Polymeric Micelles Encapsulating a Small Molecule SO2 Fluorescent

Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Polymeric Micelles Encapsulating a Small Molecule SO2 Fluorescent Probe Exhibiting Novel Analytical Performance and Enhanced Cellular Imaging Ability Chuang Jiang,† Guifeng Zhang,‡ Gongze Peng,§ Yan-Hong Liu,† Yingjun Kong,*,‡ and Bo-Lin Wang*,† †

College of Chemistry, Sichuan University, No. 29, Wangjiang Road, Chengdu 610064, P. R. China State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China § Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center of Artificial Organ and Tissue Engineering, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, P. R. China

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ABSTRACT: Because of the limited knowledge on the relationship between molecular structure and analytical performance, developing a small molecule fluorescent probe with desirable response properties is usually a laborious work. On the other hand, the application of small molecule fluorescent probe in biological samples is always limited due to the unwanted interaction between dyes and biomacromolecules. Polymer micelles, thanks to its unique core−shell structure, may have the potential to improve these situations. However, utilization of polymer micelles to improve these situations is rarely explored. Herein, we engineered the first micellar SO2 nanoprobe Nano-Cz by self-assembly of a carbazole-based SO2 small molecule probe and an amphiphilic copolymer (DSPE-mPEG2000). The optical and cell imaging experiments revealed that Nano-Cz can work in 100% aqueous environment and act as an effective mitochondrial-targeting ratio SO2 nanoprobe. Compared with the single small molecule probe, Nano-Cz showed extraordinary large dynamic response range (0−0.7 mM vs 0−50 μM), eliminated signal interference from DNA and superior cellular imaging performance. These results clearly show the ability of polymer micelles in modulating sensors’ analytical performance and reducing the signal interference from the unwanted interaction between small molecule probe and biomacromolecule, indicating that polymer micelles encapsulating single small molecule probe can provide us an alternative strategy to explore sensors with various performance and promote the biological application of fluorescent sensors. In addition, we hope that more and more polymer micelles would be used to construct biosensors in the future. KEYWORDS: polymer micelles, fluorescent sensor, sensing performance, biological application, sulfur dioxide

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INTRODUCTION It is scarcely necessary to stress the importance of fluorescent sensors in modern biology. There is vast document on this topic. Small molecule fluorescent sensor, a responsive small molecule fluorophore, which shows a change in fluorescence properties in the presence of its analyte, is of particular concern. Up to now, a variety of small molecule probes are available that target ions,1,2 biomolecules,3−6 cell microenvironment,7,8 and various biological events.9−11 Benefiting from these achievements, researchers now can easily devise a responsive fluorescent sensor when they know an effective recognition unit. However, developing a small molecule fluorescent sensor that fully coordinates with biological application is still a difficult task. One difficulty is attributed to the limited knowledge on the relationship between molecular structures and analytical performance. When researchers design a new sensor, its analytical performance (such as selectivity, sensitivity, dynamic response range, etc.) is always unpredict© XXXX American Chemical Society

able. On account of this, only through the alteration of molecular structure to explore a sensor with desirable analytical performance in physiological environment is always a laborious task. Another difficulty is associated with sensors’ biocompatibility and complex biological environment, which always lead to several problems (including requirement of organic cosolvents, nonspecific interaction, unable to reach the target region, and heterogeneous signal response) when translating a sensor from controlled homogeneous environment of a cuvette to far more complex in vivo settings. Therefore, exploring effective methods capable of promoting the development and application of fluorescent sensors is significantly meaningful. Polymer micelle, a nanoscopic structure formed through the self-assembly of amphiphilic copolymer and extensively studied in drug delivery,12−14 was recently demonstrated to play great Received: October 1, 2018 Accepted: November 29, 2018

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DOI: 10.1021/acsabm.8b00576 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 1. Synthesis and characterization of micellar nanoprobe Nano-Cz. (a) Molecular of Cz-ID and schematic illustration of the preparation of Nano-Cz. (b) DLS size distribution of Nano-Cz. (c) Morphologic structure of Nano-Cz studied by transmission electron microscopy (TEM), the scale bar is 50 nm. (d) Steady absorption and fluorescence spectra of Cz-Id (red and green lines, 3.0 μM) and Nano-Cz (black and blue lines, 0.1 mg/mL) in PBS buffer, λex = 350 nm.

