Ratiometric Fluorescent Quantification of the Size-Dependent Cellular

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Ratiometric Fluorescent Quantification of the SizeDependent Cellular Toxicity of Silica Nanoparticles Xiuyan Wan, Xinhao Zhang, Wei Pan, Bo Liu, Longhai Yu, Honghong Wang, Na Li, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00633 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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Analytical Chemistry

Ratiometric Fluorescent Quantification of the Size-Dependent Cellular Toxicity of Silica Nanoparticles Xiuyan Wan,† Xinhao Zhang,† Wei Pan, Bo Liu, Longhai Yu, Honghong Wang, Na Li,* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China. ABSTRACT: Nanoscale particles are ubiquitous in the atmosphere, and the widespread use of nanoparticles may increase the risks of organ damage. Therefore, it is of great significance to investigate the toxicity of nanoparticles of different sizes toward living cells, especially lung epithelial cells. In this study, the quantitative ratiometric fluorescent detection of intracellular pH changes was utilized to evaluate the cytotoxicity of mesoporous silica nanoparticles of different sizes after the nanoparticles had entered lung epithelial cells. The results showed that with decreasing nanoparticle size, the intracellular reactive oxygen species (ROS) concentration increased, and the intracellular pH value decreased; consequently, this led to the enhanced cytotoxicity of the nanoparticles. Notably, no obvious cytotoxicity was induced by the nanoparticles when the size of the nanoparticles was larger than 135 nm. The presented strategy of using ratiometric fluorescent detection of intracellular pH to quantify the size-dependent cellular toxicity of nanoparticles provides a novel approach for investigating the cytotoxicity of nanomaterials.

The unique properties of nanoparticles make them increasingly attractive for commercial and medical applications.1-4 Unfortunately, the large-scale production and widespread utilization of nanoparticles have significantly increased the risk of human exposure to manufactured nanoparticles, which has raised concerns about nanoparticle organ toxicity.5-8 It has been reported that nanoparticles, including manufactured nanoparticles and natural airborne nanoparticles, threaten human health by causing organ damage.9-11 However, a quantitative relationship between the size and cytotoxicity of nanoparticles has not been precisely determined. In other words, the size at which nanoparticles cause negligible cytotoxicity still needs to be determined. Therefore, it is of profound guiding significance for the production and application of nanoparticles to develop a method for investigating the dependency between the size and the cytotoxicity of nanoparticles and quantifying the cytotoxicities of nanoparticles of different sizes. Since the cytotoxicity of nanoparticles has attracted increasing attentions in recent years, many studies on nanoparticleinduced cytotoxicity have been carried out.12-15 Most of these studies have concluded that reactive oxygen species (ROS)mediated oxidative stress is the key effect in the cytotoxicity of nanoparticles.16-19 Hence, many studies are based on detecting intracellular ROS to elucidate the mechanism for the cytotoxic effects of nanoparticles. However, the detection of ROS suffers from several insurmountable problems caused by the characteristics of ROS, including the species diversity and the short lifetimes of ROS.20-22 Therefore, the actual detection processes of ROS are often limited and have strict requirements for the probes.23-24 Previous studies have indicated that there is a correlation between intracellular ROS generation and pH change. An abnormal level of ROS generation may be accompanied by pH change.25-27 Unlike

