Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for

Apr 7, 2017 - We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn2+ and imidazole-2-carboxyaldehyde, to ...
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Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for ATP Imaging in Live Cells Jingjing Deng,† Kai Wang,†,‡ Ming Wang,*,†,‡ Ping Yu,*,†,‡ and Lanqun Mao*,†,‡ †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Zeolitic imidazole frameworks (ZIFs) are an emerging class of functional porous materials with promising biomedical applications such as molecular sensing and intracellular drug delivery. We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn2+ and imidazole-2carboxyaldehyde, to target subcellular mitochondria and image dynamics of mitochondrial ATP in live cells. Encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 suppresses the emission of RhB, while the competitive coordination between ATP and the metal node of ZIF-90 dissembles ZIFs, resulting in the release of RhB for ATP sensing. With this method, we are able to image mitochondrial ATP in live cells and study the ATP level fluctuation in cellular glycolysis and apoptosis processes. The strategy reported here could be further extended to tune nanoscale ZIFs inside live cells for targeted delivery of therapeutics to subcellular organelles for advanced biomedical applications.

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INTRODUCTION Zeolitic imidazole frameworks (ZIFs) are a subclass of metal− organic frameworks (MOFs) self-assembled from metal ions and imidazole linkers.1−4 The structural diversity, high surface area, tunable pore sizes, and functionalities of ZIFs5−7 have made them very attractive for biomedical applications.8−10 For example, by modulating the chemistry of the imidazole linker in ZIFs, they have been used as fluorescent sensors for formaldehyde11,12 and nucleic acid detection13 as well as intracellular imaging of hydrogen sulfide.14 Meanwhile, the high aqueous stability and large inner volume of ZIFs provide extra advantages to using ZIFs as nanocarriers for drug delivery.15−18 In particular, labile metal−ligand interactions of ZIFs enable targeted drug delivery in response to disease microenvironment. For instance, ZIF-8 is prone to decompose under an acidic condition, and, as a consequence, it could release encapsulated chemotherapeutics preferably at tumor sites because the acidic tumor microenvironment triggered ZIF-8 decomposition.19 More recently, ZIFs are emerging as a new platform to develop theranostics by supplementing ZIFs with an imaging agent.20 The biomedical applications of ZIFs such as in intracellular imaging and drug delivery, however, require better understanding of the intracellular fate and trafficking of ZIFs. Moreover, developing organelle-targeted ZIFs to transport drugs to subcellular compartments could further improve their therapeutic index. Herein, we report for the first time that nanoscale ZIF-90, self-assembled from Zn2+ and imidazole-2-carboxyaldehyde (2ICA), can target mitochondria in live cells (Figure 1). Moreover, we find that ZIF-90 is responsive to adenosine © 2017 American Chemical Society

triphosphate (ATP), showing ATP-triggered ZIF-90 decomposition. Based on these properties, we design a fluorescent nanoprobe by encapsulating a fluorescent dye, Rhodamine B (RhB), into ZIF-90 to form RhB/ZIF-90. Initially, encapsulation of RhB into ZIF-90 largely suppresses the emission of RhB probably due to the “self-quenching” effect.21 ATP-triggered RhB/ZIF-90 decomposition, however, efficiently releases RhB and thereby restores RhB emission. Such a host−guest chemistry of RhB/ZIF-90 and as-regulated RhB emission are therefore harnessed for ATP sensing and intracellular ATP imaging. As reported previously, ATP is mainly produced in mitochondria, serving as the primary energy source for cellular processes in living organisms.22−24 Rapid and reliable ATP sensing is informative for understanding the process of cellular respiration and for disease diagnosis. To date, genetically engineered protein indicator,25 fluorescent biosensors,26−29 and electrochemical sensors30 have been developed for ATP sensing with high selectivity and sensitivity. Fluorescent probes that are cell-permeable, low cytotoxic, and capable of monitoring ATP level fluctuation in specific organelles such as mitochondria,26,27 however, are still facing a challenge for understanding the role of ATP in biological events. With the nanoscale RhB/ZIF-90, we are able to image mitochondrial ATP in live cells and monitor ATP level fluctuation during cellular glycolysis and apoptosis, providing a new tool to study cell metabolism and other biological events. Received: February 4, 2017 Published: April 7, 2017 5877

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Figure 1. Schematic representation of modulating the host−guest chemistry of nanoscale RhB/ZIF-90 for fluorescent ATP sensing and mitochondrial ATP imaging.