and promote sensors’ practical application. To explore the potential talents of polymer micelles in biosensing, we constructed a micellar SO2 nanoprobe Nano-Cz (Figure 1a) by self-assembly of a carbazole-based SO2 fluorescence sensor Cz-Id and an amphiphilic copolymer (DSPE-mPEG2000). In 100% aqueous environment, Nano-Cz showed good analytical performance in vitro and improved imaging ability in HepG2 cells. Compared with the single fluorophore Cz-Id, Nano-Cz exhibited several new properties including large dynamic response range (up to 0.7 mM, only 50 μM for Cz-Id), eliminated signal interference from DNA, and superior cellular imaging performance. These results clearly showed the different analytical performance of Cz-Id after being encapsulated into polymer micelles and the ability of polymer micelles in reducing the signal interference from the unwanted interaction between small molecule probe and biomacromolecule, which suggested that polymer micelles encapsulating single small molecule probe can be an effective method to develop novel sensors with various performance and also promote the biological application of fluorescence sensors.

roles in promoting the development and application of fluorescent sensors. For example, Gao’s group developed a library of ultra-pH-sensitive fluorescent nanoprobe based on the supramolecular self-assembly of ionizable block copolymer;15 L. Gibbs and co-workers used polymeric micelles as carriers for nerve-highlighting fluorescent probe delivery;16 Nocera, et al. employed micelle-encapsulated quantum dotporphyrin assemblies as in vivo two-photon oxygen sensors;17 Klymchenko’s group developed an ultrabright FRET platform for amplified detection of nucleic acids by DNA-functionalized dye-loaded polymeric nanoparticles;18 etc.19−33 Considering the analytical performance of many organic fluorescent probe is related to the solvent environment and the core of polymer micelles has a completely different environment from aqueous solution, polymer micelles encapsulating fluorophores may provide us a new alternative to develop sensors with desirable performance, which would be a supplement to chemical structure modification. On the other hand, the hydrophilic part of polymer micelles can protect the hydrophobic part from the biological invasion, which may facilitate the application of fluorescence sensors through decreasing the unwanted interaction between biological environment and fluorophores. However, these potential roles of polymer micelles are rarely explored and utilized in biosensing. Recently, development of fluorescence sensors for SO2 derivatives has drawn great attention,34−39 especially since Guo’s pioneering work on the utilization of α,β-unsaturated compound for HSO3− detection.40 However, almost all the reported SO2 fluorescence sensors were organic small molecules-based, and many of these sensors showed poor water solubility or compromised performance in aqueous. In addition, because of the large aromatic structure, these sensors may suffer signal interference from the nonspecific interaction between fluorophores and biomacromolecules. For example, carbazole derivatives have been used as sulfite sensors,41−44 but many reports have also demonstrated that carbazole derivatives can also interact with DNA G-quadruplex (a very important biology macromolecule).45−48 As mentioned above, polymer micelles may have the potential to overcome these drawbacks

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RESULTS AND DISCUSSION Preparation and Characterization of Nano-Cz. The small molecule SO2 fluorescent probe Cz-Id was synthesized according to the literature.44 Figure 1a illustrates the major steps of Nano-Cz synthesis. The synthesis started with the preparation of a homogeneous CHCl3 solution containing DSPE-mPEG2000 (5.0 mg), Cz-Id (0.08 mg). After CHCl3 was evaporated, deionized water was added and the solution was sonicated for 30 min; in this step, DSPE-mPEG2000 tended to form micelles and encapsulated fluorescent dyes CzId into the hydrophobic core. Subsequently, the solution containing Nano-Cz was filtered through a polyvinylidene fluoride (0.22 μm) syringe, dialyzed with highly pure water and freeze-drying. Then a pink powder (Nano-Cz) was obtained. The amount of Cz-Id molecules in the synthesized Nano-Cz was determined by absorption spectra. According to the calibration curve in Figure S1a and the absorbance of Nano-Cz B


