Research Article www.acsami.org
Self-Assembled Fluorescent Bovine Serum Albumin Nanoprobes for Ratiometric pH Measurement inside Living Cells Qiaoyu Yang,† Zhongju Ye,† Meile Zhong,† Bo Chen,† Jian Chen,‡ Rongjin Zeng,‡ Lin Wei,*,† Hung-wing Li,*,§ and Lehui Xiao*,† †
Dynamic Optical Microscopic Imaging Laboratory, Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, Hunan 410081, People’s Republic of China ‡ Key Laboratory of Theoretical Organic Chemistry and Function Molecule of Ministry of Education, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan, Hunan 411201, People’s Republic of China § Department of Chemistry, Hongkong Baptist University, Kowloon Tong, Hong Kong, People’s Republic of China S Supporting Information *
ABSTRACT: In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6−8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol. KEYWORDS: protein nanoparticles, pH sensing, ratiometric, cellular imaging, fluorescent nanoparticles
■
nanoparticles.7−11 Upon sensing the pH-induced fluctuation in fluorescence intensity (i.e., enhancement or quenching), and fluorescence lifetime, the pH in solution or inside a living cell can be quantified. In spite of the promising application capability, some grand challenges still exist, which might seriously restrain the extensive applications of these techniques. For the absolute fluorescence intensity-based measurements, although the sensitivity is excellent, they typically suffer from uncertainties in the calibration of the responses, particularly when those probes are applied in in vivo studies. Fluctuations in the probe concentration, inhomogeneous of optical illumination, as well as uncertainty of the detection system, will greatly degrade the reliability of the measured results. Fluorescence lifetime measurements can bypass the majority of these limitations.11 However, the pH-induced responses from these two domains are typically minor, greatly limiting the sensitivity of the probe. For example, the reported fluorescence lifetime change range for most of fluorescent protein-based pH sensors is usually less than 1.5 ns.11 The overlap of fluorescence lifetime between the pH sensor and intrinsic cell autofluor-
INTRODUCTION Intracellular pH plays a significant role in many biological events.1−5 It can not only modulate the biological function of individual proteins but also affect the performance of subcellular organelles, such as lysosomes and mitochondria, commonly require proton gradients to function properly. In a normal mammalian cell, the intracellular pH varies in a broad range, from 4.5 in lysosome to 8.0 in mitochondrial, which is heterogeneously distributed around the cell, as the signaling process is spatially and temporally dependent.2 An abnormal deviation of the pH units even by 0.1−0.2 in either direction can lead to dysfunction of the organelle that might finally result in cardiopulmonary and neurologic problems (e.g., cancer, stroke, and Alzheimer’s disease).6 As a consequence, detection and monitoring of the pH with subcellular spatial resolution in living cells is fundamentally important, which can afford deep insight into the better understanding of the cellular metabolism process as well as provide essential knowledge for the early diagnosis of fatal diseases. Intracellular pH sensing using fluorescent materials represents a potentially useful tool for real-time and in vivo monitoring of important cellular analytes. Recently, amounts of fluorescent probes have been developed for the measurement of pH such as fluorescent dyes, proteins, inorganic and organic © 2016 American Chemical Society
