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A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during heat shock
SAA-117761; No of Pages 6 Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
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A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during heat shock Xiao-Fan Zhang b,1, Tian-Ran Wang a,1, Xiao-Qun Cao a, Shi-Li Shen a,⁎ a b
School of Chemistry and Pharmaceutical Engineering, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271016, PR China Taian Center For Food and Drug Control, Taian 271000, PR China
a r t i c l e
i n f o
Article history: Received 14 October 2019 Received in revised form 2 November 2019 Accepted 3 November 2019 Available online xxxx Keywords: Fluorescent probe Rhodamine derivative pH Near-infrared Lysosomes Heat shock
a b s t r a c t Heat shock is a potentially fatal condition characterized by high body temperature (N40 °C), which may lead to physical discomfort and dysfunctions of organ systems. Acidic pH environment in lysosomes can activate enzymes, thus facilitating the degradation of proteins in cellular metabolism. Owing to the lack of a practical research tool, it remains difficult to exploit relationship between heat shock and lysosome. Herein, a NIR lysosomal pH chemosensor (NRLH) was developed. One typical lysosome-locating group, morpholine, was incorporated into NRLH. The fluorescence intensity showed pH-dependent characteristics and responded sensitively to pH fluctuations in the pH range of 3.0–5.5. NRLH with a pKa of 4.24 displayed rapid response and high selectivity for H+ among common species. We also demonstrated NRLH was capable of targeting lysosomes. Importantly, NRLH was applied in cellular imaging and the data revealed that lysosomal pH increased but never decreased during the heat shock. Therefore, NRLH may act as an effective molecular tool for exploring the mechanisms of heat-related pathology in bio-systems. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Many cellular processes are regulated by pH values, including cell proliferation, apoptosis and endocytosis [1–3]. In eukaryotic cells, the acidic pH in lysosomes (pH 4.5–5.5) is beneficial for the activation of numerous enzymes in lysosomes [4,5]. The abnormal pH values are implicated with cellular dysfunctions, which can result in many diseases, such as cancer and Alzheimer's disease [6–8]. Heat shock, a kind of heat-related disorders, is identified as a prevalent health risk and may cause mortality [9]. Despite this, the mechanisms responsible for the direct cytotoxicity of heat are rarely explored. Recent investigations indicated that there was a certain correlation between heat shock and lysosome acidity. However, the relationship between the temperature and lysosomal pH is still unclear [10]. In the aim of studying the mechanism of heat cytotoxicity, it is fairly essential to detect lysosomal pH in heat shock process. Among various analysis methods for pH and biological species in vivo, fluorescent probe has become an indispensable tool due to the brilliant selectivity and sensitivity, nondestructive use in cells and easy ⁎ Corresponding author. E-mail address: [email protected] (S.-L. Shen). 1 These authors are equally contributed to this work.
test [11–21]. In particular, near-infrared (NIR) probes emitting in the range of 650–900 nm are especially preferred since NIR light can avoid the interference of autofluorescence and minimize photodamage to biological specimens [22–26]. So it is necessary to design nearinfrared pH sensors that possess lysosome-targeting capability. Rhodamine dyes have attracted considerable attention in biomolecular detection and intracellular imaging by virtue of the excellent biocompatibility and photophysical characteristics, including long absorption and emission wavelength as well as high fluorescence quantum yield [27,28]. Significantly, the non-fluorescent spirocyclic structure of rhodamine-based sensors could be regulated by H+-induced ring-opening process, thus resulting in dramatic fluorescence increase [29–34]. Although many lysosomal pH sensors on the basis of rhodamine framework were reported, their short absorption and emission may inevitably restrict the application in living cells [35–38]. Herein, we develop a lysosome-targeting NIR pH chemosensor (NRLH) by integrating one lysosomal locating group, morpholine [39–41], into the rhodamine framework. Compared with our previously reported pH sensors [42,43], NRLH shows longer emission wavelength (λem = 705 nm), and this would extend its biological application. And it is indicated that NRLH could target lysosome as well as monitor lysosomal pH changes. Besides, the fluorescence imaging experiments reveal that lysosomal pH increases along with the temperature rise.
https://doi.org/10.1016/j.saa.2019.117761 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: X.-F. Zhang, T.-R. Wang, X.-Q. Cao, et al., A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during h..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117761
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Scheme 1. Preparation of NRLH.
