Article pubs.acs.org/ac
Self-Assembled Supramolecular Nanoprobes for Ratiometric Fluorescence Measurement of Intracellular pH Values Leiliang He, Xiaohai Yang,* Fang Zhao, Kemin Wang,* Qing Wang, Jianbo Liu, Jin Huang, Wenshan Li, and Meng Yang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, People’s Republic of China S Supporting Information *
ABSTRACT: Self-assembly of small building blocks into functional supramolecular nanostructure has opened prospects for the design of novel materials. With this molecular engineering strategy, we have developed self-assembled supramolecular nanoprobes (SSNPs) for ratiometric fluorescence measurement of pH values in cells. The nanoprobes with a diameter of ∼30 nm could be formulated just by mixing pH-sensitive adamantane−fluorescein (Ad-F) and pH-insensitive adamantane−Rhodamine B (Ad-R) with β-cyclodextrin polymer (poly-β-CD) at one time. The nanoprobes with good biocompatibility have been successfully applied to measure intracellular pH in the pH range of 4−8 and estimate pH fluctuations associated with different stimuli in cells. Moreover, we expect that this self-assembled approach is applicable to the construction of nanoprobes for other targets in cells just by replacing the respective indicator dyes with relevant indicators.
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effects.23−25 However, self-assembled supramolecular nanoprobes derived from host−guest chemistry to intracellular biological sensing and imaging studies, to the best of our knowledge, are investigated relatively sparse and still in their infancy. Herein, we present a novel ratiometric pH-sensing supramolecular nanoprobe that is constructed by linking together functional building blocks via host−guest interactions of β-cyclodextrin polymer (poly-β-CD) and dyes. The self-assembled supramolecular nanoprobes (SSNPs) used for ratiometric pH sensing were fabricated as depicted in Scheme 1. It contains (1) β-cyclodextrin polymer (poly-β-CD) (Mn = 94 400; see the section entitled Organic Synthesis and Characterization), selected as the nanoprobe backbone based on its stability, high solubilization and biocompatibility;26 (2) derivative dyes, pH-sensitive adamantane−fluorescein (Ad-F) and pH-insensitive adamantane−Rhodamine B (Ad-R) (see the section entitled Organic Synthesis and Characterization), used as functional building blocks for ratiometric pH response. By mixing these components together in an aqueous phase, Ad-F/ Ad-R/poly-β-CD self-assembled to form nanosized particles (named SSNPs), because of cooperation of the inclusion of AdF/Ad-R guests in the cyclodextrin cavity of poly-β-CD and the strong hydrophobic interactions between the dyes. The formation mechanism of SSNPs was not completely the same
ngineering nanostructures that are capable of changing properties, in response to specific inputs generated by surrounding environments, are playing increasingly important roles in fields ranging from cell biology to biomedicine.1−3 In particular, those nanostructures enabling pH probing in cells have attracted much interest,4−8 because of the pH value playing a vital role in various cellular events, such as cell growth and apoptosis,9,10 drug resistance,11 phagocytosis,12 and endocytosis.13 These pH-sensing nanostructures are constructed by (1) covalent interactions, for example, the covalent conjugate of functional molecules with silica nanoparticles,4 carbon dots5 or dextran14 and (2) noncovalent interactions, such as embedding multiple dyes into nanogels.6 Among these interactions, self-assembly of small building blocks into functional supramolecular nanostructures via host− guest interactions receive high interest, because of the ease of synthesis/assembly and the facile implementation of targeting ligands.15−17 For instance, the inclusion of hydrophobic guests into cyclodextrins, calixarenes, or cucurbiturils18,19 have been mainly used for the stimulus response system20 and siteselective biomedical delivery vehicles.21,22 In addition, because of the easy doping of different functional molecules in matrix materials, supramolecular self-assembly could be acted as a promising and practical approach for construction of ratiometric probes. Compared to intensity-based probes, the ratiometric probes using the built-in correction of two different emission bands are more ideal in practical applications, because they can correct the probe concentration and environmental © XXXX American Chemical Society
Received: December 1, 2014 Accepted: January 22, 2015
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DOI: 10.1021/ac504458r Anal. Chem. XXXX, XXX, XXX−XXX
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RPMI 1640 without FBS. The adherent cells were incubated with SSNPs for 2 h at 37 °C under 5 wt %/vol CO2. Dynamic light scattering (DLS) measurements were made on a Zetasizer 3000Hs (Malvern, U.K.). Ultraviolet−visible (UV-vis) and fluorescence spectroscopy were recorded on a microplate reader (Infinite M1000 Pro, Switz). Dye derivatives were purified by an Agilent (USA) 1200 series HPLC system on a reverse-phase C18 column. A scanning electron microscopy (SEM) image was recorded with a JSM-6700F instrument (Japan). Cell fluorescence micrographs were performed on a Model FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan). PMT voltages of blue, green, and red channels were 750, 660, and 720 V, respectively. Image processing and analysis was also performed on Olympus software (FV10ASW). Specially, the fluorescence intensity ratio (R) images of green (Igreen) to red (Ired) of each cell were obtained from green (505−545 nm) and red (575−615 nm) channels, using Olympus software (FV10-ASW). Organic Synthesis and Characterization. β-cyclodextrin polymer (poly-β-CD) was prepared by adapting published procedures.27 As shown in section S11 in the Supporting Information (Figure S11), the molecular weight of poly-β-CD (Mn ≈ 94 400) was measured by using gel permeation chromatography (GPC, Waters-515) and the 1H NMR spectra showed that most bands of β-CD at 4.2−3.4 ppm were broadened in the spectrum of poly-β-CD. Adamantane-labeled fluorescein (Ad-F) was synthesized using the following process: 5 mg (33 μmol) 1-adamantylamine, 1.8 mg (3.3 μmol) 6-carboxyfluorescein N-hydroxysuccinimide ester, 1 mL of dimethyl sulfoxide (DMSO), and 50 μL of N,N-diisopropylethylamine (DIPEA) were mixed, then