Received: January 25, 2016 Accepted: March 25, 2016 Published: March 25, 2016 9629
DOI: 10.1021/acsami.6b00857 ACS Appl. Mater. Interfaces 2016, 8, 9629−9634
Research Article
ACS Applied Materials & Interfaces
Ltd., Japan). Size measurement in solution was characterized by dynamic light scattering (DLS) (Zetasizer Nano-ZS90, U.K.). Transmission electron microscopy (TEM) characterizations were performed on a JEM-2011 electron microscope (JEM-1230, JEOL, Japan). FT-IR spectra were obtained from a Nicolet Avatar 370 (Thermo Fisher Scientific, Inc., Waltham, MA). The fluorescence lifetime and absolute quantum yield of BNPs were measured by the fluorescence spectrometer (Edinburgh FLS920). BNPs Fabrication and Dye Conjugation. For the fabrication of fluorescent BNPs, 4 mL of ethanol solution was added to a BSA solution (2 mL, 10 mg/mL) drop by drop at room temperature. The addition of ethanol resulted in the spontaneous formation of an opalescent suspension. Thereafter, 50 μL of 8% (v/v) glutaraldehyde was added to this colloidal suspension and kept stirring for 18 h to induce cross-linking. The resulting nanoparticles were purified by centrifugation and washing 3 times. The diameter of these BNPs is around 30 ± 5 nm (TEM). Through changing the concentration of BSA from 10 to 35 mg/mL, the diameter of BNPs can be readily regulated from around 30 to 90 nm. These freshly synthesized BNPs typically have two types of functional groups on the surface: amine and carboxylate, affording versatile routes for dye conjugation. In this work, Alexa 594-NHS was cross-linked with BNPs through the amine group via succinimidyl ester. Typically, 10 μL of 0.1 mg/mL Alexa 594-NHS was slowly added to the BNPs solution in 0.1 M phosphate buffer (pH 8.3) at room temperature and kept stirring for 6 h in dark. The unreacted dyes were then removed via dialysis in DI water for 12 h. The resulted dye-conjugated BNPs were stored at 4 °C. The buffers for pH measurements (from 5 to 10) were prepared by using acetate and phosphate buffer. To control the pH of the solution, the BNPs was mixed with the desired buffer in a volume-to-volume ratio of 1:10. Microscopic Imaging. The microscopic fluorescence imaging experiments were performed on a Nikon Ti−U inverted epifluorescence microscope (Nikon, Japan). The fluorescence from the nanoparticles was collected by a 100× objective (NA 1.3) and recorded with an EMCCD (ultra 897, Andor, U.K.). Before the cell imaging experiments, HeLa cells were transferred and planted on a 35 mm Petri dish with 14 mm bottom well in culture medium until the density reach to 60−70% confluence. For the pH sensing experiments, 50 μL of Alex 594 conjugated BNPs (58 ± 6 nm) were mixed with 1 mL of cell culture medium for 30 min at room temperature. The mixture was then added to the cell culture dish and coincubated with HeLa cells for 1 h at 37 °C. Then the medium was removed, and the cells were washed two times with PBS buffer to remove the residual probes. For the drug treatment experiments, 15 μL of 1 mg/mL rifampicin was added to the cell culture dish. The cell was further incubated for 3 h for the pH measurements. All of the data recorded by the EMCCD camera were analyzed by the public image processing software, ImageJ (http://rsbweb.nih.gov/ij/).