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various concentration of HSO3− in 10 mM PBS solution. With the increasing concentration of HSO3−, the emission intensity at 585 nm decreased along with a blue-shifted emission signal at 449 nm increased, and an isoemissive point at 541 nm appeared. Moreover, the ratio of the emission intensities at 449 and 585 nm (I449/I585) was increased (∼50-fold, from 0.083 to 4.14) with the addition of HSO3− and became constant when the amount of HSO3− added up to ∼700 μM (Inset in Figure 2a). An excellent linearity (R2 = 0.9939) was obtained when the fluorescence intensity ratios were plotted against the concentration of NaHSO3 ranging from 0 to 200 μM (Figure S2). The detection limit for HSO3− was determined to be 1.1 μM based on S/N = 3, which is sufficient to probe the HSO3− concentration in cells. Next, the selective experiments of Nano-Cz to other small molecules and anions were conducted. No significant ratio fluorescence intensity changes (Figure 2b, blue bar) were observed after addition of representative anions (F−, Cl−, Br−, I−, NO3−, AcO−, H2PO4−, HCO3−, SO42−, ClO−, SCN−, S2O32−, S2−) and biothiols (Cys, GSH), implying that Nano-Cz has a high selectivity. Moreover, the ratio intensity changes caused by the addition of HSO3− were not influenced by the presence of the other relevant species (Figure 2b, red bar). In addition, Nano-Cz exhibited high photostability, only 7.6% fluorescence intensity attenuation after 50 min continuous laser irradiation (Figure 2c). Kinetics experiment (Figure 2d) revealed that the emission intensity ratio (I449 /I585) increased with time and reached a maximum plateau in about 5 min, which is as fast as the response time (5 min) of Cz-Id in PBS,45 but slower than the response time (about 1 min) in the mixed solvent (PBS: DMF = 7:3).45 It has been reported that the sensing mechanism of Cz-Id to HSO3− is via nucleophilic addition reaction. The addition reaction of Nano-Cz with HSO3− was validated by HRMS (Figure S3) where two peaks at m/z = 365.2013 (attributed to [Cz-Id − I−]+, 365.2012) and m/z = 447.1740 (attributed to [Cz-Id − I− + H2SO3]+, 447.1742) were found, the continuously evenly distributed peaks were characteristic mass spectrum peaks of PEG. Moreover, the identical retention time in Figure S4 further indicated the same reaction mechanism of Nano-Cz and Cz-Id. Previous studies have suggested that carbazole derivatives have the potential to interact with DNA G-quadruplex, which may compromise their imaging performance in biological samples. Because of the unique core−shell structure, polymer micelles may have the ability to reduce the unwanted interaction between small molecule fluorescence probe and biomacromolecules. To demonstrate this, the fluorescence titration experiments of Cz-Id and Nano-Cz to src1 (a guanine-rich DNA sequence, which can form DNA Gquadruplex at high K+ concentration) were conducted. With the gradual addition of src1, the emission intensity of Cz-Id was increased steadily, while no signal enhancement was observed for Nano-Cz (Figure 3), confirming the great value of polymer micelles in biosensing. Overall, as the first micellar-based SO2 fluorescence nanoprobe, Nano-Cz exhibited good analytical performance in 100% aqueous solution (including ratio response, wide linear response range, high selectivity, good photostability, enough sensitivity, and rapid response time). Compared with the single fluorophore Cz-Id, we can observe that polymer micelle have huge effects on the analytical performance. NanoCz showed several different sensing characteristics such as broader dynamic response range (up to 0.7 mM, only 50 μM