(TLC) on gel F254 plates. Flash chromatography was carried out on silica gel (200–300 mesh; Qingdao Ocean Chemicals). 2.2. Materials Unless specifically indicated, all reagents and solvents (analytical grade) were purchased from commercial suppliers and used without further purification. Britton-Robinson (B-R) buffer was obtained by mixing boric acid (40 mM), phosphoric acid (40 mM) and acetic acid (40 mM) in deionized water. 2.3. Synthesis of probe NRLH
Fig. 1. Absorption spectra changes of 10 μM NRLH in the buffer solution (5% EtOH) upon the addition of H+.
2. Experimental 2.1. Instrumentation NMR spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. Tetramethylsilane (TMS) was used as internal standard. A Q-TOF6510 spectrograph (Agilent) was applied to record HRMS spectra. The Hitachi U-3900 spectrometer was employed to record UV–vis spectra. A Hitachi F-4500 fluorescence spectrophotometer was used for fluorescence measurements. The PHSJ-3F digital pH meter (LeiCi, Shanghai, China) was used to detect pH. Reactions were monitored by thin-layer chromatography
Synthesis of NRLH was outlined in Scheme 1. Compound 1 was obtained according to previous paper [44]. Under N2 atmosphere, compound 1 (1.0 mmol), benzotriazol-1-yloxy)tris (dimethylamino)phosphonium hexafluorophosphate (BOP, 1.0 mmol) and 4-(2-aminoethyl)morpholine (3.0 mmol) were successively added to 50 mL ethanol. The solution was heated to reflux for 12 h, cooled to room temperature, and evaporated under reduced pressure. The resulting precipitate was purified by column chromatography (MeOH/CH2Cl2 = 1/50, v/v) to afford NRLH as a faint blue solid in 49% yield. 1H NMR (DMSO-d6, 400 MHz), δ: 1.09 (t, 6H, J = 7.2 Hz), 1.22 (s, 1H), 1.35–1.44 (m, 1H), 1.53–1.70 (m, 2H), 2.23–2.86 (m, 6H), 2.53–2.66 (m, 1H), 2.73–2.82 (m, 1H), 3.12–3.28 (m, 2H), 3.35 (br, 4H), 3.45 (br, 4H), 3.85 (s, 3H), 6.22 (d, 1H, J = 8.8 Hz), 6.36 (d, 1H, J = 8.8 Hz), 6.60 (d, 1H, J = 2.4 Hz), 7.18 (q, 2H, J = 6.8 Hz), 7.25 (t, 1H, J = 7.2 Hz), 7.48 (d, 1H, J = 8.0 Hz), 7.52 (d, 2H, J = 6.4 Hz), 7.57 (d, 1H, J = 6.8 Hz), 7.60 (s, 1H), 7.76 (d, 1H, J = 7.2 Hz), 7.91 (d, 1H, J = 7.6 Hz); 13C NMR (DMSO-d6, 100 MHz), δ: 167.0, 152.2, 150.9, 148.3, 146.9, 136.1, 132.5, 131.4, 129.3, 128.5, 128.2, 127.8, 124.7, 123.3, 122.2, 122.0, 119.4, 118.8, 114.6, 111.0, 108.4, 105.8, 104.4, 97.4,
Fig. 2. (a) Fluorescence spectra of NRLH (1 μM) in the Britton-Robinson buffer (5% ethanol as co-solvent) at various pH. (b) The fluorescence intensity at 705 nm (I705) as a function of pH according to the titration under the excitation at 650 nm.
Please cite this article as: X.-F. Zhang, T.-R. Wang, X.-Q. Cao, et al., A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during h..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117761
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Scheme 2. Chemical structure of NRLH and its response mechanism to H+.
66.0, 65.7, 56.0, 54.9, 53.2, 43.5, 36.4, 32.7, 27.9, 22.3, 21.7, 12.4; HRMS calcd for C40H45N4O3 [M + H]+: 629.3492, found: 629.3491.
the facile synthetic method (Scheme 1). The final structure of NRLH was fully characterized by NMR (1H NMR, 13C NMR) and HRMS (Figs. S1–S3, ESI†).