ROS, pH is a parameter that can be evaluated easily. The detection of pH is more accurate and has lower requirements for the probes.28-32 According to the above considerations, pH may be a more appropriate intracellular detection parameter to study the cytotoxicity mechanism of nanoparticles and to quantify the cytotoxicity. Nonetheless, there are still no reported studies on the relationship between pH and the cytotoxicity of nanoparticles. Moreover, in the reported studies, nanoparticles were first incubated with living cells to enter cells and caused concentration fluctuations in ROS; then, additional probes were utilized to detect the concentration fluctuations.16-17 Undoubtedly, the introduction of additional probes causes the role played by nanoparticles in cytotoxicity to be unclear. This method is unable to determine whether the concentration fluctuations of the ROS and the resultant cytotoxicity are induced by the nanoparticles or the additional probes, which makes these studies on the cytotoxicity of nanoparticles unreliable and does not enable the quantification of the cytotoxicity. For the reasons discussed above, there is great practical value in developing new materials that can quantitatively detect intracellular pH without the need for additional probes to study the pH-related mechanism of nanoparticle-associated cytotoxicity and to quantify cytotoxicity. In this study, we successfully synthesized a series of differentsized ratiometric fluorescent nanoprobes for precisely imaging the intracellular pH in live human normal lung epithelial (BEAS-2B) cells. The nanoprobes were constructed from variously sized mesoporous silica nanoparticles (MSN), which were modified with a pH-sensitive dye, aminofluorescein (AF), and a reference dye, ethidium bromide (EB), to ratiometrically detect the intracellular pH value. After entering living cells, the designed nanoprobes can induce intracellular pH changes and simultaneously detect those pH changes. Then, the

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relationship between the pH change and the cell viability was studied. The results showed that the smaller the nanoparticle size is, the more ROS is generated, the lower the intracellular pH value is, and the higher the cytotoxicity that the nanoparticles exhibit (Scheme 1). When the size of the nanoparticles was increased to 135 nm, there was no obvious cytotoxicity of the nanoparticles. This work provides a new method for determining the exact correlation between the size and cytotoxicity of nanoparticles and to quantify the cytotoxicity of nanoparticles by ratiometrically detecting pH values via fluorescence. Scheme 1. Illustration of the formation of different-sized nanoprobes and their application in the ratiometric detection of intracellular pH and quantifying the cytotoxicity of nanoparticles AF

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EXPERIMENTAL SECTION Synthesis of different-sized MSNs and MSNs-based nanoprobes. (a) Different-sized MSNs: Different-sized MSNs were synthesized according to the reported literature.33-34 Briefly, to obtain 10 nm MSNs, 0.23 mmol hexadecyltrimethylammonium bromide (CTAB) and 2 ml 0.02 M NH3•H2O were mixed in 8 mL water at 30 °C. 0.43 mmol tetramethylorthosilicate (TMOS) was added into the solution and stirred for 24 h, then solution was heated to 80 °C and stirred for another 24 h. Next, the solution was transferred in a dialysis bag and dialyzed for 6 days. Finally, 10 nm MSNs were collected from the dialysis bag and frozen at -20 °C overnight. The other size MSNs (25 nm, 50 nm, 85 nm, 135 nm and 180 nm) were synthesized by a different method, and the size was controlled by changing the amount of triethanolamine (TEOA). 0.5 g hexadecyltrimethyl ammonium chloride (CTAC) and 0.08 g/0.06 g/0.04 g/0.02 g/0.01 g TEOA were dissolved into 20 mL water and mixed up with 1.5 mL of tetraethyl orthosilicate (TEOS) to get MSNs of sizes of 25 nm, 50nm, 85 nm, 135 nm and 180 nm, respectively. The products were collected with the solution cooled to room temperature. (b) MSN-COOH: 10 mg MSNs were dispersed in 20 mL ethanol, then 8 mL of water, 200 μL of NH3•H2O and 50 μL of carboxyethylsilanetriol disodium salt (CETS) were added in order. MSN-COOH were collected after centrifugation and dispersed in 5 mL ethanol. (c) MSN-EB-AF: 3 mL 1-ethyl-3-(3-dimethly aminopropyl) carbodiimide (EDC) solution (10 mM) was added to 3 mL MSN-COOH solution

