Ratiometric Fluorescence Nanoprobes for Subcellular pH Imaging

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Ratiometric Fluorescence Nanoprobes for Subcellular pH Imaging with a Single-Wavelength Excitation in Living Cells Wei Pan, Honghong Wang, Limin Yang, Zhengze Yu, Na Li, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01010 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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Analytical Chemistry

Ratiometric Fluorescence Nanoprobes for Subcellular pH Imaging with a Single-Wavelength Excitation in Living Cells Wei Pan, Honghong Wang, Limin Yang, Zhengze Yu, Na Li,* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China. ABSTRACT: Abnormal pH values in the organelles are closely associated with inappropriate cellular functions and many diseases. Monitoring subcellular pH values and their variations is significant in biological processes occurring in living cells and tissues. Herein, we develop a series of ratiometric fluorescence nanoprobes for quantification and imaging of pH values with a single-wavelength excitation in cytoplasm, lysosomes, and mitochondria. The nanoprobes consist of mesoporous silica nanoparticles assembled with aminofluorescein as recognition unit for pH measurement and ethidium bromide as reference fluorophore. Further conjugation of subcellular targeting moiety enable the nanoprobes to specifically target lysosome and mitochondria. Confocal fluorescence imaging demonstrated that the nanoprobes could effectively monitor the pH fluctuations from 5.0 to 8.3 in living cells by ratio imaging with 488 nm excitation. Subcellular pH determination and imaging in lysosome and mitochondria could also be achieved in different conditions. The current method can offer a general strategy to determine subcellular analytes and investigate the interactions in biological samples.

Intracellular pH balance plays many vital roles in various biological processes, including cell proliferation, apoptosis,1-4 multidrug resistance,5 phagocytosis,6 and endocytosis.7,8 Anomalous pH values in cells and tissues are associated with the cellular dysfunctions, which are correlated with many severe diseases such as cancer9-11 and Alzheimer’s disease.12 Moreover, intracellular pH distribution is heterogeneous in different subcellular compartments and the unique functions of organelles determine the pH values of each separate compartment. For instance, lysosomes and endosomes are acidic ranging from 4.7 to 6.5,1315 the cytoplasm and nucleus have a neutral pH of 7.27.4,16-18 and mitochondria display a slightly basic pH about 8.0.19-21 To better understand the roles of pH in physiology and pathology, it is of great significance to exploit effective approaches for monitoring subcellular pH values and their fluctuations in living cells. Fluorescence imaging has served as an indispensable tool for the investigation of intracellular pH due to the ability in offering spatial-temporal information.22 A number of fluorescent probes have been explored for determining intracellular pH values.22-25 Since the fluorescence intensity could be influenced by the microenvironment, localized probe distribution and imaging parameters,26 the slight pH fluctuations could be ignored when using turn-on fluorescent probes. Ratiometric self-calibration using dual emission bands is more favorable in decreasing the artefacts caused by the above factors, which permits

more accurate quantification and determination of intracellular pH values. Recently, many efforts have been made to develop ratiometric fluorescent probes for intracellular pH measurements.27-29 However, most of these ratiometric probes only report the pH values in cytoplasm or a single organelle in cells. Moreover, the singlewavelength excitation mode could avoid complicated operation, the interference of autofluorescence from the cells and the background noise from multiple excitations, Scheme 1. Schematic illustration of the ratiometric fluorescence nanoprobes for imaging of subcellular pH.

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which is beneficial for biological applications. Therefore, the development of a general strategy for monitoring subcellular pH variations in different organelles with a singlewavelength excitation is urgently needed. Nanoparticle-based ratiometric pH sensors have attracted more and more attention owing to their remarkable advantages,30-34 the most important of these being that it is easy to simultaneously assemble diverse dyes on the same nanoparticle to acquire ratiometric fluorescent sensors with tunable pH response range. Based on this, we assemble two dyes into one nanoparticle to construct a variety of ratiometric fluorescent nanoprobes. After further conjugation of the targeting moiety, quantitatively detecting and imaging of the subcellular pH fluctuations in different organelles using a single-wavelength excitation could be achieved in living cells. Mesoporous silica nanoparticles (MSN) were selected as the carrier due to their good biocompatibility, and easy functionalization.3538 The carboxyl-coated mesoporous silica nanoparticles were conjugated with aminofluorescein for pH measurements and ethidium bromide for reference. Fluorescein has been widely used to determine intracellular pH values owing to the good biocompatibility and high sensitivity under physiological conditions. The ethidium bromide was served as the reference to form a ratiometric fluorescent sensor for pH detection with good stability and high selectivity. Further conjugation of the triphenylphosphonium (TPP) or 3-(4-morpholinyl) propanoic acid hydrochloride (MPP) moiety on the surface of aminofunctionalized silica by amide bonds endows the nanoprobes with specific mitochondria or lysosome targeting. The designed nanoprobes are capable of accurately monitoring the changes of pH in cytoplasm, lysosomes, and mitochondria of living cells by ratio imaging. The details of the strategy are shown in Scheme 1 and Scheme 2. Scheme 2. Schematic diagram of the procedures for preparing the nanoprobes.