subjected to vibration treatment overnight at 700 rpm (25 °C). The initial products (Ad-F) were purified by HPLC (section S1 in the Supporting Information (Table S1)). As shown in section S11 in the Supporting Information (Figure S9), ESI-MS, m/z: 508.1 ([M−H]−); 1H NMR (400 MHz, DMSO, ppm) δ: 7.99 (1H, s), 7.79 (1H, s), 7.62 (1H, s), 6.41 (6H, m), 3.02 (6H, s), 2.03 (3H, s), 1.62 (6H, s). Adamantane-labeled Rhodamine B (Ad-R) was synthesized using the following process: 5 mg (33 μmol) 1-adamantylamine, 1.6 mg (3.3 μmol) Rhodamine B isothiocyanate, 400 μL DMSO, 100 μL DIPEA, and 400 μL 60 mM carbonate buffer solution (pH 8.5) were mixed and subjected to vibrational treatment overnight at 700 rpm (25 °C). The initial products (Ad-R) were purified by HPLC (see section S1 in the Supporting Information (Table S2)). As shown in section S11 in the Supporting Information (Figure S10)), ESI-MS, m/ z: 651.2 ([M−Cl]+); 1H NMR (400 MHz, DMSO, ppm) δ: 6.98 (3H, m), 6.43 (6H, m), 3.26 (8H, m), 3.02 (6H, s), 1.97 (3H, s), 1.61 (6H, s), 1.24(12H, m). Determination of the pKa Value. The pKa value for AdF/poly-β-CD (10 μM Ad-F with 500 μM poly-β-CD) was estimated from the changes in the fluorescence intensity with various pH values, using the Henderson−Hasselbalch28 equation:
Scheme 1. (a) Schematic Presentation of the Self-Assembled Supramolecular Nanoprobes (SSNPs) Formation and the Fluorescence Changes of the SSNPs upon pH Variation (Inset: Photographs Showing the Fluorescence Responses of SSNPs between pH 4 (Left) and pH 9 (Right)). (b) Responsive Guest Molecules (Ad-F and Ad-R) and Supramolecular Host (poly-β-CD) Involved in SSNPs Formationa
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Legend: adamantane−fluorescein (Ad-F), adamantane−Rhodamine B (Ad-R), and β-cyclodextrin polymer (poly-β-CD).
as electrostatic interactions in the formation of siRNAcontaining delivery vehicles.21 SSNPs can give green and red fluorescence emission at basic pH and at acidic pH, respectively.
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EXPERIMENTAL SECTION Chemicals, Materials, and Instruments. β-cyclodextrin (β-CD) was obtained from J&K Scientific (China). Fluorescein N-hydroxysuccinimide ester was obtained from China Langchem, Inc. (China). 1-Adamantylamine, Rhodamine B isothiocyanate, nigericin, nacytelcysteine (NAC), chloroquine (CQ), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma−Aldrich (Germany). LysoTracker Blue DND-22 (LTB) was purchased from Molecular Probes (Invitrogen, USA). Anhydrous dimethyl sulfoxide (DMSO) and N,N-diisopropylethylamine (DIPEA) were obtained by molecular sieve dehydrating. Other chemicals and solvents were analytical grade and used without further purification. All solutions were prepared with Milli-Q ultrapure water (grade: 18.2 MΩ). Different pH buffer solutions (pH 3− 9) were prepared by using 0.1 M of citric acid and 0.2 M of disodium hydrogen phosphate, and the pH was adjusted by adding 2 M NaOH or HCl solutions. HeLa (human cervical cancer) cell lines were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum (FBS) and 100 IU/mL penicillin−streptomycin, and grown on glass-bottom culture dishes (MatTek Co.) in a humidified incubator containing 5 wt %/vol CO2 at 37 °C. For self-assembled supramolecular nanoprobes (SSNPs) loaded HeLa cells, the growth medium was removed and replaced with
⎛ FI − FI ⎞ log⎜ max ⎟ = pH − pKa ⎝ FI − FI min ⎠
where FI is the observed fluorescence intensity at a given pH, and FImax and FImin are the corresponding maximum and minimum, respectively. As shown in section S2 in the B
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Ad-F without poly-β-CD migrated (channel 3), but Ad-F in the presence of poly-β-CD remained (channel 2). As such, free AdR without poly-β-CD migrated (channel 5), but Ad-F in the presence of poly-β-CD remained (channel 4). Therefore, we could demonstrate that the inclusion complex of poly-β-CD/ dyes was stable. At the same time, SSNPs (2 μM Ad-F/8 μM Ad-R/500 μM poly-β-CD) remained (channel 1), which also demonstrated that the SSNPs was stable. Furthermore, compared with tube 2 and tube 3 (or compared with tube 4 and tube 5), it showed the fluorescence intensities of the inclusion complex were shown to increase, because of the microenvironmental changes of dyes after combination with poly-β-CD. The stability of the SSNPs in a pH 7.4 PBS buffer solution, a blood serum-containing (15% v/v) solution, and a HeLa cell lysate-contained solution was investigated, as well as the storage stability of the SSNPs in water. These data were obtained by excitation at 488 nm on a microplate reader (Infinite M1000 Pro, Switz). As shown later in this work in Figures 2a, 2b, and 2c, the corresponding solution was irradiated for different times (0, 10, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600 min) by using a 6 W hand-held UV lamp as the radiation source. In Figure 2d, the storage time of the SSNPs was 0, 1, 2, 3, 4, 6, 8, and 10 days, respectively. Spectroscopic Studied of Self-Assembled Supramolecular Nanoprobes (SSNPs) versus pH Value In Vitro. A series of different pH buffers (pH values of 3.0, 4.0, 4.6, 5.0, 5.6, 6.0, 6.6, 7.0, 7.6, 8.0, 9.0, 10.0) were prepared by mixing 0.1 M of citric acid and 0.2 M of disodium hydrogen phosphate at varied volume ratios. The pH value was measured usign a pH-meter. Then, 50 μL of the corresponding pH buffer and 50 μL of 2×SSNPs solution (4 μM Ad-F/16 μM Ad-R/1 mM poly-β-CD) were mixed in a 96-well Elisa plates to measure fluorescence spectrum (Ex = 488 nm). Toward fluorescence reversibility of SSNPs with pH, the pH of SSNPs solution between pH 4 and pH 9 was adjusted by adding 2 M NaOH or HCl solution, and then measured by pHmeter. The fluorescence spectra were obtained by excitation at 488 nm. Meanwhile, the antidilution performance of SSNPs under various pH values was estimated from the changes in the fluorescence ratio (F520/F575) signal. F520 and F575 indicate the fluorescence intensity at 520 and 575 nm, respectively. Interference Study and Cytotoxicity Assay. To study the effects of intracellular species on the fluorescence response of SSNPs, the changes in the fluorescence ratio (F520/F575) signal were investigated in the presence of metal ions (Na+, K+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+, Fe3+), anions (F−, ClO−, CO32−, NO2−, I−, NO3−, PO43−, Br−, SO32−, SO42−, Cl−, OAc−, HCO3−), oxidative-stress-associated redox chemicals (including glutathione (GSH), cysteine (Cys), H2O2) under physiological conditions (PBS buffer, pH 7.4, 37 °C) and different ionic strength. For more details, see section S8 in the Supporting Information. The cytotoxicity of SSNPs was evaluated by the standard MTT assay. Briefly, HeLa cells were seeded into 96-well Ubottom plates at a density of 7000 cells/well, and incubated with free dyes (Ad-F and Ad-R) and dyes/poly-β-CD complex at varied concentrations ([dyes] = 0, 5, 10, 20, and 50 μM; [poly-β-CD] = 0, 0.25, 0.5, 1, and 2.5 mM, respectively, as shown later in this work in Figure 4). The cells incubated with the culture medium only were served as the controls. After incubation for 24 h at 37 °C, the cells were washed with PBS for three times, and 0.1 mL MTT solution (0.5 mg/mL in PBS)