escence further complicates the accurate interpretation of the measured results. Ratiometric measurement uses the ratio of two fluorescent peaks (one is pH sensitive and the other is a pH independent reference peak) instead of the absolute intensity of one peak.12,13 This detection scheme effectively overcomes the limitations in absolute intensity measurements. The signal output is no more affected by the fluctuations of excitation light source and the noneven distribution of local concentration of the probe. Owing to these attractive merits, several interesting approaches have been proposed, including dye/dye, dye/ protein, and dye/nanoparticle.4,6,8,9,12,14−16 As a fluorescent probe for monitoring metabolic processes in living cells, three fundamental concerns need to be considered carefully. One is the biocompatibility of the matrix. The physical or chemical disturbance of the biological function of proteins or organelles might mislead the native mechanism of the interested biological process. The second issue is the fluorescence stability and brightness of the probe. Because most biological events do not occur transiently, dynamic and long-term observation is typically required for complete and accurate understanding of the metabolic mechanism. Furthermore, the cellular translocation efficiency of the probe is also an important issue that needs to be considered, which is usually determined by the physical dimension (typically less than 100 nm) and surface chemistry of the probe. From these considerations, the simple dye/dye conjugate is not an appropriate candidate for dynamic and long-term intracellular sensing because of the easy of photobleaching. Gene-coded fluorescent protein and semiconducting quantum dots are two commonly used fluorescence probes and exhibit better optical performance over individual dyes in pH sensing.17−19 However, the complicated procedures for the generation of fluorescent proteins make the first one hardly assessable in common chemical laboratory. The biological toxic effect from semiconducting quantum dots remains a critical issue under studying until now.20 In this work, we present the synthesis and characterization of newly developed fluorescent protein nanoparticles for the sensitive and robust ratiometric pH sensing. The fluorescent protein nanoparticles were synthesized through cross-linking the denatured BSA in alcohol solution. The as-synthesized BSA nanoparticles (BNPs) display bright fluorescence at 535 nm with an excitation wavelength at 495 nm. Importantly, the BNPs exhibit a direct and reversible optical response to pH change in the range of 6−8, indicative of a good candidate for pH sensing in cellular system. Through covalently cross-linking reference dye Alexa 594 to the surface of BNPs, a ratiometric pH sensor for cellular environment was readily achieved. In comparison with other nanocomposites for pH sensing, this approach combines the advantages as noted for the ratiometric measurement and avoids the limitations as discussed for the later two types of ratiometric sensor.
■
■
RESULTS AND DISCUSSION
Fluorescent BSA Nanoparticles Fabrication and Spectroscopic Characterizations. The native fluorescence of proteins is normally originated from the primary amino acid sequence containing aromatic groups such as phenylalanine, tryptophan, tyrosine and so on.17,21 Owing to the flexible structure, the π electrons in the native proteins are mainly localized, resulting rigid and large energy gap for electron transitions in the UV region. For example, the excitation and emission spectrum of BSA proteins is located at 295 and 355 nm, respectively. To generate fluorescence in the visible or even NIR region, we usually altered parts of the chemical structure of the native amino acid sequence to form large chromophores under appropriate chemical and biological reactions. For example, the chromophore from green fluorescent protein is a p-hydroxybenzylideneimidazolinone formed by Ser-Tyr-Gly in the native protein.17 Inspired by this concept, we anticipate that reorganization of the denatured proteins into a more rigid
EXPERIMENTAL SECTION
Materials and Reagents. Bovine serum albumin (BSA), glutaraldehyde, acetic acid, sodium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, rifampicin, and other reagents not mentioned were purchased from Sigma−Aldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), trypsin and penicillin-streptomycin were purchased from HyClone (Beijing, China). Alexa 594-N-Hydroxysuccinimide (Alexa 594-NHS) ester was obtained from life technologies (Eugene, Oregon). Apparatus. Fluorescence spectroscopy measurements were performed on a Hitachi F-4600 fluorescence spectrometer (Hitachi, 9630