RESULTS AND DISCUSSION Design and Synthesis of the Ratiometric Fluorescence Nanoprobes. For the construction of a ratiometric sensing device, the selection of dyes is important, which should be based on their spectroscopic properties. We chose two fluorophores aminofluorescein (AF) and ethidium bromide (EB). The organic molecule AF plays as the response group for recognition of pH because the fluorescence intensity of AF could increase with the increase

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Figure 1. TEM image of MSNs (a) and nanoprobes (b). Scale bars are 100 nm.

of pH value (Figure S1). While EB is chosen as the reference signal element based on its good stability in solutions with different pH values. Importantly, the two dyes possess a single excitation wavelength at 488 nm and different emissions at 515nm and 595 nm (Figure S2). This dual-emission ratiometric probe upon one excitation provided an internal reference for correcting the environmental errors, thereby improving the detection accuracy. Mesoporous silica nanoparticles (MSNs) were first synthesized as a start point of the study.39 Then the surface of the MSNs was functionalized by carboxyl. The pHsensitive dye AF and pH-insensitive dye EB were modified onto the MSNs by the formation of the amido bond (MSNs-EB-AF). In order to prevent the dyes from leaking and further modify organelles targeted group on the nanoprobes, the MSNs-EB-AF was coated by aminofunctionalized mesoporous silica (MSNs-EB-AF@SiO2NH2). Finally, the organelles targeted groups (4carboxybutyl) triphenylphosphonium bromide (TPP) targeting mitochondria or 3-(4-morpholinyl) propanoic acid hydrochloride (MPP) targeting lysosome were fabricated through the amidation reaction. The TEM image in Figure 1a showed that MSNs were well monodispersed in solution with an average diameter about 30nm and the morphology of the nanoprobes (MSNs-EB-AF@SiO2-NH2TPP) did not show obvious change compared with MSNs (Figure 1b). Zeta potential experiments were then employed to confirm the successful synthesis of the nanoprobes at different stages (Figure S3). The zeta potential of the as-prepared MSNs was −17.5 ± 0.3 mV. After carboxyl functionalization, the zeta potential of MSNsCOOH was changed to −30.5 ± 0.23 mV. When AF and EB were modified, the zeta potential was +2.04 ±0.24 mV. While the zeta potential of MSNs-EB-AF@SiO2-NH2 was changed to +15.9 ± 0.55 mV. With further modification of TPP and MPP (MSNs-EB-AF@SiO2-TPP, MSNs-EBAF@SiO2-MPP), the zeta potentials were +17.6 ± 0.64 and +15.6 ± 0.96 mV, respectively. The results confirmed that the nanoprobes were successfully assembled at different stages. The amounts of connected AF and EB in the nanoprobes were calculated to be 0.72 mg/g and 5.9 mg/g, respectively (Figure S4, Figure S5). Determination of pH Response of the Nanoprobes. The fluorescence response of the nanoprobes for different pH values are shown in Figure 2a. With the increase of pH values, the emission intensity at 515 nm for AF gradually enhanced, while the emission at 595 nm for EB changed


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Figure 2. (a) Fluorescence emission spectra of the nanoprobes with various pH values from 5.6 to 8.3 (λex = 488 nm, 10 mM PBS). (b) The linear relationship between R and pH values in the range pH 5.6–8.3. R is the ratio (I515/I595) of the fluorescence intensity of nanoprobes at 515 nm and 595 nm; λex = 488 nm.