and stirred for 30 min. Subsequently, 3 mM EB and 300 μM AF were added to the mixture and stirred for 24 h. MSN-EBAF was collected after centrifugation and dispersed in 5 mL ethanol. (d) MSN-EB-AF@SiO2: 8 ml water and 200 μL NH3•H2O were added to the MSN-EB-AF ethanol solution (0.5 mg/mL) in order and stirred for 30 min. Then, 20 μL (3aminopropyl) triethoxysilane (APTES) was added into the solution, then heated to 30 °C for 24 h to form the mesoporous silica shell. The obtained nanoprobes were centrifuged and washed before dispersed in 5 mL ethanol. Fluorescence spectra measurements. A series of standard pH buffers were prepared. Then, 1 mL of the standard pH buffer and 20 μL of nanoprobe solution (0.2 mg/mL) were mixed and measured fluorescence spectra with λex = 488 nm. The signal ratio (R = IAF/IEB) was calculated from the fluorescence intensities at 515 nm and 595 nm. Intracellular pH calibration and ratiometric imaging of pH in Living Cells. The cells were plated on chamber slides for 24 h before imaging studies. Next, the cells were incubated in culture media containing 0.2 mg/mL nanoprobes for 6 h. And the nanoprobes-loaded cells were washed three times with PBS (pH 7.4) in order to remove the nanoprobes that were not taken up into the cells. (a) Intracellular pH calibration: The nanoprobes-loaded cells were incubated at 37 °C for 15 min in high K+ buffer with various pH values and 10 μM nigericin. Then the cells were imaged by confocal laser scanning microscopy (CLSM) with 488 nm excitation. The fluorescence emissions were collected in the ranges of 500550 nm (AF, green fluorescence channel) and 570-630 nm (EB, red fluorescence channel), respectively. Pseudocolor ratiometric images were obtained by mediating the green channel image with the red channel at the same pH. The pH calibration was finally obtained based on the average intracellular ratio values shown in the ratiometric images. (b) Ratiometric imaging: The cells loaded with different-sized nanoprobes were imaged by CLSM with 488 nm excitation. Then the fluorescence images and pseudocolor images were collected as mentioned above. The pH values were calculated based on the ratio values shown in the ratiometric images. MTT Assay. BEAS-2B cells were cultured in 96-well microtiter plates and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 h. The cells were incubated with the different-sized nanoprobes (0.2 mg/mL) for 12 h after the original medium was removed. Next, 150 μL of MTT solution (0.5 mg/mL) was added to each well. 4 h later, the remaining MTT solution was removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. After shaking the plates for 10 min, the absorbance was measured at 490 nm with the RT 6000 microplate reader. ROS detection. After plated on chamber slides for 24 h, the cells were incubated in culture media containing 0.2 mg/mL nanoprobes and 10 μmol/L hydroethidine (HE) for 6 h. The cells were washed three times with PBS (pH 7.4) in order to remove the nanoprobes that were not taken up into the cells. The cells were examined by CLSM with 518 nm excitation, and the fluorescence emissions were collected in the ranges of 595-615 nm (HE, red fluorescence channel). And for ROS scavenging experiments, the cells incubated with 0.2 mg/mL nanoprobes, 10 μM HE, and 25 mM N-acetyl-L-cysteine (NAC) (or 300μM acetovanillone) were examined by CLSM. Then the fluorescence images were collected as mentioned above.

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RESULTS AND DISCUSSION Preparation and characterization of nanoprobes with different sizes. MSNs were utilized as the nanoplatforms to design the nanoprobes and were synthesized as previously reports. As shown in the transmission electron microscopy (TEM) images (Figure 1), the MSNs exhibited uniform spherical morphologies with average sizes of approximately 10 ± 2 nm, 25 ± 5 nm, 50 ± 5 nm, 85 ± 10 nm, 135 ±10 nm, and 180 ± 10 nm (Figure S1) and showed no substantial aggregation. (a)

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EB are 11.64 mg/g, 10.61 mg/g, 9.63 mg/g, 8.07 mg/g, 6.65 mg/g, and 5.67 mg/g, respectively (Figure S3). The zeta potential results confirmed the successful synthesis of the nanoprobes (Figure S4). Taking 10 nm MSN-EBAF@SiO2 nanoprobes as an example, the zeta potential of the MSNs was -9.3 ± 0.3 mV, which was consistent with the negative charge of the silanol surface. After carboxylation, the zeta potential further decreased to -28.8 ± 0.2 mV because of the negative charge carried by the -COOH group. Then, due to modification with the positively charged dyes, the zeta potential changed to -8.5 ± 0.2 mV. Finally, after coating with the positively charged aminated SiO2 layer, the zeta potential increased to +4.2 ± 0.6 mV. (a) 10000 Fluorescence Intensity / a.u.