2.4. Sample preparation 3.2. Capability of NRLH to respond to H+ in buffer solution The stock solution of probe NRLH was prepared by dissolving the probe compound in ethanol. All optical spectra of NRLH were measured in B-R buffer (5% ethanol was needed as a co-solvent). A standard 1 cm quartz cuvette was utilized for detection. 2.5. Cell culture HeLa cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (HyClone, USA) in a humidified atmosphere of 5/ 95 (v/v) of CO2/air at 37 °C. For confocal laser scanning microscopy imaging (Leica SP8), the medium was replaced with serum-free DMEM. Subsequently, cells were treated with NRLH at 37 °C. 3. Results and discussion 3.1. Design and preparation of NRLH Design of NRLH was based on the excellent photophysical properties of rhodamine fluorophore and the structural change between spirocyclic (non-fluorescent) and ring-opened (fluorescent) form responding to pH. Furthermore, morpholine, which was identified as one typical lysosomal locating moiety, was integrated into NRLH to realize the lysosome-targeting capability. Probe NRLH was obtained by
Fig. 3. Fluorescence intensity (I705) of NRLH (1 μM) in presence of various analytes in the Britton-Robinson buffer (5% EtOH as co-solvent) at pH 3.00 and 7.40 under the excitation at 650 nm. (1) blank, (2) Glu, (3) HS−, (4) Cys, (5) Hcy, (6) K+, (7) Zn2+, (8) Fe2+, (9) Ag+, (10) Ca2+, (11) Mg2+, (12) Co2+, (13) Na+, (14) Ni2+, (15) Cu2+, (16) Cd2+, − 2− (17) Cr3+, (18) Mn2+, (19) AcO−, (20) H2PO− 4 , (21) NO3 , (22) SO4 . Concentration: 1 mM for (6), (10), (11) and (13); 50 μM for others.
UV–vis absorption and fluorescence emission spectroscopy of NRLH was investigated in B-R buffer (5% (v/v) ethanol was needed as the cosolvent) at different pH. As depicted in Fig. 1, NRLH exhibited almost no absorption bands at 500–750 nm when pH was 7.40. As the pH decreased from 7.40 to 2.00, the absorption peak of rhodamine fluorophore at 650 nm enhanced greatly. The phenomena were attributed to the structural transformation of the rhodamine group in response to pH. The addition of H+ induced the ring-opening process of rhodamine in NRLH. As displayed in Fig. 2a, probe NRLH showed very weak fluorescence in weakly basic aqueous solution upon excitation at 650 nm. When pH varied from 7.40 to 2.00, the emission maxima of NRLH (I705) increased, in agreement with the absorption spectra changes. In basic or neutral pH conditions, rhodamine fluorophore was in the spirocyclic form, which was non-fluorescent. The spirocyclic form would convert to the fluorescent ring-opened form due to the interaction between H+ and NRLH, as outlined in Scheme 2. Most notably, the pKa was determined to be 4.24, demonstrating that NRLH may be used for the pH detection in lysosomes (Fig. 2b). To elucidate the above proposed mechanism of NRLH, 1H NMR titration was conducted in DMSO-d6 with addition of CF3COOD and then NaOH. We could see from Fig. S4 (ESI†) that compared with NRLH alone (Fig. S4a), the aromatic proton signals were down-field shifted, which was a result of ring-opening process of rhodamine framework
Fig. 4. The time course of I705 of NRLH in Britton-Robinson buffer (5% EtOH as co-solvent) at pH 3.00 and 7.40, respectively. λex = 650 nm.
Please cite this article as: X.-F. Zhang, T.-R. Wang, X.-Q. Cao, et al., A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during h..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117761
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Fig. 5. (a) Confocal images of cells treated with different dose of bafilomycin A1 (0–100 nM) for 6 h, and then with NRLH (1 μM) for 1 h. (b) Fluorescence intensity quantitation was analyzed by the Image J. Results were presented as means ± SE with replicates n = 3. *, p b 0.05; **, p b 0.01, ***, p b 0.001.
after H+ addition (Fig. S4b). Meanwhile, the addition of CF3COOD resulted in the down-field shift of protons in methylene group, which connected to the nitrogen atom of morpholine. The changes confirmed that H+ could induce the protonation of nitrogen atom of morpholine moiety. And the results were in perfect agreement with our previous paper [43]. Notably, after NaOH was added (Fig. S4c), the 1H NMR spectra were almost the same as NRLH alone (Fig. S4a). The data indicated that NRLH possessed excellent reversibility, which was essential for pH detection in real samples. For a fluorescent probe, the anti-interference ability is a key parameter. Then the effect of other biological components (anions, metal cations and amino acids) on the pH detection was studied. As depicted in Fig. 3, the change of the emission intensity (I705) was almost negligible upon addition of diverse analytes at pH 3.00. Similarly, the experiment was also conducted at pH 7.40. The results showed that NRLH displayed high selectivity to pH and the fluorescence was basically unperturbed by the above interfering species. To further study practical applications of NRLH, we proceeded to investigate its reversibility between pH 3.00 and 7.40 (Fig. S5, ESI†). The data manifested that the probe possessed desirable reversibility. In
addition, the time courses of fluorescence intensity (I705) of NRLH at pH 3.00 and 7.40 (Fig. 4) were examined. Notably, the probe could rapidly respond to pH, which was beneficial for its biological application. 3.3. Fluorescence imaging of probe NRLH The spectroscopic characteristics of NRLH, including short response time, outstanding anti-interference ability, specific response to H+ and good reversibility, proved that NRLH could serve as a useful tool for pH detection in vitro. Nevertheless, some endogenous substances that may cause auto fluorescence background often exist in the complicated biological specimens, thus making common chemosensors ineffective in cellular fluorescence imaging [45]. To our delight, probe NRLH was a near-infrared probe. So it was facile to differentiate the probe signals from background fluorescence (around the range of 400–500 nm). To verify this, bafilomycin A1 (a selective inhibitor of the vacuolartype H+-ATPase) was employed for cellular imaging. Bafilomycin A1 was able to inhibit lysosomal acidification to enhance the pH. Firstly, HeLa cells were incubated with various dose of bafilomycin A1 for 6 h. Subsequently, the cells were incubated with 1 μM NRLH for an hour.