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2 mM ATP for 3 min. The resulting RhB/ZIF-90 with the ATP exposure was then used for SEM and TEM measurements. ATP Assay in Cell Lysates. HeLa cells (ATCC, Manassas, VA) were maintained in high-glucose Dulbecco’s modified eagle’s medium (DMEM, HyClone, UT) supplemented with 10% fetal bovine serum (FBS, HyClone, UT) and 1% penicillin-streptomycin (GIBCO, CA). To prepare cell lysates for ATP sensing, HeLa cells were lysed with a cold 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 0.5% CHAPS, and 10% glycerol) supplemented with 20 μM cetrimonium bromide at a density of 1 × 106 cells/mL. The cell suspension was incubated for 30 min on ice, followed by centrifugation at 4 °C for 20 min to remove cell debris. The supernatant was flash frozen and stored at −20 °C before use. In general, 2 mL of ATP spiked cell lysates were incubated with 4 mg/mL of RhB/ZIF-90 nanocrystals (in DPBS) for 5 min, the suspensions were then centrifuged at 13000 rpm for 5 min at room temperature, and RhB fluorescence intensity of supernatant was measured. Cytotoxicity Assay of RhB/ZIF-90 Nanocrystals. To evaluate the biocompatibility of RhB/ZIF-90 nanocrystals for ATP imaging in live cells, HeLa cells were seeded in a 96-well plate at a density of 1 × 104 per well and incubated with RhB/ZIF-90 nanocrystals with concentrations ranging from 100 to 20 μg/mL. The cell viability was measured by Cell Counting Kit-8 (CCK-8) assay (Dojondo, Japan) after incubation with the nanocrystals for 24 h. Mitochondrial ATP Imaging. To conduct the fluorescent imaging of mitochondrial ATP, HeLa cells were first seeded on glass-bottom culture dishes (Nest, China) for 24 h at a density of 1 × 105 cells/mL. RhB/ZIF-90 nanocrystals (10 μL, 4 mg/mL) were mixed with 390 μL cell culture medium and then incubated with HeLa cells for 2 h. At the end of incubation, the cells were washed with DPBS and covered with fresh DMEM for CLSM imaging. For the colocalization study of RhB/ ZIF-90 with mitochondria, the cells were incubated with 1 μM rhodamine 123 or 50 nM MitoTracker@Green (Molecular Probes, USA) for 15 min at the end of RhB/ZIF-90 incubation. The cells were then washed thoroughly with DPBS prior to CLSM imaging. To further understand the mechanism of intracellular fluorophore release from ZIF-90 nanocrystals, we performed the colocalization study of Dox/ZIF-90 nanocrystals with mitochondria. To do this, Dox/ZIF-90 nanocrystals (10 μL, 4 mg/mL) were mixed with 390 μL cell culture medium and then incubated with HeLa cells for 2 h. At the end of incubation, rhodamine 123 was added to 1 μM for another 15 min incubation prior to CLSM imaging. To study the time-dependent cellular uptake of Dox/ZIF-90 nanocrystals, HeLa cells were incubated with 100 μg/mL of Dox/ZIF-90 nanocrystals, followed by CLSM imaging at indicated time without washing cells. To demonstrate the feasibility of RhB/ZIF-90 nanocrystals for monitoring the fluctuation of mitochondrial ATP, HeLa cells were first incubated with DMEM containing pentachlorophenol (PCP, 20 μM), sodium azide (10 mM), or Ca2+ (5 mM) for 2 h. The cells were then incubated with the mixture of RhB/ZIF-90 nanocrystals (10 μL, 4 mg/ mL) and DMEM (390 μL) for another 2 h, finally washed with DPBS, and covered with fresh DMEM for CLSM imaging. The CLSM imaging data were collected, and the fluorescence intensity was