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Figure 1 TEM images of MSNs with average sizes of approximately 10 ± 2 nm (a), 25 ± 5 nm (b), 50 ± 5 nm (c), 85 ± 10 nm (d), 135 ± 10 nm (e), and 180 ± 10 nm (f). Scheme 2 Synthetic process for the MSN-EB-AF@SiO2 nanoprobes

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Carboxyl groups were introduced onto the surface of the MSNs to obtain carboxyl-functionalized MSN-COOH to be further modified with the pH-sensitive dye and reference dye. After carboxylation of the MSNs, the pH-sensitive dye, AF, and reference dye, EB, were connected to the surface through an amide bond to obtain MSN-EB-AF. Finally, the MSN-EBAF@SiO2 nanoprobes were obtained after coating the MSNEB-AF with a thin protective layer of aminated silicon dioxide to prevent the leakage of dyes (Scheme 2).35 Based on our previous studies, we confirmed that the nanoprobes size exhibited no obvious change compared to the MSNs after modifying with dyes and coating with silicon dioxide layer.36 Therefore, in this work, the size of nanoprobes can be represented by the size of the MSNs. The calculated connected amounts of AF for the different-sized nanoprobes (10 nm, 25 nm, 50 nm, 85 nm, 135 nm and 180 nm) are 0.60 mg/g, 0.65 mg/g, 0.58 mg/g, 0.50 mg/g, 0.40 mg/g, and 0.31 mg/g, respectively (Figure S2). The calculated connected amounts of

Fluorescent responses of the nanoprobes to pH and selectivity tests. To test the validity of the different-sized ratiometric fluorescent nanoprobes, we first investigated their fluorescent responses to pH. Spectrometry measurements were carried out in an aqueous buffered solution with different pH values that covered a wide pH range of physiological activity (pH = 5.60, 6.00, 6.30, 6.60, 7.00, 7.40, 7.70, 8.00, and 8.30) (Figure 2 and Figure S5-S9). Since the fluorescent response behaviors of different-sized nanoprobes to pH are very similar, here we chose the 50 nm nanoprobe as an example to characterize their fluorescent properties. As shown in Figure 2a, upon being excited at 488 nm, the pH-sensitive AF exhibited an emission band centered at 515 nm, and the pHinsensitive EB exhibited an emission band centered at 595 nm. With increasing pH, the intensity of the AF emission band gradually increased, and the intensity of the EB emission band showed no obvious change. The fluorescence intensity ratio, R (R = IAF/IEB, where IAF and IEB are the fluorescence intensities

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Analytical Chemistry of the emission bands centered at 515 nm and 595 nm, respectively), increased with increasing pH. As shown in Figure 2b, the R values showed a good linear relationship with pH value over the pH range of 5.60−8.30. The linear relationships between R and pH for different-sized nanoprobes are presented in Figure 3, which shows that all as-prepared nanoprobes constructed from variously sized silica nanoparticles exhibit approximately the same pH-responsive behavior. Furthermore, the results of repeated experiments indicate that all the nanoprobes exhibit good reversibility in the processes of pH detection (Figure S10). Before the application of these nanoprobes in living cells, the selectivity of the nanoprobes against possible interfering biological species was tested. We investigated the interference from metal ions, including Na+, K+, Mg2+, Ca2+, Zn2+, Mn2+, Fe3+ and Co2+, and oxidative-stress-associated redox chemicals, including glutathione and H2O2. As the results (Figure S11) illustrated, there was no obvious fluorescent change observed when the interfering species are present, which means that these substances have negligible interference. These results indicate that the as-synthesized nanoprobes can specifically respond to pH over other biologically relevant interfering analytes. (a)