Fig. 6. Co-localization images of HeLa cells stained with NRLH and LysoSensor Green DND-189. Cells were incubated with NRLH (1 μM) for 30 min, and then the medium was replaced with fresh medium containing LysoSensor Green DND-189 (0.5 μM) and further incubated for 30 min. (a) Confocal images from NRLH (red emission); (b) confocal images from LysoSensor Green DND-189 (green emission); (c) overlay of (a) and (b); (d) bright-field images; (e) an intensity scatter plot of red and green channels. Excitation wavelength of NRLH and LysoSensor Green DND-189 was 650 nm and 488 nm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: X.-F. Zhang, T.-R. Wang, X.-Q. Cao, et al., A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during h..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117761
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Fig. 7. Relationship between fluorescence intensity and heat shock in HeLa cells monitored by NRLH. (a) Fluorescence images of NRLH-loaded cells under heat shock at 37 °C, 41 °C and 45 °C for 20 min. (b) Results were expressed as the mean of three separate measurements ± SE. Fluorescence intensity statistical analyses were conducted using Image J. Scale bars = 20 μm. *, p b 0.05; **, p b 0.01, ***, p b 0.001.
As described in Fig. 5a, increasing dose of bafilomycin A1 led to significant fluorescence intensity decrease, and phenomena were apparent in intensity quantitation experiment (Fig. 5b). On this basis, we proposed that NRLH was able to sensitively monitor cellular pH changes. Considering morpholine was a lysosome-targeting functional group, it was anticipated that NRLH could target lysosomes in cells. We subsequently performed the co-localization assays with a commercial lysosome tracker (LysoSensor Green DND-189). Fig. 6 indicated that NRLH had significant overlap with LysoSensor Green DND-189 and the corresponding co-localization coefficient was 0.96. Hence, the probe could selectively stain lysosomes due to the presence of morpholine moiety. To further explore relationship between lysosomal pH and temperature during heat shock, we carried out the experiment to monitor dynamic changes of fluorescence intensity during heat shock. HeLa cells were first incubated with NRLH (1 μM) for 5 min, and then washed three times with PBS (pH 7.4). Fluorescence imaging assays were conducted at 37 °C (control), 41 °C and 45 °C, respectively (Fig. 7). It could be seen that the control group (37 °C) showed brighter fluorescence in comparison to the other two groups undergoing heat treatment (41 °C and 45 °C). The corresponding fluorescence intensity obviously declined during the heat shock (Fig. 7b). It may be speculated that heat treatment may result in the rise of lysosomal pH, which was consistent with the previous literature [24]. Finally, toxicity of probe NRLH on living cells was evaluated via SRB assays (Fig. S6, ESI†). After HeLa cells were treated with NRLH (1, 2, 5 μM) for 12 h, it was apparently noticed that the probe caused negligible cytotoxicity to cells. The experiments demonstrated its low cytotoxicity and excellent biocompatibility.
4. Conclusions In summary, we reported a rhodamine-based near-infrared fluorescent chemosensor for lysosomal pH. The probe displayed dramatic fluorescence enhancement from pH 7.40 to 2.00 owing to the ring-opening of spirolactam induced by the interaction between the probe and H+. Moreover, the probe displayed fast response, prominent reversibility as well as high sensitivity and selectivity. Meanwhile, the sensor was able to target lysosomes on account of morpholine (a typical lysosome-targetable moiety). Notably, we applied it to explore the effect of temperature on lysosomal acidity in heat shock and it was speculated that heat treatment may cause the increase of lysosomal pH. Hence, it was anticipated that the chemosensor would be beneficial for our further research about pH-related biology and pathology, especially about the heat-related diseases.