in chloroform (Figure S1b), the loading amount of Cz-Id in Nano-Cz was estimated to be 30 nmol/mg. The size of NanoCz was investigated by dynamic light scattering (DLS), which suggested that the Z-average hydrodynamic diameter is about 35.6 nm. Figure 1b shows the size distribution of Nano-Cz in water. The morphology of Nano-Cz was further investigated using transmission electron microscopy (Figure 1c), which indicated that they are in spherical shape with an average size of about 37.9 nm. Furthermore, the optical properties of NanoCz were studies in PBS buffer (10 mM, pH = 7.4). Compared with Cz-Id, the maximum absorption and fluorescence peaks of Nano-Cz showed slightly red and blue shift (Figure 1d), from ∼483 and ∼593 nm for Cz-Id to ∼490 and 585 nm for NanoCz, indicative of the poor environment sensitivity of Cz-Id. By contrast, the extinction coefficient at the absorption maximum and fluorescence quantum yield of Nano-Cz (ε = 3.23 × 104 M−1 cm−1, ΦF = 0.26) is ∼1.54- and ∼2.36-fold larger than that of Cz-Id (ε= 2.10 × 104 M−1 cm−1, ΦF = 0.11), clearly demonstrating that micellar-based sensor Nano-Cz exhibited superior brightness than Cz-Id. Since luminophor’s extinction coefficient and fluorescence quantum yield are highly environment-dependent, the larger ε and ΦF may attributed to the different environment (such as, more hydrophobic, lower polarity and restricted molecules rotate) of micellar interior. General Analytical Performance of Nano-Cz in Vitro. To evaluate the analytical performance of the micellar nanoprobe Nano-Cz, a series of fluorescence experiments were conducted. First, the fluorescence titration experiments of Nano-Cz for HSO3− were conducted. Figure 2a shows the fluorescence titration spectra of 0.1 mg/mL Nano-Cz with

Figure 2. (a) Fluorescence spectra changes of Nano-Cz (0.1 mg/mL) upon gradual addition of NaHSO3 (0−700 μM) in 7.4 PBS buffer excited at 350 nm. Inset: relative intensity ratio (I449/I585) versus sulfite concentration. The spectra were recorded after incubation of the probe with sulfite for 5 min. (b) Fluorescence intensity ratio of 0.1 mg/mL Nano-Cz (at 449 and 585 nm) in 10 mM PBS buffer (pH = 7.40) to various species (1.0 mM Cys, 5.0 mM GSH, 200 μM Na2S, 50 μM NaHSO3, and 1.0 mM for other analysts. Black bar: Nano-Cz + various species. Red bar: Nano-Cz + various species + NaHSO3. Data are expressed as mean ± S.D., ∗p < 0.05 versus Nano-Cz (blank); ∧p > 0.05 versus Nano-Cz (blank); #p > 0.05 versus NanoCz+NaHSO3. (c) Time-dependent fluorescence intensity@585 nm changes of Nano-Cz (0.1 mg/mL) in 10 mM PBS. (d) Timedependent fluorescence intensity ratio (I449/I585) changes of 0.1 mg/ mL in the presence of 20 μM NaHSO3. λex = 350 nm. C


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Id (15.67), indicating the micellar sensor Nano-Cz also exhibited superior brightness than Cz-Id in living HepG2 cells. It is well established that SO2 can be enzymatically generated by AAT2 (aspartate aminotransferase 2) in mitochondria. Fluorescence sensor for HSO3− with mitochondria targeting is important. Although Cz-Id is a mitochondriatargeted fluorescence sensor, the cellular localization of NanoCz may be different. In general, the mitochondria targeting of small molecules is attributed to the molecular positive charge. After self-assembly, the charge density would be changed. On the other hand, small molecules usually enter cell by free diffusion, while nanoparticles enter cell by endocytosis. To test the localization of Nano-Cz, mitochondrial colocalization experiment of Nano-Cz was conducted. As seen from Figure 5, the signal of Nano-Cz merged very well with that of Mito

Figure 3. Emission spectra of Cz-Id (left) and Nano-Cz (right) increasing src1 concentration in 50 mM Tris-HCl buffering solution (pH = 7.2, containing 50 mM KCl). Src1 is a (G)-rich DNA sequence which can form noncanonical four-stranded helical structures Gquadruplexes in high K+ concentration. The sequence of src1:5′GGGCGGCGGGCTGGGCGGGG-3′. λex = 350 nm.