EXPERIMENTAL SECTION

General Materials. All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO) or from Beijing Chemical Reagent Co. (Beijing, China) and used as received. Scanning electron microscopy (SEM) images were recorded using S-4300 and S4800 microscopy (Hitachi, Japan). Transmission electron microscopy (TEM) images were recorded with JEM-2100F microscopy (JEOL, Japan). Confocal laser scanning microscopy (CLSM) images were performed on Olympus FV-IX81 confocal system. Fluorescence and UV−vis spectra were recorded on Hitachi F-4600 and Shimadzu UV2600 spectrophotometers, respectively. Synthesis and Characterization of ZIFs. Briefly, RhB/ZIF-90 nanocrystals were synthesized by adding a DMF solution of Zn(CH3COOH)2·2H2O (2 mL, 0.1 M) into a DMF solution of imidazolate-2-carboxyaldehyde (ICA) (2 mL, 0.2 M) containing 1 mM RhB under vigorous stirring. After 5 min, another 10 mL of DMF was added into the reaction mixture to further stabilize the spheres, followed by centrifugation and washing with DMF (once) and ethanol until no significant fluorescence signal was detected in the supernatant. To synthesize Dox/ZIF-90 nanocrystals, RhB was substituted with doxorubicin (Sangon Biotechology, China), followed by the procedures as described for the synthesis of RhB/ZIF-90. The nanocrystals were then dried in vacuum overnight and stored at 4 °C with protection from light. To further clarify the location of RhB in ZIF-90 nanocrystals, we synthesized RhB/ZIF-90 microcrystals and characterize them with CLSM because the size of RhB/ZIF-90 nanocrystals is below the resolution limit of CLSM. To synthesize RhB/ZIF-90 microcrystals, 1 mL of DMF solution of Zn(CH3COOH)2·2H2O (0.2 M) was first diluted with 1 mL of water before adding to a mixture of imidazolate2-carboxyaldehyde (1 mL of DMF, 0.4 M) and RhB (1 mL of H2O, 2 mM). After 5 min of reaction, DMF (10 mL) was added into the reaction mixture. The resulting RhB/ZIF-90 microcrystals were purified and stored in the same way as that of RhB/ZIF-90 nanocrystals. To study the pH stability of RhB/ZIF-90 nanocrystals, an aqueous dispersion of RhB/ZIF-90 nanocrystals (2 mg/mL) was adjusted to different pH values with HCl or sodium hydroxide, and the resulting dispersions with different values were first allowed to stand by for 5 min at room temperature and then used for the measurement of emission of RhB possibly released from ZIF-90. ATP Sensing with RhB/ZIF-90 Nanocrystals. To investigate the validity of RhB/ZIF-90 nanocrystals as the nanoprobe for fluorescent ATP sensing, a stock dispersion of RhB/ZIF-90 nanocrystals was prepared by dispersing RhB/ZIF-90 into DPBS (4 mg/mL). Different concentrations of ATP were mixed into the stock dispersions of RhB/ ZIF-90 nanocrystals, and the resulting mixtures were then incubated for 5 min, followed by centrifugation at 13000 rpm for 5 min at room temperature. The fluorescence spectra of RhB in the supernatants were recorded on the fluorescence spectrophotometer. To characterize the morphology change of RhB/ZIF-90 nanocrystals after the exposure of ATP, 4 mg/mL of RhB/ZIF-90 nanocrystals in DPBS was treated with 5878

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Journal of the American Chemical Society quantified using ImageJ (NIH, MD). The pixel intensity of at least 25 cells was averaged and compared.