Declaration of competing 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. Acknowledgments This study was supported by the National Natural Science Foundation of China (21807078), Medical Science and Technology Development Plan Project of Shandong Province (2015WS0104), Natural Science Foundation of Shandong Province (ZR2016BB35, ZR2018LB014). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117761.
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A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during heat shock Xiao-Fan Zhang b,1, Tian-Ran Wang a,1, Xiao-Qun Cao a, Shi-Li Shen a,⁎ a b
School of Chemistry and Pharmaceutical Engineering, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271016, PR China Taian Center For Food and Drug Control, Taian 271000, PR China
a r t i c l e
i n f o
Article history: Received 14 October 2019 Received in revised form 2 November 2019 Accepted 3 November 2019 Available online xxxx Keywords: Fluorescent probe Rhodamine derivative pH Near-infrared Lysosomes Heat shock
a b s t r a c t Heat shock is a potentially fatal condition characterized by high body temperature (N40 °C), which may lead to physical discomfort and dysfunctions of organ systems. Acidic pH environment in lysosomes can activate enzymes, thus facilitating the degradation of proteins in cellular metabolism. Owing to the lack of a practical research tool, it remains difficult to exploit relationship between heat shock and lysosome. Herein, a NIR lysosomal pH chemosensor (NRLH) was developed. One typical lysosome-locating group, morpholine, was incorporated into NRLH. The fluorescence intensity showed pH-dependent characteristics and responded sensitively to pH fluctuations in the pH range of 3.0–5.5. NRLH with a pKa of 4.24 displayed rapid response and high selectivity for H+ among common species. We also demonstrated NRLH was capable of targeting lysosomes. Importantly, NRLH was applied in cellular imaging and the data revealed that lysosomal pH increased but never decreased during the heat shock. Therefore, NRLH may act as an effective molecular tool for exploring the mechanisms of heat-related pathology in bio-systems. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Many cellular processes are regulated by pH values, including cell proliferation, apoptosis and endocytosis [1–3]. In eukaryotic cells, the acidic pH in lysosomes (pH 4.5–5.5) is beneficial for the activation of numerous enzymes in lysosomes [4,5]. The abnormal pH values are implicated with cellular dysfunctions, which can result in many diseases, such as cancer and Alzheimer's disease [6–8]. Heat shock, a kind of heat-related disorders, is identified as a prevalent health risk and may cause mortality [9]. Despite this, the mechanisms responsible for the direct cytotoxicity of heat are rarely explored. Recent investigations indicated that there was a certain correlation between heat shock and lysosome acidity. However, the relationship between the temperature and lysosomal pH is still unclear [10]. In the aim of studying the mechanism of heat cytotoxicity, it is fairly essential to detect lysosomal pH in heat shock process. Among various analysis methods for pH and biological species in vivo, fluorescent probe has become an indispensable tool due to the brilliant selectivity and sensitivity, nondestructive use in cells and easy ⁎ Corresponding author. E-mail address: [email protected] (S.-L. Shen). 1 These authors are equally contributed to this work.
test [11–21]. In particular, near-infrared (NIR) probes emitting in the range of 650–900 nm are especially preferred since NIR light can avoid the interference of autofluorescence and minimize photodamage to biological specimens [22–26]. So it is necessary to design nearinfrared pH sensors that possess lysosome-targeting capability. Rhodamine dyes have attracted considerable attention in biomolecular detection and intracellular imaging by virtue of the excellent biocompatibility and photophysical characteristics, including long absorption and emission wavelength as well as high fluorescence quantum yield [27,28]. Significantly, the non-fluorescent spirocyclic structure of rhodamine-based sensors could be regulated by H+-induced ring-opening process, thus resulting in dramatic fluorescence increase [29–34]. Although many lysosomal pH sensors on the basis of rhodamine framework were reported, their short absorption and emission may inevitably restrict the application in living cells [35–38]. Herein, we develop a lysosome-targeting NIR pH chemosensor (NRLH) by integrating one lysosomal locating group, morpholine [39–41], into the rhodamine framework. Compared with our previously reported pH sensors [42,43], NRLH shows longer emission wavelength (λem = 705 nm), and this would extend its biological application. And it is indicated that NRLH could target lysosome as well as monitor lysosomal pH changes. Besides, the fluorescence imaging experiments reveal that lysosomal pH increases along with the temperature rise.
https://doi.org/10.1016/j.saa.2019.117761 1386-1425/© 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Preparation of NRLH.
(TLC) on gel F254 plates. Flash chromatography was carried out on silica gel (200–300 mesh; Qingdao Ocean Chemicals). 2.2. Materials Unless specifically indicated, all reagents and solvents (analytical grade) were purchased from commercial suppliers and used without further purification. Britton-Robinson (B-R) buffer was obtained by mixing boric acid (40 mM), phosphoric acid (40 mM) and acetic acid (40 mM) in deionized water. 2.3. Synthesis of probe NRLH
Fig. 1. Absorption spectra changes of 10 μM NRLH in the buffer solution (5% EtOH) upon the addition of H+.