for Cz-Id), compromised sensitivity, and slower response time. Though not all the new sensing characteristics were better than the single small molecule fluorophore, this example provided us a direct view on the ability of polymer micelle in modulating single fluorophore’s analytical performance and reducing the interaction between small molecule sensor and biomacromolecule. We hope more and more polymer micelles would be used to explore novel sensor with novel performance. Considering the response mechanism of Cz-Id to HSO3− is based on nucleophilic addition, the extended dynamic response range may attribute to the changed electronic state of Cz-Id in the interior of polymer micelle. Confocal Imaging of Nano-Cz in Living Cells. Before living cells imaging, the cytotoxicity of Nano-Cz against HepG2 cells was investigated. After treating HepG-2 cells with different concentrations (0−0.20 mg/mL) of Nano-Cz for 24 h, the cell viability were determined by a CCK-8 assay. The results in Figure S5 revealed that Nano-Cz exhibited low toxicity when the concentration of Nano-Cz is below 0.15 mg/ mL. The cell viability decreased only ∼5% after 24 h at a concentration of 0.1 mg/mL, supporting this concentration of Nano-Cz could be used for imaging in living cells. For living cells imaging, fluorescent sensors require not only low toxicity, but also high brightness. The previous optical studies have clearly demonstrated that Nano-Cz exhibited superior brightness than Cz-Id in PBS buffer. Herein, we further investigated the brightness of Nano-Cz and Cz-Id in living HepG2 cells. Figure 4a and b show the confocal

Figure 5. Mitochondrial colocalization experiment of Nano-Cz in HepG2 cells. (a) Fluorescence image from Nano-Cz (0.1 mg/mL). Ex = 488 nm, Em = 500−580 nm. (b) Fluorescence image from Mito Tracker@ Deep Red FM (200 nM). Ex = 633 nm, Em = 650−710 nm. (c) Overlay of panels a and b. (d) Pearson’s coefficient of Nano-Cz (green channel) and Mito Tracker@ Deep Red FM (red channel) is 0.9403. Scale bar: 10 μm.

Tracker@ Deep Red FM. The Pearson’s coefficient of the Nano-Cz channel to the Mito-Tracker channel was calculated to be 0.9403, indicating that Nano-Cz is still an effective mitochondria-targeted sensor. The optical titration experiments in Figure 3 have demonstrated that polymer micelle (DSPE-mPEG200) encapsulating small molecule sensor Cz-Id is an efficient method to reduce the unwanted interaction between Cz-Id and DNA G-quadruplex in vitro. To test this method can also play a role in cells, DNA digest experiments were conducted. For Cz-Id, after incubation of the fixed and permeabilized HepG2 cells with Cz-Id, moderate fluorescence signal in the whole cells was observed (Figure 6a). However, after incubation DNase (50 unit/mL) treated HepG2 cells with Cz-Id, the brightness of fluorescence signal was decreased obviously (Figure 6b). For Nano-Cz, a strong fluorescence signal was observed after incubation of the fixed and permeabilized HepG2 cells (Figure 6c). And the fluorescence intensity of Nano-Cz in DNase (50 unit/mL) treated HepG2 cells was slightly changed (Figure 6d). To quantitatively evaluate the DNase digest experiments, the mean fluorescence intensity (represent by the average gray value) were further estimated by image processing. In comparison with in HepG2 cells without

Figure 4. (a) Fluorescence image of Cz-Id (3.0 μM). (b) Fluorescence image of Nano-Cz (0.1 mg/mL). (c) Average gray value of blue channel in panels a and b, data are expressed as mean ± S.D., ∗∗∗∗p < 0.0001 by Student’s t test. Ex = 405 nm, Em = 420−680 nm.

fluorescence imaging of Cz-Id (3.0 μM) and Nano-Cz (0.1 mg/mL) in HepG2 cells. Under the same experimental condition, we can observe that the brightness of Nano-Cz in living HepG2 cells is much brighter than Cz-Id. Image processing by Image pro plus 6.0 shows that the average gray value of Nano-Cz (89.84) is ∼5.73 fold larger than that of CzD