DOI: 10.1021/acsami.6b00857 ACS Appl. Mater. Interfaces 2016, 8, 9629−9634
Research Article
ACS Applied Materials & Interfaces
the pH of the solution, we mixed the BNPs sample with appropriate acetate or phosphate buffer in a volume-to-volume ratio of 1:10. The measured fluorescence response in different pH buffers is shown in Figure 3a. A sigmodal curve was observed and showed a sharp transition in the range of pH 6 to 8. Previous reports have demonstrated that albumins exhibit a tendency to undergo reversible conformation variation in either native or denatured forms.10,22 The pH-induced fluorescence intensity variation might be largely caused by ionization produced chemical structure or morphological variation, which results in a slightly changed physical-chemical environment.22 On this account, the fluorescent BNPs could be served as a promising candidate for pH sensing owing to the reversible feature of ionization process.10,22 From the fluorescence spectra, no discernible spectrum shift in the acidic or basic buffer solution was noted. More importantly, the pH induced fluorescence fluctuation was indeed a fully reversible and fast process (Figure 3b and Figure S1). For biological pH sensing, the pH probes can be generally classified into two groups: one for cytosol measurement, which works in the pH range of 6.8− 7.4, and one for acidic organelles detection such as lysosomes and endosomes which functions over the pH range of 4.5−6.0.6 On this account, the fluorescent BNPs prepared herein present a good candidate for cytosol pH assay in cellular system. Ratiometric pH Sensor Fabrication and Spectroscopic Characterizations. To establish a ratiometric pH sensor, a reference dye insensitive to pH change is typically required as an inner reference.13 So far, two routes are commonly adopted for the fabrication of fluorescent ratiometric sensor: (1) particles with dyes physically embedded in a polymer network and (2) particles with dyes covalently conjugated on the nanoparticle surface. Although physical trapping is the most straightforward method, the undesired leakage effect will seriously degrade the performance of the probe owing to the weak interaction between the dye and matrix. As a consequence, to form a stable sensor for intracellular pH measurements, we covalently conjugated the reference dye to the surface of BNPs. As Alexa 594 is insensitive to pH change and exhibits high fluorescence quantum yield in biological surroundings, Alexa 594-NHS was thus chosen as the reference dye. Figure S2 shows the FT-IR spectrum of BNPs, typical peaks ascribed to the stretching of −NH, −OH, as well as −COO− can be discerned (3299, 2960, and 1656 cm−1 correspond to the typical stretching modes of −NH, −OH, and −COO−, respectively). The cross-linking can thus be realized through the efficient and specific reaction between the −NHS and −NH2 group. After the cross-linking process, excessive Alexa 594-NHS dyes were removed by dialysis. As shown in Figure 3c, when the nanocomposite was excited at 490 nm, only single discernible fluorescence peak corresponding to BNPs could be found. The characteristic emission at 610 nm assigned to Alexa 594 has to be excited at 590 nm, indicative of a low or negligible FRET effect in this nanocomposite. The leakage effect of covalently conjugated dye from BNPs was tested by transferring the purified particles into a dialysis cassette (MWCO: 10K) and immersing the sample in a continuously stirred DI water solution. The fluorescence intensity at 610 nm from the solution outside of the dialysis cassette was measured as a function of time. To compare the leaching effect via physical association, we used Alexa 594 without NHS group as a control. In this case, Alexa 594 was trapped inside BNPs through mixing the two samples together
structure through chemical cross-linking might induce the overlapping or reformation of chromophore groups along the amino acid sequence, resulting in altered electronic energy gap for visible wavelength fluorescence emission. In this regard, we first denatured the native structure of BSA proteins in ethanol solution for 10 min, which effectively broke the tertiary or even secondary structure of the protein, making those amino, carboxyl and hydroxyl groups available. Glutaraldehyde (8%, v/v) was then gradually added to initiate the cross-linking of functional groups among the denatured BSA proteins. The solution gradually turned to light yellow after the cross-linking process as displayed in Figure 1.
Figure 1. (a) Schematic diagram for fluorescent BNPs fabrication. (b, dashed lines) Excitation and (solid lines) emission spectra of (blue) BSA and (green) BNPs.