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Figure 4 (a) Confocal microscopy images of the 50 nm nanoprobe-loaded BEAS-2B cells imaged at pH 5.60, 6.30, 7.00, 7.70, and 8.30. From top to bottom, AF channel, λem = 500-550 nm; EB channel, λem = 570-630 nm; merged images of the AF channel and EB channel; pseudocolored ratiometric images; the color strip represents the pseudocolor change with respect to pH. (b) Plot of R as a function of pH in the cell system.

Figure 5 Plots of R values as a function of pH of differentsized nanoprobes in the cell system. Ratiometric imaging of pH in living cells treated with different-sized nanoprobes. The cellular uptake pattern of nanoprobes were tested in BEAS-2B cell firstly. The results indicated that different sized nanoprobes (25 nm and 180 nm)

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can be internalized by cells mainly through energy-dependent endocytosis (Figure S12-S13). To show that the nanoprobes can quantitatively detect pH in living cells, we first calibrated the measurement of intracellular pH with nigericin-treated BEAS-2B cells in high-K+ buffers with different pH values. Nigericin was used to homogenize the intracellular pH and culture medium.37-38 The confocal fluorescence imaging of the pH in BEAS-2B cells treated with different-sized nanoprobes is shown in Figure 4 and Figure S14-S18. Since the detection behaviors of the different-sized nanoprobes for imaging intracellular pH are nearly identical, we chose the 50 nm nanoprobe as an example to describe the intracellular experimental calibration results (Figure 4). As shown in Figure 4a, as the pH increases from 5.60 to 8.30, the fluorescent intensity of the AF channel (green) increases gradually, while the fluorescent intensity of the EB channel (red) exhibits no obvious change. Moreover, a good linear calibration curve can be established between the emission intensity ratio, R (R= IAF/IEB), of the two channels and the pH variations over the pH range of 5.60 to 8.30 (Figure 4b). The linear calibration curves of different-sized nanprobes are shown in Figure 5, indicating that all different-sized nanoprobes exhibit nearly identical detection behavior in living cells. After confirming that the as-prepared nanoprobes can be used to quantify pH in living cells, we employed the nanoprobes in living BEAS-2B cells to detect intracellular pH. The pH measurements were performed in living cells treated with different-sized nanoprobes (10 nm, 25 nm, 50 nm, 85 nm, 135 nm and 180 nm). As shown in the confocal fluorescence images of these living cells (Figure 6), cells incubated with different-sized nanoprobes exhibited different colors in the pseudocolored ratiometric images, which indicates that the intracellular pH values of these groups of cells are different from one another. Based on the ratio of the emission signals, we calculated the intracellular pH of the living cells of each group treated with different-sized nanoprobes. Interestingly, we found that cells incubated with differently sized nanoprobes exhibit different pH values. The intracellular pH values of the cells incubated with the 135 nm and 180 nm nanoprobes, which are 7.37 ± 0.02 and 7.37 ± 0.04, respectively, are nearly identical and are consistent with the pH of normal cells.39 The pH value of cells incubated with 85 nm nanoprobes slightly decreases to 7.35 ± 0.03. Then, as the nanoprobe size decreases, the intracellular pH values decrease. As shown in Figure 6, when the nanoprobe size is reduced from 50 nm to 25 nm to 10 nm, the pH values decrease from 7.31 ± 0.05 to 7.24 ± 0.03 to 7.17 ± 0.07, correspondingly. In the intracellular calibration experiments, we confirmed that differently sized nanoprobes exhibit nearly identical detection behavior in living cells. However, in the detection experiments, with the unique variable being the nanoprobe size, the pH values were quite different from one another. Inspired by these intriguing experimental results, we speculate that the nanoprobes, which can be expanded to nanoparticles in general, have an impact on the intracellular pH of living cells. Specifically, nanoparticles entering cells will induce a reduction of the pH value, and the smaller the nanoparticle size is, the lower the intracellular pH value will be. It is worth noting that when the nanoprobe size was increased to 135 nm, the intracellular pH showed no obvious difference from that of normal cells. In addition, as the nanoparticle size continued to increase, the intracellular pH remained unchanged.

