined to detect the pH of subcellular organelles,[5] such as acidic organelle lysosome (pH = 4.5–5.5),[6] alkaline organelle mitochondria (pH ~8.0),[7] etc. However, detection of pH at the plasma membrane is rarely reported, due to the challenge to fabricate such probes.[8] To achieve this goal, fluorescent probes should firstly target membrane stably and specifically, and secondly quantify pH change accurately. Currently, fluorescence ratio imaging and FLIM are two widely used techniques for the quantification of microenvironmental factors.[9] From the perspective of molecular design, ratiometric imaging often involves complex and fine molecular pair construction and is difficult to further design targeting, while FLIM imaging is usually based on sensitive small molecules and is easy to design membrane targeting. Moreover, FLIM does not suffer from spectral crosstalk, fluorophore concentration, etc.[10] Therefore, FLIM is a preferable technique for targeting plus quantitative detection.

Herein, we present a pH-sensitive probe Mem-C0C18 for quantitative detection of plasma membrane pH with fluorescence lifetime imaging technique. Mem-C0C18 is based on a rigid xanthene-analogous ring composed of an electron-rich bisthiophene and electron-deficient ketocarbonyl and phosphoryl groups. The inclusion of methyl piperazine as a pH-responsive group ensures a high sensitivity of its fluorescence lifetime to environmental pH. The introduction of long carbon chain and the natural cation forms the amphiphilic lipid-like structure that allows specific, stable targeting in the plasma membrane. Notably, the suitable hydrophilic/lipophilic nature gives it the advantage of high signal-to-noise ratio even without washing. We quantitatively measured membrane pH by fluorescence lifetime imaging using this probe in several pathological and physiological processes, which discloses pH fluctuations and self-regulatory mechanism of cancer cells.

Molecular Design of Mem-C0C18
In order to achieve pH detection on plasma membranes, the molecular design considers two main aspects: pH sensitivity and targeting stability. Firstly, a rigid framework composed of electron-rich dithiophene structure and strong electron-withdrawing carbonyl and phosphoryl groups is obtained. The introduction of quaternary ammonium piperazine groups at both ends of this highly lipophilic fluorophore can effectively inhibit the fluorescence quenching due to TICT.[11] By skillfully using symmetrical two piperazine rings, the pH response of the molecule is ensured by retaining the sensitivity of the nitrogen atom to protons on one side; the carbon chain is introduced on the other side to make the molecule self-charged, constituting the amphiphilic phospholipid-like structure Mem-C0C18 to achieve membrane targeting (Scheme [1]). According to our previously reported polar probes, the installation of a single long chain can generate a stable membrane targeting effect.[12] Furthermore, Mem-C0C18 shows almost no fluorescence due to aggregation in the aqueous phase but disperses in the membrane to recover intense fluorescence, which means high signal-to-noise ratio.

Zoom Image
Scheme 1 Synthesis route of Mem-C0C18

Spectral Properties in Solvents
We first assessed the pH response of Mem-C0C18 against different pH. Saturated cetyltrimethylammonium bromide (CTAB) is used to simulate membrane structure. We observed a significant increase in fluorescence intensity peak at 624 nm as the decrease of pH from 8.50 to 3.00 (Figure [1]A). Importantly, the fluorescence lifetime of Mem-C0C18 increased from 2.03 ns to 4.65 ns in the same pH range, which can be used for quantifying local pH (Figure [1]B, C). The above fluorescence response toward pH may ascribe to the protonation of piperazine ‘N’ in acidic medium that decreases the electron giving ability of N and therefore reduces the formation of TICT effect. Specifically, the pK a (the acidity coefficient) value was calculated as 5.00.

Zoom Image
Figure 1 Spectral properties of Mem-C0C18 in saturated CTAB aqueous solutions at different pH: (A) fluorescence emission diagram of Mem-C0C18 ; (B) fluorescence lifetime decay curve of Mem-C0C18 ; (C) fluorescence lifetime variation trend graph of Mem-C0C18 ; (D) fluorescence lifetime of Mem-C0C18 in THF–MeOH solvent system in relation to the polarity parameter Δf.