Interestingly, upon exposure to UV irradiation, the cross-linked BNPs displayed noticeable green fluorescence. Figure 1 shows the measured fluorescence spectra of BSA proteins before and after cross-linking. A significant red shift in both excitation (from 295 to 495 nm) and emission peaks (from 355 to 535 nm) was readily observed. The absolute fluorescence quantum yield of these BNPs was estimated to be 20% and fluorescence lifetime was 2.73 ns. The morphology of the cross-linked proteins was revealed by transmission electron microscopy (TEM) measurements as illustrated in Figure 2a. Generally, the cross-linked BSA proteins were spherical in shape and welldispersed in aqueous solution with a diameter of about 30 ± 5 nm (about 41.1 nm by DLS measurement). This is in good agreement with the fluorescence microscopic characterizations where the BNPs showed bright and homogeneous fluorescence after being fixed on a glass slide surface, Figure 2b. Interestingly, the size of the resulted BNPs was BSAconcentration dependent. By varying the concentration of BSA from 10 mg/mL to 35 mg/mL, the size of the fluorescent BNPs could be readily modulated from around 30 to 90 nm in diameter, yet the excitation and emission properties remained unchanged, suggesting its potential use in drug delivery applications. The fluorescence response of BNPs at different pH values was evaluated in the pH range of 5−10. To precisely control 9631
DOI: 10.1021/acsami.6b00857 ACS Appl. Mater. Interfaces 2016, 8, 9629−9634
Research Article
ACS Applied Materials & Interfaces
Figure 2. (a) TEM images of different size BNPs synthesized from BSA solution with concentrations from 10 to 35 mg/mL; diameters of the BNPs are (left to right) 30 ± 5, 58 ± 6, and 90 ± 11 nm. (b) Fluorescent microscopic images of the corresponding BNPs shown in panel a. (c) Corresponding DLS measurements of BNPs with hydrodynamic diameters of 41.1 ± 3, 69.2 ± 8, and 97.6 ± 10 nm, respectively.
on the microscope and then filled with the probes under different pH solutions. A calibration curve comparable to that determined by the spectrometer was then achieved by adjusting the intensity of the laser sources. The cell imaging experiments were then performed under the same excitation intensity. A set of typical fluorescence images from the cell loaded with pH probes is shown in Figure 4. The pH distribution within the cell was mapped by calculating the fluorescence intensity ratio between the green and red channels. Without additional chemical or biological stimulation, the pH of the cell was around 7, indicative of a healthy state of the cell. For chemical treatment of cancer, the drugs typically initiate a programmable cell death (i.e., apoptosis). Acidification of the cytosol is commonly accompanied by this process.1,3,5 With this pH sensor, it would be possible to measure the pH change of the cell after the addition of anticancer drugs. As a proof of concept experiment, we treated the cell with rifampicin, a kind of drug with anticancer capability. Figure 4e−h shows the microscopic results of the cell treated with rifampicin for 3 h. As demonstrated in the ratiometric image, noticeable acidification was observed inside the cell. Importantly, it is evident that the pH was not evenly distributed around the cell in contrast to the case when the cell was not treated with drug. This heterogeneous pH distribution would be associated with the process of cell death.
overnight. Those unloaded dyes were removed through ultrafiltration. The time-dependent fluorescence intensity tracks from these two samples are shown in Figure S3. Obviously, around 50% of the dyes under physical embedding leached out within 200 min whereas no detectable dyes could be observed from the covalently conjugated sample. The pH response of this ratiometric sensor was characterized by immersing the samples in a set of buffers with different pH values (from 5.5 to 9). The fluorescence of BNPs and Alexa 594 was measured independently by exciting the sample at 495 and 590 nm respectively, Figure 3c. As expected, the fluorescence from BNPs decreased gradually when the pH of the solution was increased while the peak assigned to Alexa 594 still maintained constantly. The fluorescence ratio of these two peaks I535/ I610 in the range of pH 6 to pH 8 can be described with a linear curve y = −0.19x + 3.3. On the basis of this intensity ratio calibration curve, we can reliably and accurately determine the pH of the surrounding where the sensor is located. pH Measurements inside Living Cells. To explore the capability of this probe for biological sample measurement, we characterized the pH value inside living cells. The pH probe was first coincubated with the cell for 1 h and then washed out. To maximize the density of the probes inside the cytosol, we continuously cultured the cells for additional 3 h. Before the cell imaging experiments, the intensity of the light sources was corrected. To this end, a micro flow channel was first installed 9632
DOI: 10.1021/acsami.6b00857 ACS Appl. Mater. Interfaces 2016, 8, 9629−9634
Research Article
ACS Applied Materials & Interfaces
Figure 3. (a) The pH-dependent fluorescence response curve (at the peak emission wavelength) of BNPs in different pH buffers. (b) The fluorescence response curve of BNPs when the pH of the solution was toggled between 6 and 8 repeatedly. (c) The pH-dependent fluorescence response curves of BNPs/Alexa 594 composite with excitation at 495 and 590 nm, respectively. (d) The fluorescence intensity ratio of I535/I610 against the pH of the solution.