slightly under a single excitation wavelength at 488 nm. As plotted in Figure 2b, the fluorescence intensity ratio R of aminofluorescein at 515 nm and ethidium bromide at 595 nm increased with the increase in pH. It showed good linearity with the pH in the range from 5.6 to 8.3, which covers most of the physiological pH ranges. Next, the reversibility of the nanoprobes was evaluated. The results showed that the ﬂuorescence could reversible for the alteration of pH 4 and pH 9, suggesting that the nanoprobes possess an excellent reversibility between pH 4 and pH 9 (Figure S6). Selectivity. The selectivity of the nanoprobes was evaluated using fluorescence spectroscopic analysis before it was applied in living cells. To assess the selectivity of the nanoprobes toward to H+, the response of the ratiometric fluorescent nanoprobes to different biological species was investigated in PBS buffer (pH 7.4). Metal ions such as Na+, K+, Mg2+, Cu2+, Co2+, and so on, as well as oxidativestress-associated redox chemicals, including glutathione (GSH) and H2O2, and amino acids that may coexist in the living cells were examined under the same conditions. The results showed that no noticeable fluorescence change was observed in the presence of these substances, which was of comparable magnitude to background fluorescence (Figure S7), demonstrating that the nanoprobes responded specifically toward pH over other interfering agents. Cytotoxicity. The cytotoxicity of the nanoprobes was evaluated by an MTT assay in human breast cancer cell line (MCF-7). The activation degree of the cells was responsible for the absorbance of MTT at 490 nm. The cell viability could be determined by the comparison of the MTT absorbance for the nanoprobes treated cells and the control cells. Figure S8 showed that the viability of MCF-7 cells were more than 85% after incubation for different time (6h, 12h and 24 h), with the high concentration of nanoprobes (2.0 mg/mL), which is 10 times higher than that used in the following cell experiments. The results revealed that the nanoprobes were low toxicity for live cells detection. Co-staining Imaging in Living Cells. Next, the targeting performance of the nanoprobes to different organelle were evaluated by colocalization imaging experiments. Mito-Tracker Green (a commercially available mitochondria-targeting dye, MTG) and Lyso-Tracker DND-26 (a

Figure 3. Mitochondria-targeting properties. Confocal fluorescence image of MCF-7 cells treated with MSNs-EB@SiO2TPP (A), MSNs-EB@SiO2-NH2 (B), and MSNs-EB-AF@SiO2TPP (C). (A and B) Green channel for MTG and the red channel for EB, respectively. (C) Green channel for AF and the red channel for EB. (c) Overlay image of (a) and (b). (d) The colocalization areas of the red and green channels selected. (e) Fluorescence intensity profile of regions of interest (white line in c) across the lines. Green channels and red channels are excited at 488 nm.

commercially available lysosome-targeting dye, LTD) were employed to co-stain with the nanoprobes in MCF-7 cells. The excitation and emission spectra of MTG, LTD and aminofluorescein are overlapped. To avoid the interference from aminofluorescein, only EB was covalently conjugated onto the carboxyl-functionalized MSNs in the following co-staining experiments. MSN-EB@SiO2-TPP and MSN-EB@SiO2-MPP nanocomposites were prepared by modification of TPP and MPP on the surface of MSNEB@SiO2-NH2. The MSN-EB@SiO2-NH2 nanocomposite without TPP or MPP was also synthesized, which will stay in the cytoplasm. After MCF-7 cells incubated with MSNEB@SiO2-TPP or MSN-EB@SiO2-NH2, the cells were additionally stained with MTG to label the mitochondria. As shown in Figure 3, a well overlapped imaging (Pearson’s correlation coefficient, ρ = 0.83) was obtained for MSNEB@SiO2-TPP and MTG as evidenced by the clear yellow signals. It confirmed that the superior mitochondriatargeting ability of MSN-EB@SiO2-TPP. In contrast, a poor overlap between the fluorescence of the MSNEB@SiO2-NH2 and MTG was found (ρ = 0.39) under the same conditions. To evaluate the lysosome-targeting performance of MSN-EB@SiO2-MPP, MCF-7 cells were costained with MSN-EB@SiO2-MPP or MSN-EB@SiO2-NH2. Then, the cells were additionally stained with LTD to label lysosome. Figure 4 showed that both MSN-EB@SiO2MPP and LTD display strong localized fluorescence within lysosomes. The fluorescence images of MSN-EB@SiO2MPP and LTD can be merged rather well (Pearson’s correlation coefficient, ρ = 0.76), confirming that the MSNEB@SiO2-MPP could specifically localize in lysosome of living cells. However, the MSN-EB@SiO2-NH2 randomly distributed inside the cells without specific lysosome localization (Pearson’s correlation coefficient, ρ = 0.37). Further experiments showed that the fluorescence of aminofluorescein and ethidium bromide in the nanoprobes was well over lapped, implying that the nano