In addition, we investigated the interference of environmental polarity on the fluorescence lifetime of Mem-C0C18 in order to exclude the interference on pH detection. We used THF–MeOH solvent system to measure the fluorescence lifetime and found that the fluorescence lifetime varied within 1.5 ns with MeOH composition from 0–100% (polarity parameter Δf from 0.210–0.309) (Figure [1]D). Compared with the polarity variation in the MeOH–THF system, the polarity fluctuations in the plasma membrane is much smaller. Taking the ferroptosis process of Hela cells as an example, the membrane polarity parameter Δf varied within 0.21–0.23 throughout the process even if there was obvious membrane damage.[12] This polarity fluctuation is equivalent to a fluorescence lifetime change less than 0.28 ns according to the working plot of Mem-C0C18 . Therefore, the effect of membrane polarity on fluorescence lifetime of Mem-C0C18 during the monitoring process was negligible.

Establishment of Standard Curves at the Cellular Level
In order to achieve quantitative detection of cell surface pH, we established a calibration curve of the fluorescence lifetime of the probe Mem-C0C18 against environmental pH. To restore the real structure and substance composition of the membrane, we used the membrane of fixed Hela cells to perform pH titration assay. Hela cells were fixed with 4% paraformaldehyde to disable their ability to maintain intracellular pH homeostasis, thus keep the pH in membrane consistent with the environmental pH. The pH of the medium MEM was adjusted in gradient from 4.00 to 8.00 every other 0.5 pH unit, and the corresponding fluorescence lifetime of Mem-C0C18 in membrane (average value from three imaging views, at least 10 cells in total) was measured by fluorescence lifetime imaging. These data were used to obtain the working plot between the fluorescence lifetime and pH in membrane (Figure [2]A, B). We found that the lifetime of the probe increased from 2.44 ns to 4.10 ns as the pH increased from 8.00 to 4.00.

Zoom Image
Figure 2 (A) FLIM images of 4% paraformaldehyde-fixed Hela cells stained by 3 μM Mem-C0C18 for 15 min. (B) Standard curve of fluorescence lifetime–pH relationship of Mem-C0C18 established on fixed Hela cells. Scale bar: 20 μm.

Effect of Extracellular Fluid Acidity on Cell Surface pH
We then utilized Mem-C0C18 to evaluate the pH in live Hela cells. Several integral proteins on the surface of the plasma membrane are dedicated to the active transport of acids and bases across the membrane, maintaining homeostasis by maintaining the appropriate intracellular pH through, for example, proton exchange. We evaluated this buffering effect of the cell membrane by switching the PBS buffer in culture dish with different pH and measured the fluorescence lifetime by FLIM (Figure [3]A). As showed in Figure [3]B, the measured pH value is much smaller than the designed values. In the pH range of 4.00–8.00 (extracellular PBS), Hela cells showed normal physiological activity and self-regulation ability and keeps the actual pH in membrane within 5.32–6.85. Even if the extracellular environment increases to pH 8.00, the pH in membrane is maintained below 7.00.

Zoom Image
Figure 3 (A) FLIM images of live Hela cells stained by Mem-C0C18 for 15 min. (B) The measured pH value of the plasma membrane of living cells corresponds to the designed value (extracellular PBS); the gray diagonal line is the ideal situation where the cell membrane and environmental pH are consistent; the colored data are the actual measured plasma membrane pH. Scale bar: 20 μm.

Determination of the Effect of Glycolysis on Cell Surface pH
We further measured the pH in plasma membrane during glycolysis using our probe. Glycolysis is the main energy supply mode in cancer cells,[13] and glucose and 2-deoxy-d-glucose (2-DG) promote/inhibit glycolysis by increasing/decreasing energy supply, respectively.[14] The pH changes in the microenvironment of cancer cells triggered by monitoring the degree of glycolysis are of physiological importance for studies such as the degree of tumor malignancy and the proliferation status of cancer cells. We first measure Hela cells cultured under standard culture condition as a control group, and pH in their membrane was calculated to be 6.42 ± 0.1 (Figure [4]Aa). The cell surface pH of cancer cells (6.5–6.8) is reported to be lower than the normal physiological level (pH 7.4), while the surface pH of metastatic cancer cells such as Hela is somewhat lower compared to nonmetastatic cells.[15] Our detection results are consistent with those reported in the literature, which further validates the reliability of our standard curve established on fixed cells for pH calibration of live cells.

