Development of an Endoplasmic Reticulum-targeting Fluorescent Probe for the Imaging of Superoxide Anion in Living Cells

Superoxide anion (O2•−) is an important reactive oxygen species (ROS), and plays critical roles in biological systems. ER stress has close relation with many metabolic diseases, and could lead to the abnormal production of ROS including O2•−. Herein, we present an ER-targeting probe (ER-Tf) for the detection of O2•− in living cells. The probe ER-Tf used triflate as the response site for O2•−, and employed p-methylbenzenesulfonamide as ER-targeting moiety. In response to O2•−, the triflate of the probe ER-Tf converted to hydroxyl group, providing strong blue emission under the excitation of ultraviolet light. The probe ER-Tf exhibited high sensitivity and selectivity to O2•−. Bioimaging experiments showed that the probe ER-Tf can be applied to detect O2•− at ER, and also demonstrated that rotenone could increase the generation of O2•− in living cells, while the O2•− level at ER showed no remarkable change during ferroptosis.


Introduction
Superoxide anion(O 2 •− ) is a significant reactive oxygen species (ROS), and plays many critical roles in biological system [1][2][3][4][5][6]. Generally, O 2 •− is produced endogenously during cellular respiration, and O 2 •− levels are tightly controlled by scavenging enzymes in living organisms and closely related to homeostasis [7]. O 2 •− usually serves as a precursor to generate the other ROS including H 2 O 2 , hydroxyl radical (·OH), hypochlorous acid (HClO),and singlet oxygen ( 1 O 2 ) [8]. At the same time, O 2 •− can also be used as a signal molecule for the redox reaction in biological systems [9,10]. However, when excess O 2 •− is produced, autophagy or apoptotic signaling pathways may be activated, leading to cell death and ultimately to various diseases including atherosclerosis and neurodegenerative diseases [11]. Meanwhile, endoplasmic reticulum (ER) is widely found in various mammal cells, and plays significant roles in the synthesis processes of proteins, lipids, sugars, and also works in maintaining blood glucose levels [12][13][14][15][16]. Many studies have indicated that ER stress is bound up with obesity, insulin resistance and other metabolic diseases, which may lead to abnormal production of reactive oxygen species (ROS), including O 2 •− [17,18]. Therefore, development of sensitive and selective method to detect O 2 •− at ER is highly significant for intensively studying the pathological role of O 2 •− in living systems. Traditionally, the test methods for the detection of O 2 •− chiefly include MS, high performance liquid chromatograph (HPLC) and electron paramagnetic resonance (EPR) [19][20][21][22]. Nevertheless, the complex instruments of these methods can damage biological samples and are unsuitable for the real-time detection of O 2 •− under the normal physiological conditions. In contrast, fluorescence imaging as an emerging detection technology, has attracted widely attention due to its advantages of high sensitivity, low detection limit, real-time and non-destructive [23][24][25]. Currently, many fluorescent probes have been developed to detect cellular O 2 •− [26][27][28][29]. These probes utilized benzothiazoline, catechol, triflate, diphenyl phosphate as the selective recognition sites for O 2 •− , and studied the endogenous generation of O 2 •− in many organelles including mitochondrial and

Cell Culture and Fluorescence Imaging
HeLa cells were obtained from the College of Life Science, Nankai University (Tianjin, China). Hela cells were used to study the properties of ER-Tf, the cells were cultured in a medium containing 10% heat-inactivated fetal bovine serum and 1% antibiotics in air at 37℃ (5% CO2). Before fluorescence imaging, the HeLa cells were inoculated into a glass cover and cultured for 24 h to achieve the suitable density. The images were obtained by a Nikon A1R confocal microscope. •− : Firstly, the HeLa cells were incubated with 10 μM or 20 μM rotenone for 30 min, or treated with 10 μM erastin for 2 h, and then washed with PBS for three times. Whereafter, 5 μM ER-Tf was used to stain HeLa cells for another 20 min, and subsequently the cells were washed with PBS for three times and imaged.

Synthesis of ER-Targeting Probe (ER-Tf) for O 2 •−
In order to meet requirements for the fluorescence imaging of cellular O 2 •− at ER, the designed fluorescent probe should structurally contain three units including fluorophore, response site of O 2 •− and ER-targeting group. Accordingly, we constructed the ER-targeting fluorescent probe (ER-Tf) for detecting cellular O 2 •− at ER. In the chemical structure of ER-Tf (Scheme 1), coumarin group was used as the fluorophore, p-methylsulfonamide acted as an ER-targeting part, and triflate group was selected as a reaction site for the reorganization of O 2 •− [30].The synthetic route is revealed in Scheme 1, and the chemical structures were characterized by 1 H NMR, 13 C NMR and HRMS.