Supporting Information, the pKa values (y-intercept) of Ad-F/ poly-β-CD (pKa = 5.78 ± 0.10) was derived from the plot of pH vs log[(FImax − FI)/(FI − FImin)]. At the same time, the effect of pH on the fluorescence intensity changes of Ad-R/ poly-β-CD (10 μM Ad-R with 500 μM poly-β-CD) was also studied. The results demonstrated that the system of Ad-R/ poly-β-CD is pH-insensitive. Preparation and Characterization of Self-Assembled Supramolecular Nanoprobes (SSNPs). Preparation of the sensing 10×SSNPs (100 μM, calculated by total concentration of dyes) was as follows. The poly-β-CD was incubated with AdF and Ad-R at varied molar ratios (see section S4 in the Supporting Information) in water at one time, and subjected to vibration treatment for 4 h at 300 rpm (25 °C). 10×SSNPs composed of different feed ratios (from 4:1 to 1:19 Ad-F/AdR) could be formed. The mixtures were filtrated with an Amicon YM-10 filter to remove the free Ad-F and Ad-R. The resultant suspension was used in further experiments. As shown in section S3 in the Supporting Information, the binding constants (K-values) of Ad-F with poly-β-CD in buffer (pH 7.0) was 3.5 × 103 M−1, and that of Ad-R with poly-β-CD was 4.0 × 103 M−1. Because 1/(F − F0) vs 1/[β-CD] was a linear relationship, it indicated the formation of a 1:1 inclusion complex between the dyes with poly-β-CD. At the same time, according to our previous reports,29 the K-values of Ad-F or Ad-R with poly-β-CD under different pH values are ∼3.5 × 103 M−1, and more than 94% of dyes have combined with β-CD cavities of the poly-β-CD. Therefore, as described in section S6 in the Supporting Information, the average number of dye molecules on each nanoparticle is ∼1.7 × 103 dye molecules per nanoparticle. Single nanoparticle imaging of the SSNPs was as the follows. Glass coverslips (Fisher, 24 mm × 32 mm) were first immersed in Piranha solution (H2SO4:H2O2 = 7:3) for 60 min and then cleaned in ultrapure water in an ultrasonic bath for 30 min. The coverslips were shaken free of excess liquid and dried in a slide holder. Finally, the coverslips were vacuum plasma exposure for 2 min at 150 W. After these, the substrates were exposed to the SSNPs solution (1.0 nM, calculated by total concentration of dyes) by placing 5 μL drops onto the slides and allowing them to image at room temperature. Fluorescence micrographs were recorded on a Model FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan) with dual-wavelength excitation (488 nm for Ad-F and 550 nm for Ad-R) through a 100× oil immersion objective. To demonstrate whether the SSNPs could load two types of dyes in the same particle or not, the experiment of fluorescence resonance energy transfer (FRET) was performed in a buffer solution (pH 7.4) with excitation at 450 nm. Comparing the emission spectra of two-dye-doped inclusion complex (SSNPs) with that of single-dye-doped one, the SSNPs showed that the emission of Ad-F at 520 nm decreased and the emission of AdR at 575 nm increased (see section S5 in the Supporting Information (Figure S4b)), but the free dyes, as a control, could not show similar tendencies (see section S5 in the Supporting Information (Figure S4a)). These results demonstrated the existence of FRET between Ad-F and Ad-R in the SSNPs, and the observations also proved successful loading of the two types of dyes (Ad-F and Ad-R) in the same particle. To demonstrate whether the inclusion complex of poly-βCD/dyes was stable or not, the agarose gel electrophoresis (AGE) was performed. As shown in section S7 in the Supporting Information, under the effect of electric field, free C
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Analytical Chemistry was added to each well with incubation at 37 °C for 4 h. After the addition of DMSO (100 μL/well), the assay plate was shaken at room temperature for 10 min. Absorbance values of the wells were measured by using a microplate reader. Each concentration was tested at least three times. The cell viability was calculated based on measuring the UV-vis absorption at 570 nm (OD570), according to the following equation: cell viability =
OD570(sample) − OD570(blank) OD570(control) − OD570(blank)
Microscopic Documentation of Intracellular Uptake of Self-Assembled Supramolecular Nanoprobes (SSNPs). The HeLa Cells were incubated with SSNPs for 2h at 37 °C, and then the medium was replaced with fresh medium containing 2.0 μM LysoTracker Blue DND-22 (LTB) and incubated for 10 min. Cell fluorescence micrographs were performed on a Model FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan) with a 20× 0.75 NA objective (excitation at 488 nm for SSNPs and 405 nm for LTB). Intracellular pH Calibration. The HeLa cells were incubated with SSNPs for 2 h at 37 °C, and the growth medium without FBS was removed and replaced with high K+ buffer (30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES, and 20 mM NaOAc) at various pH values (pH = 4−8) in the presence of 10.0 μM of nigericin. After 30 min, the fluorescence images were measured, and the pH calibration curve was constructed with Olympus software (FV10-ASW). Fluorescence Imaging of Intact and Treated Cells. To explore the intracellular pH fluctuations associated with the different stimuli, the SSNPs-loaded HeLa cells treated with a redox substance (1 mM NAC or 0.1 mM H2O2,) or 0.1 mM CQ were incubated for 1 h at 37 °C in PBS (pH 7.4), and then the treated cells were subjected to fluorescence imaging. As a control, the SSNPs-loaded HeLa cells untreated with stimuli were used as intact cells.