Figure 4. Lysosome-targeting properties. Confocal fluorescence image of MCF-7 cells treated with MSNsEB@SiO2-MPP (A), MSNs-EB@SiO2-NH2 (B), and MSNs-EBAF@SiO2-MPP (C). (A and B) Green channel for LTD and the red channel for EB, respectively. (C) Green channel for AF and the red channel for EB. (c) Overlay image of (a) and (b). (d) The colocalization areas of the red and green channels selected. (e) Fluorescence intensity profile of regions of interest (white line in c) across the lines. Green channels and red channels are excitated at 488 nm.

probes possessed the ability to ratiometric detection pH at the same position of mitochondria or lysosome in living cells. The above observations are further verified by the line scanning profiles of quantifying fluorescence intensity (Figure 3e, Figure 4e). Intracellular Imaging of pH Fluctuations in Different Organelles. To demonstrate the applicability of the nanoprobes to quantifying intracellular pH in different organelles, the intracellular calibration experiment was first carried out in MCF-7 cells with high K+ buffers of different pH. Nigericin, an H+/K+ ionophorethe, was employed to homogenize the intracellular pH to the surrounding medium.29,40 As shown in Figure 5, the fluorescence intensity of the AF channel (green) gradually increased with the increase of pH values from 5.0 to 8.3, whereas that of the EB channel(red) remained hardly changed. Moreover, the fluorescence intensity ratio (R = I500-550/ I570-630) of the two channels showed a sensitive response to pH changes, which generated a good linear calibration curve in the pH range from 5.0 to 8.3 (Figure 6). Next, the three types of nanoprobes were used to investigate the pH difference in different organelles. The pH values of cytoplasm, lysosome and mitochondria in MCF7 cells were determined by confocal fluorescence imaging using MSNs-EB-AF@SiO2-NH2, MSNs-EB-AF@SiO2-MPP and MSNs-EB-AF@SiO2-TPP. Figure 7 showed that obvious ratio signal differences were obtained when the three nanoprobes targeting different organelles were incubated with the MCF-7 cells, suggesting that these three regions of MCF-7 cells had different pH environments. On the basis of intracellular calibration curve, the average intracellular pH value of cytoplasm, lysosome and mitochondria in MCF-7 cells is calculated to be 7.20 ± 0.22, 5.04 ± 0.11 and 7.91 ± 0.12, respectively. Next, the general applicability of the nanoprobes was investigated in another two types of cell lines, human cervical cancer cell line (HeLa) and human ovarian cancer cell line (SK-OV-3).

Figure 5. Confocal microscopy images of MSNs-EBAF@SiO2-NH2 nanoprobe-loaded MCF-7 cells clamped at pH 5.0, 5.6, 6.3, 7.0, 7.7, and 8.3, respectively. The concentration of nanoprobe is 0.2 mg/mL. The images of the first row (AF channel) and second row (EB channel) were collected in the ranges of 500–550 nm and 570–630 nm, respectively. The third row shows the merged image of green channel and red channel. The images of the fourth row is the pseudocolored ratiometric image.

The results showed that the ratio signal and pH values were also conformed to linear relationship in the two cell lines (Figure S9-S12). According to the intracellular calibration curve of pH, the average pH values of cytoplasm, lysosome and mitochondria are calculated to be 7.37 ± 0.20, 5.01 ± 0.14, 7.99 ± 0.09 for HeLa cells, and 7.39 ± 0.23, 5.02 ± 0.18, 8.01 ± 0.15 for SK-OV-3 cells, respectively (Figure S13 and Figure S14). To test whether the mitochondria targeted nanoprobes MSNs-EB-AF@SiO2-TPP could be used to identify mitochondrial pH changes associated with dysfunction and

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Figure 6. Intracellular pH calibration curve of MSNs-EBAF@SiO2-NH2 nanoprobe constructed by plotting Igreen/Ired vs pH. The dots are based on the average with the indicated standard error.


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cell death, a starvation model was employed. Nutrient deprivation impairs mitochondria function through metabolic inhibition.41-43 It leads to mitochondrial acidification, which in turn is correlated with an increase in mitophagy levels. MCF-7 cells were thus co-incubated with MSNs-EB-AF@SiO2-TPP in a serum-free medium. Fluorescence intensity was then acquired via confocal microscopy. As can be seen from Figure S15, the green fluorescence intensity of MSNs-EB-AF@SiO2-TPP decreased in the nutrient deprivation cells compared with the control cells. The average mitochondria pH of MCF-7 cells observed upon nutrient deprivation was determined to be 6.47 ± 0.26 based on the calibration curve shown in Figure 6 which was lower than that in the MCF-7 cells without nutrient deprivation. The observation of mitochondrial acidification under these conditions is fully consistent with previous reports in the literature,41 in other words, those cells subjected to nutrient deprivation possess mitochondria characterized by lower pH than those untreated. Therefore, MSNs-EB-AF@SiO2-TPP could detect the pH changes in mitochondria and monitor the mitophagy in living MCF-7 cells.