Optical Properties of the Probe ER-Tf
Firstly, we investigated the absorption spectra of the probe ER-Tf treated with or without O 2 •− . To investigate the water solubility of the probe, the absorption spectra of the probe with various concentrations in PBS (20 mM, pH 7.4, 5% MeOH) were conducted (Fig. S1). The results showed that the absorbance at 280 nm was correlated linearly with the concentration of the probe in the range 5-60 μM, indicating the probe showed desirable water solubility when its concentration below 60 μM. The probe ER-Tf showed no significant absorption peak in the visible region in PBS (20 mM, 5% MeOH) (Fig. 1A). However, after 20 μM O 2 •− was added to 5 μM ER-Tf solution, an absorption peak could be clearly found at 405 nm, indicating that the probe ER-Tf reacted with O 2 •− . Under 405 nm excitation, the probe ER-Tf showed nearly no fluorescence with the fluorescence quantum yield of 0.0023 using quinine sulfate as a reference (Fig. 1B). Upon the introduction of O 2 •− , the fluorescence peak at 462 nm appeared and increased continuously along with the gradual change of O 2 •− concentration from 0 μM to 90 μM (Fig. 1C). Meanwhile, the intensity of the fluorescence at 462 nm displayed the satisfactory linear relationship (R 2 = 0.9909) with O 2 •− concentration in the range 5-60 μM, and the detection limit was calculated to 65 nM (Fig. 1D). In addition, after ER-Tf reacted with O 2 •− , the fluorescence intensity reached the maximum at 462 nm within 7 min ( Figure  S2). Hence, ER-Tf could be used as a sensitive fluorescent probe to detect O 2 •− .  (Fig. S4). We proposed that O 2 •− firstly attacked S atom of triflate group, and then peroxytriflate free radical was removed to provide the emissive compound ER-Cou by protonation (Scheme 2).
Then, we tried to study the ability of ER-Tf to detect O 2 •− at ER. The MTT assay showed that ER-Tf showed very low cytotoxity below the concentration of 15 μM (Fig. S5). When the HeLa cells were only treated with 5 μM ER-Tf for 20 min, almost no fluorescence was found under the excitation at 405 nm. Nevertheless, after the cells were firstly incubated with 30 μM or 60 μM O 2 •− , the bright fluorescence can be found in blue channel (Fig. 3A). It indicated that ER-Tf can visualize O 2 •− at ER. Besides, the fluorescence intensity of the cells treated with 60 μM O 2 •− was more intense than that of the cells incubated with 30 μM O 2 •− , suggesting that the probe ER-Tf could evaluate the concentration of cellular O 2 •− (Fig. 3B). Hence, ER-Tf could act as a sensitive fluorescent probe to detect O 2 •− in living cells.
Subsequently, in order to explore the ER-targeting ability of the probe ER-Tf, we selected the commercial ER-specific dye (ER-tracker) as a reference to compare the positioning ability of the probe ER-Tf. The specific operation of the experiment was as follows: Firstly, the cells were treated with 5 μM ER-Tf and incubated at a constant temperature for 20 min, and then washed off the medium and incubated with 50 μM O 2 •− , finally treated with 1 μM ER-tracker. Then we observe the positioning of the probe ER-Tf under a confocal microscope, the blue channel signal generated by the reaction of ER-Tf with O 2 •− can better overlap with the red channel signal generated by the ER-tracker, and the Pearson correlation coefficient is as high as 0.89 (Fig. 4).Therefore, the co-localization experiment showed that ER-Tf possessed ER-targeting property.
Then, we assessed the ability of ER-Tf to image endogenous O 2 •− in living cells. Rotenone, as a mitochondrial electron transport chain complex I inhibitor, which can lead to apoptosis and increase the production of ROS [33]. When HeLa cells were coated with 5 μM ER-Tf for 20 min, almost no fluorescence was observed in the blue channel (Fig. 5). While HeLa cells were firstly treated with 10 μM   or 20 μM rotenone for 30 min and then stained with 5 μM ER-Tf for 20 min, the bright fluorescence could be found in blue channel. It indicates that HeLa cells produce O 2 •− when stimulated by rotenone. In addition, we evaluate the changes of endogenously generated cellular O 2 •− during ferroptosis. Erastin is a famous iron ion activator, which can inhibit the uptake of cystine by cystine/glutamate reverse transporter (system XC), and finally lead to ferroptosis. Erastin was employed as an activator to investigate the subtle changes of O 2 •− in living cells during ferroptosis [34]. After treatment of HeLa cells with 10 μM erastin for 2 h and then 5 μM ER-Tf for 20 min, the fluorescence intensity of blue channel showed nearly no change (Fig. 5). It suggests that the O 2 •− level at ER showed no remarkable change during ferroptosis. Hence, ER-Tf can be used for the fluorescence imaging of endogenously generated O 2 •− at ER.

Conclusion
We