Figure 1. (a) SEM image of the SSNPs and (b) the corresponding size distribution by dynamic light scattering. Single nanoparticle fluorescence imaging of the SSNPs from different channels: (c) fluorescence image from the fluorescein channel, (d) fluorescence image from the Rhodamine channel, and (e) a merger of images from the fluorescein channel and the Rhodamine channel.
loaded in the same particle (see section S5 in the Supporting Information). At the same time, the results from the agarose gel electrophoresis (AGE) could demonstrate that the inclusion complex of poly-β-CD/dyes was stable (see section S7 in the Supporting Information). Stability of the SSNPs under various environmental conditions (for example, human serum or human cell lysatecontained biological fluids) was investigated by monitoring its fluorescence ratio (F520/F575) changes (see Figure 2). The results showed that the prepared SSNPs have good photostability in complex environment and long storage time. In addition, according to our previous reports,29 Ad-F and Ad-R could form stable inclusion complexes with poly-β-CD, in which the binding constants of Ad-F or Ad-R with poly-β-CD
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RESULTS AND DISCUSSION The morphology and size of the SSNPs were first investigated by using scanning electron microscopy (SEM) and dynamic light scattering (DLS). SEM image showed that the nanoprobe was spherical in shape with uniform size distribution (∼30 nm) (see Figure 1a), and its average hydrodynamic diameter in water was ∼70 nm by DLS analysis (see Figure 1b). In order to further demonstrate that the formation of the SSNPs was involved with the spatial colocalization of the Ad-F and Ad-R in the same particle, single nanoparticle imaging of the nanoprobes was performed. As shown in Figures 1c−e, spots showed an even distribution, indicating that there was a negligible probability that spots derived from the nanoparticle overlap.30−32 In addition, the fluorescence microscopy images acquired from the green channel and the red channel showed that the nanoprobes were imaged as a combined color due to spatial colocalization of the Ad-F and Ad-R in the same particle (see Figure 1e). As shown in Figures 1c and 1d, ImageJ software analysis determined that the standard deviation of the fluorescence intensity of the green light spot was 10.2%, but that of the red light spot was 5.7%. Thus, we inferred that the standard deviation of the number of dye molecules in the SSNPs were 10.2% for Ad-F and 5.7% for Ad-R, respectively. Furthermore, the existence of FRET between Ad-F and Ad-R also could indicate that the two types of dyes were successfully
Figure 2. (a) Stability of the SSNPs in a pH 7.4 PBS buffer solution, (b) blood serum-contained (15% v/v) solution, (c) HeLa cell lysatecontained solution, and (d) the storage stability of the SSNPs in water. F520 and F575 indicate the fluorescence intensity of the SSNPs at 520 and 575 nm, respectively. These data were obtained by excitation at 488 nm on a microplate reader (Infinite M1000 Pro, Switz). In panels (a), (b), and (c), the corresponding solution was irradiated for different times (0, 10, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600 min) by using a 6 W hand-held UV lamp as the radiation source. In panel (d), the storage times of the SSNPs were 0, 1, 2, 3, 4, 6, 8, and 10 days. The error bars indicate the standard deviations of three experiments. D
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Analytical Chemistry under different pH values are ∼3.5 × 10 3 M −1, the photostability of Ad-F/poly-β-CD and Ad-R/poly-β-CD are higher than the corresponding free dyes. Fluorescence responses of the nanoprobe in vitro have been investigated as shown below. As a critical factor for the pH response of SSNPs, the molar feed ratio of Ad-F to Ad-R was first optimized. As shown in Figure 3a, the ratios of the two
Information). It was found that these species and ionic strength change scarcely affected the ratiometric fluorescent signal of SSNPs. Furthermore, the in vitro cytotoxicity of the SSNPs at varied concentrations was measured using a standard MTT assay. It was found that cell viability was not significantly affected after incubation with SSNPs for 24 h (Figure 4), clearly indicating the good biocompatibility of the SSNPs.
Figure 4. Cell viability values (%) estimated by MTT assay. HeLa cells were incubated with varied concentrations dyes/poly-β-CD (pink) and dyes (azure) at 37 °C for 24 h. Dyes contained Ad-F and Ad-R with fixed molar ratio of Ad-F to Ad-R (1:4). Cells without any addition were taken as a control, and the viability was set as 100%. The error bars indicated the standard deviations of three experiments.
Figure 3. (a) Plots of F520/F575 versus pH values for SSNPs prepared with different molar feed ratios from 4:1 to 1:19 (Ad-F/Ad-R). F520 and F575 indicate the fluorescence intensity at 520 and 575 nm, respectively. The error bars indicated the standard deviations of three experiments. (b) Fluorescence emission spectra of SSNPs (Ad-F/Ad-R = 1:4) at various pH values. (c) Reversible fluorescence ratio (F520/ F575) changes of SSNPs between pH 4 and pH 9. (d) Antidilution performance of SSNPs under various pH values (diluted with buffer). All data were obtained by excitation at 488 nm.