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sensor Cz-Id and an amphiphilic copolymer (DSPEmPEG2000). Nano-Cz exhibited good analytical performance in 100% aqueous environment and can ratio imaging of SO2 in living cells. Compared with Cz-Id, Nano-Cz showed large dynamic response range (0−0.7 mM vs 0−50 μM), eliminated signal interference from DNA and displayed superior luminance brightness, clearly demonstrated the ability of polymer micelles in modulating sensors’ analytical performance and reducing the signal interference from the unwanted interaction between small molecule probe and biomacromolecule. Therefore, these results suggested that polymer micelles encapsulating small molecule probe provide us an alternative method to develop fluorescent probe with various performance, which act as a supplement to chemical structural modification. And we believe that more and more polymer micelles would be used to explore fluorescent sensors in the future.

Figure 6. (a, b) Images of fixed and permeabilized HepG2 cells stained with 3.0 μM Cz-Id without and with DNase (50 unit/mL) treatment. (c, d) Images of fixed and permeabilized HepG2 cells stained with 0.1 mg/mL Nano-Cz without and with DNase (50 unit/ mL) treatment. Ex = 405 nm, Em = 420−680 nm. Scale bar: 25 μm. (e) Average gray values of blue channel in panels a−d, data represent mean value ± S. D., ∗∗p < 0.01, #p > 0.05 by Student’s t test.

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DNase treatment, the average gray value of Cz-Id and NanoCz in DNase treated HepG2 cells decreased ∼66% and ∼8% (Figure 6e), respectively. These results proved that polymer micelles also has the ability to reduce the negative effects which brought by the nonspecific interaction between biomacromolecules and small molecule fluorescence sensor in cells. Finally, ratiometric fluorescence imaging of Nano-Cz to HSO3− were studied and compared with Cz-Id in living HepG2 cells. After Nano-Cz (0.1 mg/mL) incubated for 30 min, the HepG2 cells showed weak fluorescence signal in blue channel (Figure 7b) and strong fluorescence signal in the red

EXPERIMENTAL SECTION

Determination of Fluorescence Quantum Yield. Rhodamine B (Φf = 0.97 in EtOH) was used as a standard to determine the quantum yield. The calculation equation can be found in the previous report.49 Fluorescence Titration Experiments of Nano-Cz to NaHSO3. For this, 0.1 mg/mL solution of Nano-Cz was freshly prepared by solubilizing a certain quality of Nano-Cz powder into 10 mM PBS buffer (pH = 7.4). Sodium bisulfite (NaHSO3) stock solutions were freshly prepared prior to each experiment. For the fluorescence titration experiments, a mixture containing the corresponding-fold molar ratio of NaHSO3 was added to the solution of Nano-Cz. After stewing for 5 min, the fluorescence spectra were measured at room temperature. Fluorescence Titration Experiments of Cz-Id and Nano-Cz to src1. Nano-Cz (0.1 mg/mL) and Cz-Id (3.0 μM) solution was freshly prepared by solubilizing a certain quality of Nano-Cz powder or Cz-Id stock solution into 50 mM Tris-HCl buffer (pH = 7.4, containing 50 mM KCl). For the fluorescence titration experiments, 10.0 μL of srcl stock solutions (100 μM) was added to 1.0 mL 50 mM Tris-HCl buffer solution containing 0.1 mg Nano-Cz or 3.0 nmol CzId each time, followed by uniform mixing, the fluorescence spectrum was collected. All the fluorescence spectra were measured at room temperature after stewing for 10 min. Cell Cultures before Confocal Imaging. The detailed experimental procedure about the HepG2 cells cultivation before confocal imaging can be seen in the experimental section of the previous report.49 Fluorescence Brightness Contrast. The prepared HepG-2 cells were washed 1× with PBS and then stained with 0.1 mg/mL Nano-Cz or 3.0 μM Cz-Id at 37 °C with 5% CO2 for 0.5 h. After rinsed with 0.01 M PBS twice, the cells were imaged, Ex = 405 nm, Em = 420−680 nm. Intracellular Fluorescence Imaging of SO2 Derivatives. The prepared HepG2 cells were seeded at confocal dishes (35 mm) for the confocal microscope imaging. After cultured for 24 h, the cells were washed with PBS, and then incubated with 0.1 mg/mL Nano-Cz or 3.0 μM Cz-Id at 37 °C with 5% CO2 for 0.5 h. Then the HepG2 cells were washed with PBS (3 × 1.0 mL/dish) to remove excess Nano-Cz or Cz-Id. Then the sensor-stained cells were treated with NaHSO3 for another 15 min and further imaged. The cells incubated with NanoCz or Cz-Id only for 0.5 h and equal volume PBS buffer without NaHSO3 for 15 min were used as the control groups. Signals were collected from the blue channel (Ex = 405 nm, Em = 450 ± 20 nm) and the red channel (Ex = 488 nm, Em = 590 ± 20 nm). Cytotoxicity Assays. Toxicity of Nano-Cz toward HepG2 cells was investigated by using a Cell Counting Kit-8. The detailed experimental description can be found in the previous paper.49 DNase Digest Tests. The prepared HepG2 cells were washed 1× with PBS then fixed in paraformaldehyde solution and again washed