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RESULTS AND DISCUSSION Synthesis and Characterization of RhB/ZIF-90 Crystals. RhB/ZIF-90 nanocrystals were synthesized by encapsulating RhB into ZIF-90 (see Experimental Section details). The efficient encapsulation of RhB into ZIF-90 was mainly due to the nonplanar structure of RhB molecule with a size around 11.9 × 10 × 6 Å in three dimension,31 which matches the pore (11.2 Å) and window (3.5 Å) size of ZIF-90 and thus facilitates diffusion of RhB into the pore channels of ZIF-90.7,32 SEM image indicates that RhB/ZIF-90 crystals were well dispersed with an average size of 75 nm (Figure 2A). The identity and

Figure 3. CLSM images of the RhB/ZIF-90 microcrystals dispersed in water (10 μg/mL). (A) fluorescence image of the RhB/ZIF-90 microcrystals. (B) Overlay of (A) with the bright-field image of the RhB/ZIF-90 microcrystals. Scale bar, 10 μm.

demonstrate the efficient encapsulation of RhB into ZIF-90 nanocrystals, and such a host−guest encapsulation had minimal effect on the retention of ZIF-90 nanostructure. Fluorescent ATP Sensing with RhB/ZIF-90 Nanocrystals. It has been previously reported that the coordination between ATP and Zn2+ is much stronger than that between imidazole and Zn2+.38,39 Therefore, addition of ATP into the aqueous dispersion of RhB/ZIF-90 would displace 2-ICA from ZIF-90, resulting in the collapse of the ZIF-90 framework and thus the release of RhB. The encapsulation and release of RhB from ZIF-90 and the consequent modulation of RhB emission in response to ATP could therefore be harnessed for ATP sensing. As shown in Figure S2, treatment of RhB/ZIF-90 nanocrystals (80 μg/mL in DPBS) with 8 mM ATP resulted in the efficient release of RhB from ZIF-90, as confirmed by increased RhB absorption in the UV−vis spectra of RhB/ZIF90 after ATP treatment. To further confirm the ATP-induced ZIF-90 collapse, RhB/ZIF-90 nanocrystals (4 mg/mL) were treated with a lower concentration of ATP (2 mM) for a short period of time (3 min), followed by SEM and TEM measurements. As displayed in Figures 4A and S3, the ATP

Figure 2. (A) SEM images of the RhB/ZIF-90 nanocrystals. (B) PXRD patterns of simulated ZIF-90 (blue), RhB/ZIF-90 nanocrystals (black), and RhB alone (red).

phase purity of RhB/ZIF-90 nanocrystals were further confirmed by powder X-ray diffraction (PXRD). As shown in Figure 2B, RhB-encapsulated ZIF-90 nanocrystals (black) have similar peaks to the simulated ones of pure ZIF-90 crystals (i.e., without RhB encapsulation) (blue curve), indicating the minimal effect of RhB encapsulation on lattice distortion of ZIF-90. In addition, we did not observe the PXRD peak of RhB in the pattern of RhB/ZIF-90 nanocrystals (black), presumably suggesting that RhB may be encapsulated inside the pore of ZIF-90, rather than physically adsorbed on the surface of ZIF90.33−35 FT-IR spectrum of RhB/ZIF-90 was also indicative for the encapsulation of RhB into ZIF-90. As displayed in Figure S1, there was minimal shift of the peaks from 790 to 1600 cm−1 that were ascribed to the transmittance of ZIF-90 nanocrystals with and without RhB encapsulation. The peaks from ZIF-90 nanocrystals at 3431 and 3120 cm−1 were slightly blue-shifted after RhB encapsulation, suggesting the retention of ZIF-90 nanostructure after RhB encapsulation. We did not observe the IR peaks of RhB at 1590, 1179, 1342, and 1645 cm−1 after its encapsulation into ZIF-90, again demonstrating that RhB was localized inside the pore of ZIF-90 and the vibration of RhB was restrained due to the shielding effect of the ZIF-90 framework.36 To further clarify the location of RhB in ZIF-90, we synthesized RhB/ZIF-90 microcrystals according to our previous report37 and characterized the microcrystals with CLSM. As shown in Figure 3, CLSM images show that RhB/ ZIF-90 microcrystals were red fluorescent under the conditions employed here, and the Z-stacked CLSM images show a rather uniform distribution of RhB in the inner layers of RhB/ZIF-90 microcrystals (see Supplementary Movie 1). Collectively, both the CLSM images and spectrometric characterizations well