2. Experimental 2.1. Instrumentation NMR spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. Tetramethylsilane (TMS) was used as internal standard. A Q-TOF6510 spectrograph (Agilent) was applied to record HRMS spectra. The Hitachi U-3900 spectrometer was employed to record UV–vis spectra. A Hitachi F-4500 fluorescence spectrophotometer was used for fluorescence measurements. The PHSJ-3F digital pH meter (LeiCi, Shanghai, China) was used to detect pH. Reactions were monitored by thin-layer chromatography
Synthesis of NRLH was outlined in Scheme 1. Compound 1 was obtained according to previous paper [44]. Under N2 atmosphere, compound 1 (1.0 mmol), benzotriazol-1-yloxy)tris (dimethylamino)phosphonium hexafluorophosphate (BOP, 1.0 mmol) and 4-(2-aminoethyl)morpholine (3.0 mmol) were successively added to 50 mL ethanol. The solution was heated to reflux for 12 h, cooled to room temperature, and evaporated under reduced pressure. The resulting precipitate was purified by column chromatography (MeOH/CH2Cl2 = 1/50, v/v) to afford NRLH as a faint blue solid in 49% yield. 1H NMR (DMSO-d6, 400 MHz), δ: 1.09 (t, 6H, J = 7.2 Hz), 1.22 (s, 1H), 1.35–1.44 (m, 1H), 1.53–1.70 (m, 2H), 2.23–2.86 (m, 6H), 2.53–2.66 (m, 1H), 2.73–2.82 (m, 1H), 3.12–3.28 (m, 2H), 3.35 (br, 4H), 3.45 (br, 4H), 3.85 (s, 3H), 6.22 (d, 1H, J = 8.8 Hz), 6.36 (d, 1H, J = 8.8 Hz), 6.60 (d, 1H, J = 2.4 Hz), 7.18 (q, 2H, J = 6.8 Hz), 7.25 (t, 1H, J = 7.2 Hz), 7.48 (d, 1H, J = 8.0 Hz), 7.52 (d, 2H, J = 6.4 Hz), 7.57 (d, 1H, J = 6.8 Hz), 7.60 (s, 1H), 7.76 (d, 1H, J = 7.2 Hz), 7.91 (d, 1H, J = 7.6 Hz); 13C NMR (DMSO-d6, 100 MHz), δ: 167.0, 152.2, 150.9, 148.3, 146.9, 136.1, 132.5, 131.4, 129.3, 128.5, 128.2, 127.8, 124.7, 123.3, 122.2, 122.0, 119.4, 118.8, 114.6, 111.0, 108.4, 105.8, 104.4, 97.4,
Fig. 2. (a) Fluorescence spectra of NRLH (1 μM) in the Britton-Robinson buffer (5% ethanol as co-solvent) at various pH. (b) The fluorescence intensity at 705 nm (I705) as a function of pH according to the titration under the excitation at 650 nm.
Please cite this article as: X.-F. Zhang, T.-R. Wang, X.-Q. Cao, et al., A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during h..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117761
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Scheme 2. Chemical structure of NRLH and its response mechanism to H+.
66.0, 65.7, 56.0, 54.9, 53.2, 43.5, 36.4, 32.7, 27.9, 22.3, 21.7, 12.4; HRMS calcd for C40H45N4O3 [M + H]+: 629.3492, found: 629.3491.
the facile synthetic method (Scheme 1). The final structure of NRLH was fully characterized by NMR (1H NMR, 13C NMR) and HRMS (Figs. S1–S3, ESI†).