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changing trend of cell viability, indicating that the intracellular pH value has a quantitative relationship with the cytotoxicity of the nanoparticles. Moreover, the pH value can be visually represented with different colors in pseudocolored ratiometric images, meaning that the size-dependent cytotoxicity can be quantified by the ratiometric fluorescent signals. Mechanism of the different-sized nanoprobes inducing intracellular pH changes. Based on the literature, we proposed a probable cellular pathway for the nanoprobes to induce a pH value reduction. Numerous studies have confirmed that, upon exposure of cells to nanoparticles, cells rapidly produce ROS.17-19, 41-42 Inspired by these results, we speculate that the nanoparticles perturb the intracellular pH through a pathway related to the generation of ROS. (a)

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Cell viability and size-dependent cytotoxicity. To investigate the effects of the differently sized nanoprobes on the viability of living cells, the cytotoxicities of the differentsized nanoprobes were evaluated by an MTT assay40 with the BEAS-2B cell line. To eliminate the effect of dye molecules on cell viability, we tested the cytotoxicity of EB and AF molecules firstly. As shown in Figure S19, the cell viability were all high than 98%, indicating that there was no obvious cytotoxicity of the utilized dye molecules. Then the cytotoxicity of nanoprobes were tested. As shown in Figure 7, after treatment with the different-sized nanoprobes for 12 h, BEAS-2B cells exhibit different cell viabilities. The cell viabilities of the 135 nm and 180 nm groups were very close to one another and were approximately 98%, which suggests that nanoprobes with sizes beyond 135 nm have negligible cytotoxicity toward cells. The cell viabilities decreased as a function of the nanoprobe size. As the nanoparticle size decreased from 85 nm to 50 nm to 25 nm, the cell viability decreased from 91.4% to 90.3% to 87.7%, correspondingly. Remarkably, the cell viability of the 10 nm group is 85.1%, suggesting they have the highest cytotoxicity. In summary, the nanoparticle cytotoxicity increases as the nanoparticle size decreases: the smaller the nanoparticle size is, the higher the nanoparticle cytotoxicity. When the nanoparticle size is larger than 135 nm, no obvious cytotoxicity is induced by the nanoparticles.

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Figure 8 (a) and (b) ROS detection with HE in BEAS-2B cells treated with different-sized nanoprobes; (c) and (d) ROS detection with HE in BEAS-2B cells treated with differentsized nanoprobes and NAC; (e) confocal fluorescence images of BEAS-2B cells treated with different-sized nanoprobes and NAC.

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Figure 7 MTT assays for the cell viability of BEAS-2B cells treated with differently sized nanoprobes for 12 h. It should be noted that the trend of changing pH values along with the changing of nanoparticle size is consistent with the

To verify the process by which nanoprobes induce an intracellular pH value reduction, the ROS concentrations in BEAS-2B cells incubated with different-sized nanoprobes were first tested with a commercial fluorescence probe, HE43. As shown in Figure 8a and 8b, different levels of ROS are generated when nanoprobes enter the cells. Compared with the ROS levels of the control group, the 10 nm nanoprobes give rise to the maximum concentration of ROS. As the size of the nanoparticles increases, the ROS levels decrease, and the 135 nm and 180 nm groups do not exhibit elevated levels of ROS compared to that of the control group. The trend in ROS