Figure 4. (A) SEM image of RhB/ZIF-90 nanocrystals treated with ATP (2 mM) for 3 min. (B) Fluorescence spectra of aqueous dispersion of RhB/ZIF-90 nanocrystals (4 mg/mL) in the presence of ATP with increasing concentrations (red arrow, 0, 25, 75, 175, 425, 915, 1885, 4235, and 8600 μM). λex = 545 nm.

treatment essentially resulted in partial swelling and melting of RhB/ZIF-90 nanocrystals, indicating the ATP-responsive nature of them, which was further confirmed by the restoration of RhB emission in the presence of ATP. As shown in Figures 4B and S4, addition of ATP with increasing concentrations into the dispersion of the RhB/ZIF-90 (4 mg/mL) resulted in gradual enhancement of RhB emission. Moreover, the fluorescence intensity of RhB released from the framework was linear with the ATP concentration within the concentration 5879

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Journal of the American Chemical Society range from 25 μM to 8.6 mM (Figure S4B), demonstrating the potential of RhB/ZIF-90 nanocrystals as the nanoprobes for the detection of intracellular ATP, which usually changes within millimolar range.40,41 To investigate the capability of the RhB/ZIF-90 nanocrystals for intracellular ATP detection, we first determined ATP concentration in cell lysate with RhB/ZIF-90 nanocrystals as the probes. To do this, HeLa cell lysates spiked with different concentrations of ATP were incubated with the aqueous dispersion of RhB/ZIF-90 nanocrystals (4 mg/mL in DPBS), followed by RhB emission monitoring. As shown in Figure S5, the fluorescence intensity of aqueous dispersion of RhB/ZIF-90 nanocrystals was enhanced after adding the HeLa cell lysate. Moreover, we observed a linear relationship between the fluorescence intensity of RhB/ZIF-90 nanocrystals and the concentration of added ATP (from 250 μM to 2.4 mM). By normalizing the fluorescence intensity of the control group without ATP spiking, the concentration of ATP in HeLa cell lysates was estimated to be 3.0 mM, which was consistent with previous reports.42,43 This result validates the RhB/ZIF-90 as a nanoprobe to determine the concentration of ATP in cell lysates. Intracellular ATP Imaging with RhB/ZIF-90 Nanocrystals. The RhB/ZIF-90 nanocrystals show low cytotoxicity and high stability over a broad range of pH values, which is highly desirable for intracellular imaging. As indicated in Figure S6, HeLa cells treated with different concentrations of RhB/ZIF-90 nanocrystals retained cell viability >80%. Additionally, RhB/ ZIF-90 nanocrystals are stable under pH values ranging from 5 to 11, as verified by the minimal change of RhB emission as pH varies (Figure S7), which excludes the potential interference from the acidic medium in subcellular organelles during the cell metabolism process for intracellular ATP imaging.44,45 Low cytotoxicity and high stability of RhB/ZIF-90 nanocrystals well validate their application for in situ fluorescent ATP imaging in live cells. To demonstrate this, we incubated HeLa cells with RhB/ZIF-90 crystals and investigated the cell imaging with CLSM. As shown in Figure 5, RhB/ZIF-90 treated cells showed strong intracellular red fluorescence (Figure 5A), indicating the high cell membrane permeability of RhB/ZIF-90 nanocrystals for ATP imaging. To further study the subcellular localization of RhB/ZIF-90 nanocrystals, the RhB/ZIF-90 treated HeLa cells were costained with rhodamine 123 or MitoTracker@Green, which were frequently used as mitochondria labeling dyes,27 for CLSM imaging. We found that RhB/ZIF-90 colocalized very well with rhodamine 123 (Figures 5B−D and S8) or MitoTracker@Green (Figure S9), indicating the preferential accumulation of RhB/ZIF-90 in mitochondria. This might be ascribed to positive surface charges of ZIF-90 that facilitate the accumulation of RhB/ZIF90 within mitochondria via the electrostatic interaction between positively charged ZIF-90 and negatively charged mitochondria inner membrane.46,47 With the aim of further understanding the mechanism of intracellular fluorophore release from ZIF-90 nanocrystals, we encapsulated doxorubicin (Dox), a fluorophore that preferentially accumulates in the cell nucleus,48,49 into ZIF-90 and incubated HeLa cells with the Dox/ZIF-90 nanocrystals. Interestingly, we found that Dox/ZIF-90 was able to enter the cells and rapidly release Dox for ATP imaging soon after 5 min of incubation (Figure S10). It is revealed that Dox was mostly localized in mitochondria with almost no accumulation in cell nucleus after 2 h of incubation (Figure 6). The