Figure 4. From left to right are the bright field, green channel fluorescence, red channel fluorescence and the ratio image between green and red channel of HeLa cells (a−d) without and (e−h) with anticancer drug treatment, respectively.
■
CONCLUSIONS
above. For example, the signal output will be no more affected by the fluctuations of excitation light source and the noneven distribution of local concentration of the probe, which is particularly suitable for the pH measurement in complex biological environment, that is, inside living cells. With this probe, the pH change of the cancer cell before and after the treatment of anticancer drugs was observed. Additional
In summary, we demonstrated a new ratiometric method for the measurement of pH value in solution and inside living cells with a biocompatible protein nanoparticle BNP as the probe. Distinct from other probes for pH sensing, such as absolute intensity measurement, lifetime imaging, and so on, this ratiometric approach displays notable advantages as noted 9633
DOI: 10.1021/acsami.6b00857 ACS Appl. Mater. Interfaces 2016, 8, 9629−9634
Research Article
ACS Applied Materials & Interfaces
(11) Orte, A.; Alvarez-Pez, J. M.; Ruedas-Rama, M. J. Fluorescence Lifetime Imaging Microscopy for the Detection of Intracellular pH with Quantum Dot Nanosensors. ACS Nano 2013, 7, 6387−6395. (12) Sun, H.; Scharff-Poulsen, A. M.; Gu, H.; Almdal, K. Synthesis and Characterization of Ratiometric, pH Sensing Nanoparticles with Covalently Attached Fluorescent Dyes. Chem. Mater. 2006, 18, 3381− 3384. (13) Grillo-Hill, B. K.; Webb, B. A.; Barber, D. L. Ratiometric Imaging of pH Probes. Methods Cell Biol. 2014, 123, 429−448. (14) Lee, M. H.; Kim, J. S.; Sessler, J. L. Small Molecule-Based Ratiometric Fluorescence Probes for Cations, Anions, and Biomolecules. Chem. Soc. Rev. 2015, 44, 4185−4191. (15) Berbasova, T.; Nosrati, M.; Vasileiou, C.; Wang, W.; Lee, K. S. S.; Yapici, I.; Geiger, J. H.; Borhan, B. Rational Design of a Colorimetric pH Sensor From a Soluble Retinoic Acid Chaperone. J. Am. Chem. Soc. 2013, 135, 16111−16119. (16) Nie, H.; Li, M.; Li, Q.; Liang, S.; Tan, Y.; Sheng, L.; Shi, W.; Zhang, S. X.-A. Carbon Dots with Continuously Tunable Full-Color Emission and Their Application in Ratiometric pH Sensing. Chem. Mater. 2014, 26, 3104−3112. (17) Tsien, R. Y. The Green Fluorescent Protein. Annu. Rev. Biochem. 1998, 67, 509−544. (18) Benčina, M. Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors. Sensors 2013, 13, 16736−16758. (19) Hanson, G. T.; McAnaney, T. B.; Park, E. S.; Rendell, M. E. P.; Yarbrough, D. K.; Chu, S.; Xi, L.; Boxer, S. G.; Montrose, M. H.; Remington, S. J. Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors: Structural Characterization and Preliminary Application. Biochemistry 2002, 41, 15477−15488. (20) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Probing the Cytotoxicity of Semiconductor Quantum Dots. Nano Lett. 2004, 4, 11−18. (21) Day, R. N.; Davidson, M. W. The Fluorescent Protein Palette: Tools for Cellular Imaging. Chem. Soc. Rev. 2009, 38, 2887−2921. (22) El Kadi, N.; Taulier, N.; Le Huérou, J. Y.; Gindre, M.; Urbach, W.; Nwigwe, I.; Kahn, P. C.; Waks, M. Unfolding and Refolding of Bovine Serum Albumin at Acid pH: Ultrasound and Structural Studies. Biophys. J. 2006, 91, 3397−3404.