2.4. Sample preparation 3.2. Capability of NRLH to respond to H+ in buffer solution The stock solution of probe NRLH was prepared by dissolving the probe compound in ethanol. All optical spectra of NRLH were measured in B-R buffer (5% ethanol was needed as a co-solvent). A standard 1 cm quartz cuvette was utilized for detection. 2.5. Cell culture HeLa cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (HyClone, USA) in a humidified atmosphere of 5/ 95 (v/v) of CO2/air at 37 °C. For confocal laser scanning microscopy imaging (Leica SP8), the medium was replaced with serum-free DMEM. Subsequently, cells were treated with NRLH at 37 °C. 3. Results and discussion 3.1. Design and preparation of NRLH Design of NRLH was based on the excellent photophysical properties of rhodamine fluorophore and the structural change between spirocyclic (non-fluorescent) and ring-opened (fluorescent) form responding to pH. Furthermore, morpholine, which was identified as one typical lysosomal locating moiety, was integrated into NRLH to realize the lysosome-targeting capability. Probe NRLH was obtained by
Fig. 3. Fluorescence intensity (I705) of NRLH (1 μM) in presence of various analytes in the Britton-Robinson buffer (5% EtOH as co-solvent) at pH 3.00 and 7.40 under the excitation at 650 nm. (1) blank, (2) Glu, (3) HS−, (4) Cys, (5) Hcy, (6) K+, (7) Zn2+, (8) Fe2+, (9) Ag+, (10) Ca2+, (11) Mg2+, (12) Co2+, (13) Na+, (14) Ni2+, (15) Cu2+, (16) Cd2+, − 2− (17) Cr3+, (18) Mn2+, (19) AcO−, (20) H2PO− 4 , (21) NO3 , (22) SO4 . Concentration: 1 mM for (6), (10), (11) and (13); 50 μM for others.
UV–vis absorption and fluorescence emission spectroscopy of NRLH was investigated in B-R buffer (5% (v/v) ethanol was needed as the cosolvent) at different pH. As depicted in Fig. 1, NRLH exhibited almost no absorption bands at 500–750 nm when pH was 7.40. As the pH decreased from 7.40 to 2.00, the absorption peak of rhodamine fluorophore at 650 nm enhanced greatly. The phenomena were attributed to the structural transformation of the rhodamine group in response to pH. The addition of H+ induced the ring-opening process of rhodamine in NRLH. As displayed in Fig. 2a, probe NRLH showed very weak fluorescence in weakly basic aqueous solution upon excitation at 650 nm. When pH varied from 7.40 to 2.00, the emission maxima of NRLH (I705) increased, in agreement with the absorption spectra changes. In basic or neutral pH conditions, rhodamine fluorophore was in the spirocyclic form, which was non-fluorescent. The spirocyclic form would convert to the fluorescent ring-opened form due to the interaction between H+ and NRLH, as outlined in Scheme 2. Most notably, the pKa was determined to be 4.24, demonstrating that NRLH may be used for the pH detection in lysosomes (Fig. 2b). To elucidate the above proposed mechanism of NRLH, 1H NMR titration was conducted in DMSO-d6 with addition of CF3COOD and then NaOH. We could see from Fig. S4 (ESI†) that compared with NRLH alone (Fig. S4a), the aromatic proton signals were down-field shifted, which was a result of ring-opening process of rhodamine framework
Fig. 4. The time course of I705 of NRLH in Britton-Robinson buffer (5% EtOH as co-solvent) at pH 3.00 and 7.40, respectively. λex = 650 nm.
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Fig. 5. (a) Confocal images of cells treated with different dose of bafilomycin A1 (0–100 nM) for 6 h, and then with NRLH (1 μM) for 1 h. (b) Fluorescence intensity quantitation was analyzed by the Image J. Results were presented as means ± SE with replicates n = 3. *, p b 0.05; **, p b 0.01, ***, p b 0.001.
after H+ addition (Fig. S4b). Meanwhile, the addition of CF3COOD resulted in the down-field shift of protons in methylene group, which connected to the nitrogen atom of morpholine. The changes confirmed that H+ could induce the protonation of nitrogen atom of morpholine moiety. And the results were in perfect agreement with our previous paper [43]. Notably, after NaOH was added (Fig. S4c), the 1H NMR spectra were almost the same as NRLH alone (Fig. S4a). The data indicated that NRLH possessed excellent reversibility, which was essential for pH detection in real samples. For a fluorescent probe, the anti-interference ability is a key parameter. Then the effect of other biological components (anions, metal cations and amino acids) on the pH detection was studied. As depicted in Fig. 3, the change of the emission intensity (I705) was almost negligible upon addition of diverse analytes at pH 3.00. Similarly, the experiment was also conducted at pH 7.40. The results showed that NRLH displayed high selectivity to pH and the fluorescence was basically unperturbed by the above interfering species. To further study practical applications of NRLH, we proceeded to investigate its reversibility between pH 3.00 and 7.40 (Fig. S5, ESI†). The data manifested that the probe possessed desirable reversibility. In
addition, the time courses of fluorescence intensity (I705) of NRLH at pH 3.00 and 7.40 (Fig. 4) were examined. Notably, the probe could rapidly respond to pH, which was beneficial for its biological application. 3.3. Fluorescence imaging of probe NRLH The spectroscopic characteristics of NRLH, including short response time, outstanding anti-interference ability, specific response to H+ and good reversibility, proved that NRLH could serve as a useful tool for pH detection in vitro. Nevertheless, some endogenous substances that may cause auto fluorescence background often exist in the complicated biological specimens, thus making common chemosensors ineffective in cellular fluorescence imaging [45]. To our delight, probe NRLH was a near-infrared probe. So it was facile to differentiate the probe signals from background fluorescence (around the range of 400–500 nm). To verify this, bafilomycin A1 (a selective inhibitor of the vacuolartype H+-ATPase) was employed for cellular imaging. Bafilomycin A1 was able to inhibit lysosomal acidification to enhance the pH. Firstly, HeLa cells were incubated with various dose of bafilomycin A1 for 6 h. Subsequently, the cells were incubated with 1 μM NRLH for an hour.