Figure 5. CLSM images of HeLa cells after 2 h of incubation with RhB/ZIF-90 nanocrystals (100 μg/mL) and 15 min of incubation with rhodamine 123 (1 μM). (A) RhB image obtained with band path of 570−670 nm upon excitation of the RhB/ZIF-90 nanocrystals at 559 nm. (B) Rhodamine 123 image obtained with band path of 500−545 nm upon excitation of rhodamine 123 at 488 nm. (C) Bright-field image of HeLa cells. (D) Overlay of (A−C). Scale bar, 20 μm.

Figure 6. Colocalization study of Dox/ZIF-90 nanocrystals with mitochondria. CLSM images of HeLa cells after 2 h of incubation with Dox/ZIF-90 nanocrystals (100 μg/mL) and 15 min of incubation with rhodamine 123 (1 μM) for mitochondria labeling. (A) Dox image obtained with band path of 570−670 nm upon excitation of Dox/ZIF90 nanocrystals at 559 nm. (B) Rhodamine 123 image obtained with band path of 500−545 nm upon excitation of rhodamine 123 at 488 nm. (C) Bright-field images of HeLa cells. (D) Overlay of (A−C). Scale bar, 20 μm.

intracellular trafficking study suggests that Dox was mainly released from ZIF-90 in mitochondria in response to ATP 5880

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Figure 7. Fluorescent monitoring of the mitochondrial ATP level fluctuation. CLSM images of HeLa cells alone (A1 and A2); treated with RhB/ ZIF-90 nanocrystals (B1 and B2); treated with 20 μM PCP (C1 and C2) or 5 mM Ca2+ (D1 and D2) before ATP imaging. The cells were incubated with RhB/ZIF-90 nanocrystals (100 μg/mL) for 2 h before CLSM imaging. The images in the top row are the fluorescent images of the RhB/ZIF90-incubated cells, while those in the bottom row are overlays of fluorescent RhB images with corresponding bright-field image of HeLa cells. Scale bar, 20 μm.

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CONCLUSIONS In summary, by tuning the encapsulation and release of RhB from ZIF-90 nanocrystals, we have successfully developed a fluorescent probe for mitochondrial ATP sensing and imaging in live cells. The RhB/ZIF-90 nanoprobe has low cytotoxicity and high cell permeability and is then able to monitor the ATP level fluctuation in a diverse range of cellular processes such as cellular glycolysis and apoptosis. The facile self-assembly of nanoscale ZIFs avoids the complication of laborious synthesis of small molecule probes for ATP sensing and mitochondrial ATP imaging, enabling the study of various ATP-related biological processes in a simple way. In addition, the strategy employed here to tune ZIFs for cargo release inside cells using intracellular microenvironment would be further explored for the development of other fluorescent sensors for biological implications. Moreover, the targeting capability of ZIFs to mitochondria essentially provides a new platform to develop spatiotemporally controlled delivery to subcellular organelles for treating mitochondria malfunction related diseases.