To demonstrate particle uptake of the SSNPs by cells and to investigate the intracellular distribution of the nanoprobe, colocalization experiments using the nanoprobe and organellespecific fluorescent dyes (LysoTracker Blue) have been performed. Particle uptake was achieved by exposing the cells to the SSNPs for 2 h, presumably by vesicular uptake mechanisms. The blue fluorescence from LysoTracker Blue could overlap with the green or red fluorescence from the nanoprobe, which indicated that the nanoprobe was localized to lysosomes (see section S9 in the Supporting Information). Therefore, we inferred that the nanoprobe could be internalized by cells and used to measure lysosomal pH value in cells. SSNPs have been further used to measure pH in cells. The intracellular pH was set to different values with the H+/K+ ionophore nigericin, which was well used for homogenizing the intracellular pH and culture medium.33 As shown in Figure 5, the fluorescence intensity of the fluorescein unit (green channel) in cells increased with pH value (4−8), whereas that of the Rhodamine unit (red channel) was almost unchanged. The ratio images of green intensity to red intensity, which showed a characteristic pH-dependent signal, demonstrated that the SSNPs could be effectively used to measure intracellular pH with a good linear calibration curve in the pH range from 4.0 to 8.0 (see Figure 6a). To demonstrate the utility of the newly constructed SSNPs, the average pH values of HeLa cells were measured when the cells were exposed to the different stimuli, such as NAC (Nacetylcysteine, a GSH precursor), H2O2, and chloroquine (CQ) (see Figure 6). As shown in the calibration curve in Figure 6a. The pH values for the untreated cells, as well as the NAC-, H2O2-, and CQ-treated cells, were determined to be 5.8 ± 0.1, 5.4 ± 0.2, 6.6 ± 0.2, and 6.9 ± 0.3, respectively. Because of the generation of reduced GSH intracellularly,34 NAC could decrease the intracellular pH to 5.4 ± 0.2, which was in agreement with previous reports that reducing substances
dopants ranging from 4:1 to 1:19 make the pH-responsive sensitivity of the nanoprobe different. The SSNPs prepared with a molar ratio of 1:4 (Ad-F to Ad-R) showed distinct fluorescence ratio (F520/F575) changes at pH = 4−8, which could cover most of the pH ranges for biological applications. The fluorescence response of SSNPs in a wide pH range (pH 4−8) can be explained by the features of pH-sensitive adamantane−fluorescein (Ad-F)/poly-β-CD (pKa = 5.78 ± 0.10) and pH-insensitive adamantane−Rhodamine B (Ad-R)/ poly-β-CD (see section S2 in the Supporting Information). Therefore, the SSNPs with a molar feed ratio of 1:4 (Ad-F to Ad-R) were selected in this work, unless otherwise noted. At the same time, fluorescence emission spectra of the nanoprobe under single-wavelength excitation (at 488 nm) were explored in buffer solution at various pH values. As shown in Figure 3b, the fluorescence (520 nm) of fluorescein increased dramatically as the pH increased whereas that (575 nm) of Rhodamine B increased little. Most notably, the fluorescence emission of SSNPs was reversible between pH 4 and pH 9 (Figure 3c), and the SSNPs showed good antidilution performance, because of the ratiometric self-calibration of the probe, avoiding the concentration difference (Figure 3d). To determine the influences of intracellular species on the fluorescence response of SSNPs, fluorescence intensity ratios of the nanoprobes were measured in the presence of essential metal ions, different anions, oxidative-stress-associated redox substances (glutathione (GSH), cysteine (Cys), H2O2) under physiological conditions (PBS buffer, pH 7.4, 37 °C) and different ionic strength (see section S8 in the Supporting E
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could be successfully applied to estimation of pH fluctuations associated with different stimuli.
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CONCLUSIONS In summary, a self-assembled supramolecular fluorescent pH nanoprobe has been engineered by linking together fluorescein/rhodamine units via noncovalent interactions with poly-β-CD. First, compared to pH-sensing nanostructures constructed by covalent interactions, this self-assembly method for fabricating a functional nanostructure involves one step, is simple, and is easily tunable, with regard to dyes ratios, to obtain optimal ratiometric pH response. Second, cytotoxicity studies have demonstrated its good biocompatibility. Third, the plot of the fluorescence intensity ratio versus intracellular pH values ranging from 4.0 to 8.0 was linear, which covers most of the pH ranges for biological applications. Fourth, the nanoprobe has been successfully applied for measurements of intracellular pH of intact HeLa cells and estimation of pH fluctuations associated with different stimuli in cells. Importantly, we expect that the self-assembly preparation procedure of the supramolecular nanoprobe shown here can be used for the construction of other sensing nanoprobes by replacing the respective indicator dyes by indicators for other biologically relevant ions and small molecules.
Figure 5. Confocal microscopy images of SSNPs in HeLa cells clamped at (a−d) pH 4, (e−h) pH 5, (i−l) pH 6, (m−p) pH 7, and (q−t) pH 8, respectively. The excitation wavelength was 488 nm and the images were collected in the ranges of 505−545 nm (first row, fluorescein) and 575−615 nm (second row, Rhodamine). The corresponding differential interference contrast images (third row) and the ratio images of green (Igreen) to red (Ired) fluorescence intensity of each cell were obtained from green (505−545 nm) and red (575− 615 nm) channels by Olympus software (FV10-ASW) (fourth row). The bottom-right color strip represents the pseudo-color change with pH. Scale bar = 20 μm.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected] (X. Yang). *E-mail: [email protected] (K. Wang). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the Natural Science Foundation of China (Nos. 21190040, 21175035, and 21375034), National Basic Research Program (No. 2011CB911002), and International Science & Technology Cooperation Program of China (No. 2010DFB30300).
Figure 6. (a) Intracellular pH calibration curve of SSNPs in HeLa cells. R, generated by Olympus software (FV10-ASW), indicates the ratio of green (Igreen) to red (Ired) fluorescence intensity of each cell. The error bars indicated the standard deviations of three experiments. (b) Ratiometric images of green (Igreen) to red (Ired) fluorescence intensity of SSNPs loaded HeLa cells in PBS (pH 7.4). Intact cells, NAC (1.0 mM) treated, H2O2 (100 μM) treated, and CQ (100 μM) treated cells were incubated for 1 h at 37 °C, respectively. Scale bar = 20 μm. (For more details, see section S10 in the Supporting Information.)