Fig. 6. Co-localization images of HeLa cells stained with NRLH and LysoSensor Green DND-189. Cells were incubated with NRLH (1 μM) for 30 min, and then the medium was replaced with fresh medium containing LysoSensor Green DND-189 (0.5 μM) and further incubated for 30 min. (a) Confocal images from NRLH (red emission); (b) confocal images from LysoSensor Green DND-189 (green emission); (c) overlay of (a) and (b); (d) bright-field images; (e) an intensity scatter plot of red and green channels. Excitation wavelength of NRLH and LysoSensor Green DND-189 was 650 nm and 488 nm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. Relationship between fluorescence intensity and heat shock in HeLa cells monitored by NRLH. (a) Fluorescence images of NRLH-loaded cells under heat shock at 37 °C, 41 °C and 45 °C for 20 min. (b) Results were expressed as the mean of three separate measurements ± SE. Fluorescence intensity statistical analyses were conducted using Image J. Scale bars = 20 μm. *, p b 0.05; **, p b 0.01, ***, p b 0.001.
As described in Fig. 5a, increasing dose of bafilomycin A1 led to significant fluorescence intensity decrease, and phenomena were apparent in intensity quantitation experiment (Fig. 5b). On this basis, we proposed that NRLH was able to sensitively monitor cellular pH changes. Considering morpholine was a lysosome-targeting functional group, it was anticipated that NRLH could target lysosomes in cells. We subsequently performed the co-localization assays with a commercial lysosome tracker (LysoSensor Green DND-189). Fig. 6 indicated that NRLH had significant overlap with LysoSensor Green DND-189 and the corresponding co-localization coefficient was 0.96. Hence, the probe could selectively stain lysosomes due to the presence of morpholine moiety. To further explore relationship between lysosomal pH and temperature during heat shock, we carried out the experiment to monitor dynamic changes of fluorescence intensity during heat shock. HeLa cells were first incubated with NRLH (1 μM) for 5 min, and then washed three times with PBS (pH 7.4). Fluorescence imaging assays were conducted at 37 °C (control), 41 °C and 45 °C, respectively (Fig. 7). It could be seen that the control group (37 °C) showed brighter fluorescence in comparison to the other two groups undergoing heat treatment (41 °C and 45 °C). The corresponding fluorescence intensity obviously declined during the heat shock (Fig. 7b). It may be speculated that heat treatment may result in the rise of lysosomal pH, which was consistent with the previous literature [24]. Finally, toxicity of probe NRLH on living cells was evaluated via SRB assays (Fig. S6, ESI†). After HeLa cells were treated with NRLH (1, 2, 5 μM) for 12 h, it was apparently noticed that the probe caused negligible cytotoxicity to cells. The experiments demonstrated its low cytotoxicity and excellent biocompatibility.
4. Conclusions In summary, we reported a rhodamine-based near-infrared fluorescent chemosensor for lysosomal pH. The probe displayed dramatic fluorescence enhancement from pH 7.40 to 2.00 owing to the ring-opening of spirolactam induced by the interaction between the probe and H+. Moreover, the probe displayed fast response, prominent reversibility as well as high sensitivity and selectivity. Meanwhile, the sensor was able to target lysosomes on account of morpholine (a typical lysosome-targetable moiety). Notably, we applied it to explore the effect of temperature on lysosomal acidity in heat shock and it was speculated that heat treatment may cause the increase of lysosomal pH. Hence, it was anticipated that the chemosensor would be beneficial for our further research about pH-related biology and pathology, especially about the heat-related diseases.
Declaration of competing 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. Acknowledgments This study was supported by the National Natural Science Foundation of China (21807078), Medical Science and Technology Development Plan Project of Shandong Province (2015WS0104), Natural Science Foundation of Shandong Province (ZR2016BB35, ZR2018LB014). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117761.
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Please cite this article as: X.-F. Zhang, T.-R. Wang, X.-Q. Cao, et al., A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during h..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117761