because the release of Dox in cytosol would result in accumulation of Dox in the nucleus due to the nuclear targeting capability of free Dox. Interestingly, we found that the incubation of Dox/ZIF-90 nanocrystals with cells for a longer period (4 h of incubation) resulted in partial accumulation of Dox in the cell nucleus (Figure S11), suggesting that a long time of incubation of the Dox/ZIF-90 may lead to partial release of Dox from mitochondria to enter the nucleus. Collectively, the good response of RhB/ZIF-90 nanocrystals toward intracellular ATP and targeted imaging of mitochondrial ATP eventually enable the study of ATP metabolism dynamic in live cells with RhB/ZIF-90 as the nanoprobe, as demonstrated below. Mitochondrial ATP Level Fluctuation Monitoring. At last, we demonstrated the use of RhB/ZIF-90 nanocrystals for studying the change of mitochondrial ATP level in live cells. To do this, HeLa cells were first treated with pentachlorophenol (PCP) and sodium azide, both of which regulate intracellular ATP generation. PCP and azide uncouple mitochondrial oxidative phosphorylation (OXPHOS), inhibit mitochondria ATPase, and thus induce an abrupt decrease of ATP concentration during cell apoptosis.50,51 As shown in Figure 7, treatment of HeLa cells with 20 μM PCP resulted in significant decrease of the intracellular ATP level, as evidenced by weak RhB fluorescence compared to that in the cells without PCP treatment. Similarly, HeLa cells treated with sodium azide exhibited decreased intracellular RhB emission (Figure S12), indicating a decreased generation of ATP inside cells. In contrast, treatment of HeLa cells with 5 mM Ca2+ resulted in significant enhancement of RhB fluorescence compared to that without Ca2+ stimulation (Figure 7D), because Ca2+ activates dehydrogenases in mitochondria and thus levels up NADH and hence ATP production.52 The mitochondrial ATP level fluctuation was further quantified by measuring cellular fluorescence intensity change after different treatments. As shown in Figure S13, addition of PCP and sodium azide resulted in the decrease of mitochondria ATP up to 51.3 ± 15% and 40.7 ± 12.7%, respectively, compared to those without any treatment, while treatment of cells with Ca2+ increased the ATP concentration up to 148.5 ± 31.8%, which is consistent with previous reported value.53 Taken together, the fluorescent ATP imaging results well demonstrated the capability and advantages of using RhB/ZIF-90 nanocrystals for monitoring dynamics of mitochondrial ATP and cellular metabolism process.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b01229. Absorption and FT-IR spectra of RhB/ZIF-90; characterization of RhB/ZIF-90 nanocrystals with and without ATP addition; CLSM images of HeLa cells incubated with RhB/ZIF-90 and Dox/ZIF-90 nanocrystals and mitochondria staining using rhodamine 123 or MitoTracker@Green (PDF) distribution of RhB in the inner layers of RhB/ZIF-90 microcrystals (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] *[email protected] ORCID

Lanqun Mao: 0000-0001-8286-9321 Notes

The authors declare no competing financial interest. 5881

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ACKNOWLEDGMENTS This work is financially supported by National Science Foundation of China (grant nos. 21621062, 21435007, and 21210007 for L.M. and 21475138 and 21322503 for P.Y.), the National Basic Research Program of China (2013CB933704 and 2016YFA0200104), and the Chinese Academy of Sciences. The authors thank Dr. Fei Wu and Xiaoti Yang for their great help in the experiments.

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DOI: 10.1021/jacs.7b01229 J. Am. Chem. Soc. 2017, 139, 5877−5882