Figure 7. Fluorescence images of HepG2 cells with probe Nano-Cz by confocal fluorescence imaging: the cells were stained with 0.1 mg/ mL Nano-Cz for 30 min, followed by incubation with NaHSO3. (a, e) Bright field; (b, f) images from the blue channel, Ex = 405 nm, Em = 430−470 nm; (c, g) images from the red channel, Ex = 488 nm, Em = 570−610 nm; (d) merged images of panels b and c; (h) merged images of panels f and g. Scale bar = 20 μm.

channel (Figure 7c). By contrast, when the Nano-Cz treated cells were further incubated with HSO3−, the brightness of blue channel was obviously enhanced (Figure 7f) accompanied with the decrease of red channel (Figure 7g), indicating Nano-Cz can image HSO3− in living cells. Different from Nano-Cz, Figure S6 shows that the addition of HSO3− to Cz-Id stained HepG2 cells only caused slight signal variation. These results demonstrated that polymer micelles can effectively improve the fluorescence imaging ability of small molecule fluorescence sensor in living cells.

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CONCLUSION In this work, to explore the roles of polymer micelles in biosensing, we constructed a micellar nanoprobe Nano-Cz through self-assembly of a carbazole-based SO2 fluorescence E


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2× in PBS then permeabilized with Triton X-100 in PBS buffer. After rinsed with 0.01 M PBS twice, PBS (as a control experiment), or 50 units mL−1 of DNase was added into cell culture dishes and then incubated at 37 °C in 5% CO2 for 3 h. After washed 3× in PBS, staining cells with 3 μM of Cz-Id or 0.1 mg/mL Nano-Cz in PBS at 37 °C. Following 3× thorough washing with PBS, the cells were imaged, Ex = 405 nm, Em = 420−680 nm.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00576. 1 H NMR and 13C NMR spectra of all Cz-Id; line relationship between fluorescent intensity ratio of NanoCz (I449/I585) and concentration of bisulfite; cytotoxicity of Nano-Cz to HepG2 cells; confocal fluorescence imaging of HepG2 cells with Cz-Id; loading amount of Cz-Id in Nano-Cz estimated by absorption spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Bo-Lin Wang: 0000-0003-1433-3896 Author Contributions

The manuscript was written by B.-L.W. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from National Key R&D Program of China (2017YFF0207800). ABBREVIATIONS BODIPY, fluoroboron dipyrrole; CCK-8, Cell Counting Kit-8; DLS, dynamic light scattering; FRET, fluorescence resonance energy transfer; HRMS, high-resolution mass spectroscopy; Φ, fluorescence quantum yield.

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Polymeric Micelles Encapsulating a Small Molecule SO2 Fluorescent

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