Zoom Image
Figure 4 (A) FLIM images of Hela cells cultured in suitable conditions with MEM medium containing 100 mM and 200 mM glucose solution for 12 h and MEM medium containing 10 mM D-Glu for 4 h, respectively. (B) pH of the abnormal glycolysis group compared with the control group. (C) Average fluorescence lifetime decay curves of the four groups of cells. Scale bar: 20 μμm.

We next investigated the effects of glucose and 2-DG on the acidity of the plasma membrane of Hela cells. We incubated Hela cells with MEM medium containing 100 mM and 200 mM glucose solution for 12 h. As the glucose concentration increased, the amount of lactic acid produced by the cells increased, we found that the pH in the membrane of these two groups were 6.28 and 6.25, respectively, which is lower than control statistically significant (Figure [4]Ab,c). Further on, we cultured Hela cells with MEM medium containing 10 mM of the glycolysis inhibitor 2-DG for 4 h, which would inhibit proton production and reduce proton flux.[16] We found the pH in membrane of these cells was 6.86, which is 0.44 pH units higher than the control group (Figure [4]Ad, B, C). It is worth mentioning that this study showed high imaging quality without washing the stained cells in order to maintain the pH of the extracellular fluid.

In conclusion, we designed a pH-sensitive membrane-targeted probe Mem-C0C18 based on a novel heterocyclic xanthene-analogous backbone.[17] Its fluorescence lifetime shows high sensitivity against pH over a wide range. The amphiphilic structure of Mem-C0C18 enables its targeting in plasma membranes stably and rapidly. By using Mem-C0C18 we were able to measure pH in plasma membrane in live cells. We found the difference between extracellular fluid and cell surface pH due to tumor cell self-regulation and quantified fluctuations in membrane pH of HeLa cell triggered by aberrant glycolysis. In this study, we demonstrate a pH probe for quantifying pH fluctuations within cell membrane that can be a potential tool for biology study.


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Conflict of Interest

The authors declare no conflict of interest.

Supporting Information

    Supporting information for this article is available online at https://doi-org.accesdistant.sorbonne-universite.fr/10.1055/a-2012-0232.
  • Supporting Information

Corresponding Authors

Xinfu Zhang
State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology
Dalian, 116024
P. R. of China   

Xiaoxi Li
Central Laboratory, Liaoning Cancer Hospital & Institute, Cancer Hospital of Dalian University of Technology
Shenyang, 110042
P. R. of China   

Yi Xiao
State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology
Dalian, 116024
P. R. of China   

Publication History

Received: 08 December 2022

Accepted after revision: 12 January 2023

Accepted Manuscript online:
12 January 2023

Article published online:
14 March 2023

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Zoom Image
Scheme 1 Synthesis route of Mem-C0C18
Zoom Image
Figure 1 Spectral properties of Mem-C0C18 in saturated CTAB aqueous solutions at different pH: (A) fluorescence emission diagram of Mem-C0C18 ; (B) fluorescence lifetime decay curve of Mem-C0C18 ; (C) fluorescence lifetime variation trend graph of Mem-C0C18 ; (D) fluorescence lifetime of Mem-C0C18 in THF–MeOH solvent system in relation to the polarity parameter Δf.
Zoom Image
Figure 2 (A) FLIM images of 4% paraformaldehyde-fixed Hela cells stained by 3 μM Mem-C0C18 for 15 min. (B) Standard curve of fluorescence lifetime–pH relationship of Mem-C0C18 established on fixed Hela cells. Scale bar: 20 μm.
Zoom Image
Figure 3 (A) FLIM images of live Hela cells stained by Mem-C0C18 for 15 min. (B) The measured pH value of the plasma membrane of living cells corresponds to the designed value (extracellular PBS); the gray diagonal line is the ideal situation where the cell membrane and environmental pH are consistent; the colored data are the actual measured plasma membrane pH. Scale bar: 20 μm.
Zoom Image
Figure 4 (A) FLIM images of Hela cells cultured in suitable conditions with MEM medium containing 100 mM and 200 mM glucose solution for 12 h and MEM medium containing 10 mM D-Glu for 4 h, respectively. (B) pH of the abnormal glycolysis group compared with the control group. (C) Average fluorescence lifetime decay curves of the four groups of cells. Scale bar: 20 μμm.