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REFERENCES
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induce lysosomal acidification.35 In contrast, H2O2 could make the organelles (possibly the lysosomes) to be basic in cells, because the inactivation of lysosomal V-ATPase by oxidative stress led to the lysosomal pH increase.36,37 The application of SSNPs to monitor the effect of the antimalarial drug CQ on the intracellular pH was also discussed here. CQ has been shown to induce an increase of the pH value, from 5.8 ± 0.1 to 6.9 ± 0.3, in HeLa cells. The reason may be that CQ is acted as a cellpermeable base and can neutralize the acidic environment in lysosomes.38−40 The results demonstrated that the nanoprobe F
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DOI: 10.1021/ac504458r Anal. Chem. XXXX, XXX, XXX−XXX
Self-Assembled Supramolecular Nanoprobes for Ratiometric Fluorescence Measurement of Intracellular pH Values Leiliang He, Xiaohai Yang,* Fang Zhao, Kemin Wang,* Qing Wang, Jianbo Liu, Jin Huang, Wenshan Li, and Meng Yang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, People’s Republic of China S Supporting Information *
ABSTRACT: Self-assembly of small building blocks into functional supramolecular nanostructure has opened prospects for the design of novel materials. With this molecular engineering strategy, we have developed self-assembled supramolecular nanoprobes (SSNPs) for ratiometric fluorescence measurement of pH values in cells. The nanoprobes with a diameter of ∼30 nm could be formulated just by mixing pH-sensitive adamantane−fluorescein (Ad-F) and pH-insensitive adamantane−Rhodamine B (Ad-R) with β-cyclodextrin polymer (poly-β-CD) at one time. The nanoprobes with good biocompatibility have been successfully applied to measure intracellular pH in the pH range of 4−8 and estimate pH fluctuations associated with different stimuli in cells. Moreover, we expect that this self-assembled approach is applicable to the construction of nanoprobes for other targets in cells just by replacing the respective indicator dyes with relevant indicators.
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effects.23−25 However, self-assembled supramolecular nanoprobes derived from host−guest chemistry to intracellular biological sensing and imaging studies, to the best of our knowledge, are investigated relatively sparse and still in their infancy. Herein, we present a novel ratiometric pH-sensing supramolecular nanoprobe that is constructed by linking together functional building blocks via host−guest interactions of β-cyclodextrin polymer (poly-β-CD) and dyes. The self-assembled supramolecular nanoprobes (SSNPs) used for ratiometric pH sensing were fabricated as depicted in Scheme 1. It contains (1) β-cyclodextrin polymer (poly-β-CD) (Mn = 94 400; see the section entitled Organic Synthesis and Characterization), selected as the nanoprobe backbone based on its stability, high solubilization and biocompatibility;26 (2) derivative dyes, pH-sensitive adamantane−fluorescein (Ad-F) and pH-insensitive adamantane−Rhodamine B (Ad-R) (see the section entitled Organic Synthesis and Characterization), used as functional building blocks for ratiometric pH response. By mixing these components together in an aqueous phase, Ad-F/ Ad-R/poly-β-CD self-assembled to form nanosized particles (named SSNPs), because of cooperation of the inclusion of AdF/Ad-R guests in the cyclodextrin cavity of poly-β-CD and the strong hydrophobic interactions between the dyes. The formation mechanism of SSNPs was not completely the same
ngineering nanostructures that are capable of changing properties, in response to specific inputs generated by surrounding environments, are playing increasingly important roles in fields ranging from cell biology to biomedicine.1−3 In particular, those nanostructures enabling pH probing in cells have attracted much interest,4−8 because of the pH value playing a vital role in various cellular events, such as cell growth and apoptosis,9,10 drug resistance,11 phagocytosis,12 and endocytosis.13 These pH-sensing nanostructures are constructed by (1) covalent interactions, for example, the covalent conjugate of functional molecules with silica nanoparticles,4 carbon dots5 or dextran14 and (2) noncovalent interactions, such as embedding multiple dyes into nanogels.6 Among these interactions, self-assembly of small building blocks into functional supramolecular nanostructures via host− guest interactions receive high interest, because of the ease of synthesis/assembly and the facile implementation of targeting ligands.15−17 For instance, the inclusion of hydrophobic guests into cyclodextrins, calixarenes, or cucurbiturils18,19 have been mainly used for the stimulus response system20 and siteselective biomedical delivery vehicles.21,22 In addition, because of the easy doping of different functional molecules in matrix materials, supramolecular self-assembly could be acted as a promising and practical approach for construction of ratiometric probes. Compared to intensity-based probes, the ratiometric probes using the built-in correction of two different emission bands are more ideal in practical applications, because they can correct the probe concentration and environmental © XXXX American Chemical Society
Received: December 1, 2014 Accepted: January 22, 2015
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RPMI 1640 without FBS. The adherent cells were incubated with SSNPs for 2 h at 37 °C under 5 wt %/vol CO2. Dynamic light scattering (DLS) measurements were made on a Zetasizer 3000Hs (Malvern, U.K.). Ultraviolet−visible (UV-vis) and fluorescence spectroscopy were recorded on a microplate reader (Infinite M1000 Pro, Switz). Dye derivatives were purified by an Agilent (USA) 1200 series HPLC system on a reverse-phase C18 column. A scanning electron microscopy (SEM) image was recorded with a JSM-6700F instrument (Japan). Cell fluorescence micrographs were performed on a Model FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan). PMT voltages of blue, green, and red channels were 750, 660, and 720 V, respectively. Image processing and analysis was also performed on Olympus software (FV10ASW). Specially, the fluorescence intensity ratio (R) images of green (Igreen) to red (Ired) of each