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Analytical Chemistry generation is correlated with the pH change, which shows that our hypothesis is credible. Moreover, the trend in ROS generation is also correlated with the changing trend of cell viability, indicating that the mechanism of the size-dependent cytotoxicity of nanoparticles is also related to ROS generation. To further confirm the correlation between ROS generation and pH perturbation, we incubated BEAS-2B cells with both nanoprobes and an ROS scavenger, NAC, and then detected the intracellular ROS concentrations and pH values. For NACincubated living cells, all the cells incubated with differentsized nanoprobes exhibited nearly the same ROS concentration (Figure 8c and 8d). The pH detection results show that after scavenging of the ROS with NAC, the intracellular pH values of all the groups are consistent with the pH values of normal cells, which is approximately 7.37 (Figure 8e). From the above experimental results, we confirm that the size-dependent intracellular pH perturbation induced by the nanoparticles is related to ROS generation. There is strong evidence that the nanoparticles induce intracellular ROS generation via NADPH oxidase activation. NADPH oxidases are the major sources of ROS in cells.44-45 To further confirm that the pH change is related to ROS generation, we incubated living cells with an NADPH enzyme inhibitor, acetovanillone, to inhibit ROS generation46 and then tested the intracellular pH. As shown in Figure 9a and 9b, after incubation with acetovanillone and different-sized nanoprobes, the cells in different groups exhibited nearly identical ROS levels that are consistent with the ROS levels of the control group, suggesting that ROS generation was inhibited. As illustrated in Figure 9c, since ROS generation is inhibited, the cells in different groups all show pH values consistent with that of normal cells. These results confirm our hypothesis that the generation of ROS can be regarded as the likely mechanism responsible for the nanoparticles inducing an intracellular pH change.

CONCLUSION In conclusion, quantitative ratiometric fluorescent detection of intracellular pH changes was realized and the toxicity of silica nanoparticles of different sizes toward living cells was evaluated. The results showed that when nanoparticles entered cells, different-sized nanoparticles activated NADPH oxidase to different degrees, leading to the generation of different levels of ROS, which in turn resulted in different intracellular pH values. The smaller the nanoparticle size is, the higher the level of ROS that is generated, the lower the intracellular pH value, and the higher the cytotoxicity of the nanoparticles. Notably, when the nanoparticle size was larger than 135 nm, there was no obvious cytotoxicity. Furthermore, the pHrelated change in the ratiometric fluorescence signal during the ratiometric pH-detection process can be explored to quantify the cytotoxicity of nanoparticles. The current study could provide a new strategy for studying the cytotoxicity of nanoparticles.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the internet at http://pubs.acs.org. Materials and instruments; Experimental details on synthesis of nanoprobes, quantitation of AF and EB loaded on the nanoprobe, the pH response of the nanoprobe, fluorescence reversibility of nanoprobe to pH, Selectivity, cell culture, cytotoxicity, intracellular pH calibration, ratiometric imaging of intracellular pH with different-sized nanoprobes, and pH changes and ROS detection in living cells after incubated with the nanoprobes; Supporting figures. (PDF)

AUTHOR INFORMATION Corresponding Author

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*Fax: (86)-531-86180017. E-mail: [email protected]. *Fax: (86)-531-86180017. E-mail: [email protected].

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† X.W. and X.Z. contributed equally to this work.

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The authors declare no competing financial interest. Control 10

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ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21535004, 91753111, 21874086, 21775094) and the Key Research and Development Program of Shandong Province (2018YFJH0502).

REFERENCES

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7.36±0.03

7.37±0.05

7.37±0.05

7.38±0.03

7.37±0.02

Figure 9 (a) and (b) ROS detection with HE in BEAS-2B cells treated with differently sized nanoprobes and acetovanillone; and (c) confocal fluorescence images of BEAS-2B cells treated with differently sized nanoprobes and acetovanillone.

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Analytical Chemistry

Table of Contents

8.0

Size

7.7

Size ↓

7.0

pH ↓

6.3

Cell viability ↓

5.6 pH

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