insightful mechanistic information for the anticancer effect of the drugs could therefore be elucidated by exploring the dynamic pH change of the cell in a spatially resolved route. More importantly, owing to the polymeric structure of this nanostructure, additional chemical reagents or stimulus could be integrated into the nanocomposite, opening a new way for imaging guided treatment in nanomedicine.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00857. Supplementary figures. (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions
Q. Y. and Z. Y. contributed equally. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by NSFC (21205037, 21405045, 21522502, 51373002), Program for New Century Excellent Talents in University (China, NCET-13-0789), Hunan Natural Science Funds for Distinguished Young Scholar (14JJ1017) and Project funded by China Postdoctoral Science Foundation (2014M550418, 2015T80867).
■
REFERENCES
(1) Webb, B. A.; Chimenti, M.; Jacobson, M. P.; Barber, D. L. Dysregulated pH: a Perfect Storm for Cancer Progression. Nat. Rev. Cancer 2011, 11, 671−677. (2) Casey, J. R.; Grinstein, S.; Orlowski, J. Sensors and Regulators of Intracellular pH. Nat. Rev. Mol. Cell Biol. 2010, 11, 50−61. (3) Damaghi, M.; Wojtkowiak, J. W.; Gillies, R. J. pH Sensing and Regulation in Cancer. Front. Physiol. 2013, 4, 370. (4) Wan, Q.; Chen, S.; Shi, W.; Li, L.; Ma, H. Lysosomal pH Rise During Heat Shock Monitored by a Lysosome-Targeting NearInfrared Ratiometric Fluorescent Probe. Angew. Chem., Int. Ed. 2014, 53, 10916−10920. (5) Lagadic-Gossmann, D.; Huc, L.; Lecureur, V. Alterations of Intracellular pH Homeostasis in Apoptosis: Origins and Roles. Cell Death Differ. 2004, 11, 953−961. (6) Tang, B.; Yu, F.; Li, P.; Tong, L.; Duan, X.; Xie, T.; Wang, X. A Near-Infrared Neutral pH Fluorescent Probe for Monitoring Minor pH Changes: Imaging in Living HepG2 and HL-7702 Cells. J. Am. Chem. Soc. 2009, 131, 3016−3023. (7) Shi, W.; Li, X.; Ma, H. Fluorescent Probes and Nanoparticles for Intracellular Sensing of pH Values. Methods Appl. Fluoresc. 2014, 2, 042001. (8) Han, J.; Burgess, K. Fluorescent Indicators for Intracellular pH. Chem. Rev. 2010, 110, 2709−2728. (9) Chan, Y.-H.; Wu, C.; Ye, F.; Jin, Y.; Smith, P. B.; Chiu, D. T. Development of Ultrabright Semiconducting Polymer Dots for Ratiometric pH Sensing. Anal. Chem. 2011, 83, 1448−1455. (10) Wu, Y.; Chakrabortty, S.; Gropeanu, R. A.; Wilhelmi, J.; Xu, Y.; Er, K. S.; Kuan, S. L.; Koynov, K.; Chan, Y.; Weil, T. pH-Responsive Quantum Dots via an Albumin Polymer Surface Coating. J. Am. Chem. Soc. 2010, 132, 5012−5014. 9634
DOI: 10.1021/acsami.6b00857 ACS Appl. Mater. Interfaces 2016, 8, 9629−9634