Figure 7. Confocal fluorescence image of MCF-7 cells treated with MSNs-EB-AF@SiO2-MPP (A), MSNs-EB-AF@SiO2-NH2 (B), and MSNs-EB-AF@SiO2-TPP (C).

We further investigated the application of MSNs-EBAF@SiO2-MPP for monitoring lysosomal pH changes in cultured cells. NH4Cl can alkalify the lysosomes to regulate the intracellular lysosomes H+ level. After incubation with MSNs-EB-AF@SiO2-MPP at 37 °C for 6 h, 10 mM NH4Cl was incubated with MCF-7 cells to regulate the intracellular H+ level. On the basis of the calibration curve, the average pH value of the lysosome treated with 10 mM NH4Cl in MCF-7 cells was determined to be 6.02 ± 0.15 (Figure S16). This result indicates that NH4Cl causes the lysosomes to become more basic in MCF-7 cells, which is consistent with previous reports in the literature.44 The experimentation implying that MSNs- EB-AF@SiO2-MPP is capable of monitoring lysosomal pH changes in living MCF-7 cells. Finally, H2O2 was applied to explore the relationship between the redox substance and intracellular pH fluctuations in MCF-7 cells. H2O2 could initiate the

Figure 8. Confocal fluorescent images of nanoprobes-loaded (MSNs-EB-AF@SiO2-MPP (A), MSNs-EB-AF@SiO2-NH2 (B), MSNs-EB-AF@SiO2-TPP (C)) MCF-7 cells treated with 100 μM H2O2.

redistribution of H+ from acidified organelles to cytosolic compartments by impairing the vacuolar proton pump (H+-ATPase), which imports H+ at the expense of ATP hydrolysis.44 It was reported that oxidation could induce alkalization of lysosomes, acidification of cytoplasm and mitochondria.44,45 In the following experiments, the cells were treated with 100 μM H2O2 for generation of oxidative stress. The average pH values of the different organelles in MCF-7 cells were determined to be 5.51± 0.26 for lysosome, 7.62 ± 0.29 for mitochondria and 7.09 ± 0.16 for cytoplasm (Figure 8) based on the calibration curve, while the pH values in untreated MCF-7 cells were calculated to be 5.04 ± 0.11 for lysosome, 7.91 ± 0.12 for mitochondria and 7.20 ± 0.22 for cytoplasm in the above experiments. The results indicated that H2O2 could induce the lysosome to be more basic and it could cause the mitochondria and cytoplasm to become more acid. The results demonstrated that the nanoprobes were capable of visualizing the pH fluctuations in different in living cells, implying that the nanoprobes could be appealing imaging tools to analyze the dynamic changes of pH in organelles.

CONCLUSIONS In conclusion, a variety of ratiometric fluorescence nanoprobes have been developed for subcellular pH imaging under a single-wavelength excitation in living cells. The nanoprobes compose of mesoporous silica nanoparticles incorporated with aminofluorescein and ethidium bromide. Mesoporous silica nanoparticles were selected as the carrier, aminofluorescein was served as pH response unit and ethidium bromide was used as reference fluorophore. MPP and TPP moiety were modified to realize the specific lysosome or mitochondria targeting. The nanoprobes could accurately monitor the changes of pH without interference. The fluorescence intensity ratio (R = I500-550/ I570-630) from the two channels generates a good linear calibration curve in the pH range from 5.0 to 8.3, which covers most of the physiological pH ranges in biological samples. Quantitative determination



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of pH in cytoplasm, lysosomes, and mitochondria in intact MCF-7 cells and the pH fluctuations associated with oxidative stress have been successfully achieved. Confocal fluorescence imaging experiments indicated that the nanoprobes are capable of reliably reporting and selectively discriminating the pH fluctuations in different organelles. The current nanoprobes give an obviously change over a wide pH range and could be an ideal diagnostic method for measuring pH fluctuations in living cells. We anticipate that the design strategy could provide new possibilities in detecting other analytes in organelles.

ASSOCIATED CONTENT Supporting Information Experimental details and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by 973 Program (2013CB933800) and National Natural Science Foundation of China (21390411, 21535004, 21227005, 21422505, 21375081, 21505087), and Natural Science Foundation of Shandong Province (JQ201503, ZR2015BQ003).

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Ratiometric Fluorescence Nanoprobes for Subcellular pH Imaging

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