cell were obtained from green (505−545 nm) and red (575−615 nm) channels, using Olympus software (FV10-ASW). Organic Synthesis and Characterization. β-cyclodextrin polymer (poly-β-CD) was prepared by adapting published procedures.27 As shown in section S11 in the Supporting Information (Figure S11), the molecular weight of poly-β-CD (Mn ≈ 94 400) was measured by using gel permeation chromatography (GPC, Waters-515) and the 1H NMR spectra showed that most bands of β-CD at 4.2−3.4 ppm were broadened in the spectrum of poly-β-CD. Adamantane-labeled fluorescein (Ad-F) was synthesized using the following process: 5 mg (33 μmol) 1-adamantylamine, 1.8 mg (3.3 μmol) 6-carboxyfluorescein N-hydroxysuccinimide ester, 1 mL of dimethyl sulfoxide (DMSO), and 50 μL of N,N-diisopropylethylamine (DIPEA) were mixed, then subjected to vibration treatment overnight at 700 rpm (25 °C). The initial products (Ad-F) were purified by HPLC (section S1 in the Supporting Information (Table S1)). As shown in section S11 in the Supporting Information (Figure S9), ESI-MS, m/z: 508.1 ([M−H]−); 1H NMR (400 MHz, DMSO, ppm) δ: 7.99 (1H, s), 7.79 (1H, s), 7.62 (1H, s), 6.41 (6H, m), 3.02 (6H, s), 2.03 (3H, s), 1.62 (6H, s). Adamantane-labeled Rhodamine B (Ad-R) was synthesized using the following process: 5 mg (33 μmol) 1-adamantylamine, 1.6 mg (3.3 μmol) Rhodamine B isothiocyanate, 400 μL DMSO, 100 μL DIPEA, and 400 μL 60 mM carbonate buffer solution (pH 8.5) were mixed and subjected to vibrational treatment overnight at 700 rpm (25 °C). The initial products (Ad-R) were purified by HPLC (see section S1 in the Supporting Information (Table S2)). As shown in section S11 in the Supporting Information (Figure S10)), ESI-MS, m/ z: 651.2 ([M−Cl]+); 1H NMR (400 MHz, DMSO, ppm) δ: 6.98 (3H, m), 6.43 (6H, m), 3.26 (8H, m), 3.02 (6H, s), 1.97 (3H, s), 1.61 (6H, s), 1.24(12H, m). Determination of the pKa Value. The pKa value for AdF/poly-β-CD (10 μM Ad-F with 500 μM poly-β-CD) was estimated from the changes in the fluorescence intensity with various pH values, using the Henderson−Hasselbalch28 equation:
Scheme 1. (a) Schematic Presentation of the Self-Assembled Supramolecular Nanoprobes (SSNPs) Formation and the Fluorescence Changes of the SSNPs upon pH Variation (Inset: Photographs Showing the Fluorescence Responses of SSNPs between pH 4 (Left) and pH 9 (Right)). (b) Responsive Guest Molecules (Ad-F and Ad-R) and Supramolecular Host (poly-β-CD) Involved in SSNPs Formationa
a
Legend: adamantane−fluorescein (Ad-F), adamantane−Rhodamine B (Ad-R), and β-cyclodextrin polymer (poly-β-CD).
as electrostatic interactions in the formation of siRNAcontaining delivery vehicles.21 SSNPs can give green and red fluorescence emission at basic pH and at acidic pH, respectively.
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EXPERIMENTAL SECTION Chemicals, Materials, and Instruments. β-cyclodextrin (β-CD) was obtained from J&K Scientific (China). Fluorescein N-hydroxysuccinimide ester was obtained from China Langchem, Inc. (China). 1-Adamantylamine, Rhodamine B isothiocyanate, nigericin, nacytelcysteine (NAC), chloroquine (CQ), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma−Aldrich (Germany). LysoTracker Blue DND-22 (LTB) was purchased from Molecular Probes (Invitrogen, USA). Anhydrous dimethyl sulfoxide (DMSO) and N,N-diisopropylethylamine (DIPEA) were obtained by molecular sieve dehydrating. Other chemicals and solvents were analytical grade and used without further purification. All solutions were prepared with Milli-Q ultrapure water (grade: 18.2 MΩ). Different pH buffer solutions (pH 3− 9) were prepared by using 0.1 M of citric acid and 0.2 M of disodium hydrogen phosphate, and the pH was adjusted by adding 2 M NaOH or HCl solutions. HeLa (human cervical cancer) cell lines were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum (FBS) and 100 IU/mL penicillin−streptomycin, and grown on glass-bottom culture dishes (MatTek Co.) in a humidified incubator containing 5 wt %/vol CO2 at 37 °C. For self-assembled supramolecular nanoprobes (SSNPs) loaded HeLa cells, the growth medium was removed and replaced with
⎛ FI − FI ⎞ log⎜ max ⎟ = pH − pKa ⎝ FI − FI min ⎠
where FI is the observed fluorescence intensity at a given pH, and FImax and FImin are the corresponding maximum and minimum, respectively. As shown in section S2 in the B
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Ad-F without poly-β-CD migrated (channel 3), but Ad-F in the presence of poly-β-CD remained (channel 2). As such, free AdR without poly-β-CD migrated (channel 5), but Ad-F in the presence of poly-β-CD remained (channel 4). Therefore, we could demonstrate that the inclusion complex of poly-β-CD/ dyes was stable. At the same time, SSNPs (2 μM Ad-F/8 μM Ad-R/500 μM poly-β-CD) remained (channel 1), which also demonstrated that the SSNPs was stable. Furthermore, compared with tube 2 and tube 3 (or compared with tube 4 and tube 5), it showed the fluorescence intensities of the inclusion complex were shown to increase, because of the microenvironmental changes of dyes after combination with poly-β-CD. The stability of the SSNPs in a pH 7.4 PBS buffer solution, a blood serum-containing (15% v/v) solution, and a HeLa cell lysate-contained solution was investigated, as well as the storage stability of the SSNPs in water. These data were obtained by excitation at 488 nm on a microplate reader (Infinite M1000 Pro, Switz). As shown later in this work in Figures 2a, 2b, and 2c, the corresponding solution was irradiated for different times (0, 10, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600 min) by using a 6 W hand-held UV lamp as the radiation source. In Figure 2d, the storage time of the SSNPs was 0, 1, 2, 3, 4, 6, 8, and 10 days, respectively. Spectroscopic Studied of Self-Assembled Supramolecular Nanoprobes (SSNPs) versus pH Value In Vitro. A series of different pH buffers (pH values of 3.0, 4.0, 4.6, 5.0, 5.6, 6.0, 6.6, 7.0, 7.6, 8.0, 9.0, 10.0) were prepared by mixing 0.1 M of citric acid and 0.2 M of disodium hydrogen phosphate at varied volume ratios. The pH value was measured usign a pH-meter. Then, 50 μL of the corresponding pH buffer and 50 μL of 2×SSNPs solution (4 μM Ad-F/16 μM Ad-R/1 mM poly-β-CD) were mixed in a 96-well Elisa plates to measure fluorescence spectrum (Ex = 488 nm). Toward fluorescence reversibility of SSNPs with pH, the pH of SSNPs solution between pH 4 and pH 9 was adjusted by adding 2 M NaOH or HCl solution, and then measured by pHmeter. The fluorescence spectra were obtained by excitation at 488 nm. Meanwhile, the antidilution performance of SSNPs under various pH values was estimated from the changes in the fluorescence ratio (F520/F575) signal. F520 and F575 indicate the fluorescence intensity at 520 and 575 nm, respectively. Interference Study and Cytotoxicity Assay. To study the effects of intracellular species on the fluorescence response of SSNPs, the changes in the fluorescence ratio (F520/F575) signal were investigated in the presence of metal ions (Na+, K+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+, Fe3+), anions (F−, ClO−, CO32−, NO2−, I−, NO3−, PO43−, Br−, SO32−, SO42−, Cl−, OAc−, HCO3−), oxidative-stress-associated redox chemicals (including glutathione (GSH), cysteine (Cys), H2O2) under physiological conditions (PBS buffer, pH 7.4, 37 °C) and different ionic strength. For more details, see section S8 in the Supporting Information. The cytotoxicity of SSNPs was evaluated by the standard MTT assay. Briefly, HeLa cells were seeded into 96-well Ubottom plates at a density of 7000 cells/well, and incubated with free dyes (Ad-F and Ad-R) and dyes/poly-β-CD complex at varied concentrations ([dyes] = 0, 5, 10, 20, and 50 μM; [poly-β-CD] = 0, 0.25, 0.5, 1, and 2.5 mM, respectively, as shown later in this work in Figure 4). The cells incubated with the culture medium only were served as the controls. After incubation for 24 h at 37 °C, the cells were washed with PBS for three times, and 0.1 mL MTT solution (0.5 mg/mL in PBS)
Supporting Information, the pKa values (y-intercept) of Ad-F/ poly-β-CD (pKa = 5.78 ± 0.10) was derived from the plot of pH vs log[(FImax − FI)/(FI − FImin)]. At the same time, the effect of pH on the fluorescence intensity changes of Ad-R/ poly-β-CD (10 μM Ad-R with 500 μM poly-β-CD) was also studied. The results demonstrated that the system of Ad-R/ poly-β-CD is pH-insensitive. Preparation and Characterization of Self-Assembled Supramolecular Nanoprobes (SSNPs). Preparation of the sensing 10×SSNPs (100 μM, calculated by total concentration of dyes) was as follows. The poly-β-CD was incubated with AdF and Ad-R at varied molar ratios (see section S4 in the Supporting Information) in water at one time, and subjected to vibration treatment for 4 h at 300 rpm (25 °C). 10×SSNPs composed of different feed ratios (from 4:1 to 1:19 Ad-F/AdR) could be formed. The mixtures were filtrated with an Amicon YM-10 filter to remove the free Ad-F and Ad-R. The resultant suspension was used in further experiments. As shown in section S3 in the Supporting Information, the binding constants (K-values) of Ad-F with poly-β-CD in buffer (pH 7.0) was 3.5 × 103 M−1, and that of Ad-R with poly-β-CD was 4.0 × 103 M−1. Because 1/(F − F0) vs 1/[β-CD] was a linear relationship, it indicated the formation of a 1:1 inclusion complex between the dyes with poly-β-CD. At the same time, according to our previous reports,29 the K-values of Ad-F or Ad-R with poly-β-CD under different pH values are ∼3.5 × 103 M−1, and more than 94% of dyes have combined with β-CD cavities of the poly-β-CD. Therefore, as described in section S6 in the Supporting Information, the average number of dye molecules on each nanoparticle is ∼1.7 × 103 dye molecules per nanoparticle. Single nanoparticle imaging of the SSNPs was as the follows. Glass coverslips (Fisher, 24 mm × 32 mm) were first immersed in Piranha solution (H2SO4:H2O2 = 7:3) for 60 min and then cleaned in ultrapure water in an ultrasonic bath for 30 min. The coverslips were shaken free of excess liquid and dried in a slide holder. Finally, the coverslips were vacuum plasma exposure for 2 min at 150 W. After these, the substrates were exposed to the SSNPs solution (1.0 nM, calculated by total concentration of dyes) by placing 5 μL drops onto the slides and allowing them to image at room temperature. Fluorescence micrographs were recorded on a Model FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan) with dual-wavelength excitation (488 nm for Ad-F and 550 nm for Ad-R) through a 100× oil immersion objective. To demonstrate whether the SSNPs could load two types of dyes in the same particle or not, the experiment of fluorescence resonance energy transfer (FRET) was performed in a buffer solution (pH 7.4) with excitation at 450 nm. Comparing the emission spectra of two-dye-doped inclusion complex (SSNPs) with that of single-dye-doped one, the SSNPs showed that the emission of Ad-F at 520 nm decreased and the emission of AdR at 575 nm increased (see section S5 in the Supporting Information (Figure S4b)), but the free dyes, as a control, could not show similar tendencies (see section S5 in the Supporting Information (Figure S4a)). These results demonstrated the existence of FRET between Ad-F and Ad-R in the SSNPs, and the observations also proved successful loading of the two types of dyes (Ad-F and Ad-R) in the same particle. To demonstrate whether the inclusion complex of poly-βCD/dyes was stable or not, the agarose gel electrophoresis (AGE) was performed. As shown in section S7 in the Supporting Information, under the effect of electric field, free C
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Analytical Chemistry was added to each well with incubation at 37 °C for 4 h. After the addition of DMSO (100 μL/well), the assay plate was shaken at room temperature for 10 min. Absorbance values of the wells were measured by using a microplate reader. Each concentration was tested at least three times. The cell viability was calculated based on measuring the UV-vis absorption at 570 nm (OD570), according to the following equation: cell viability =
OD570(sample) − OD570(blank) OD570(control) − OD570(blank)
Microscopic Documentation of Intracellular Uptake of Self-Assembled Supramolecular Nanoprobes (SSNPs). The HeLa Cells were incubated with SSNPs for 2h at 37 °C, and then the medium was replaced with fresh medium containing 2.0 μM LysoTracker Blue DND-22 (LTB) and incubated for 10 min. Cell fluorescence micrographs were performed on a Model FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan) with a 20× 0.75 NA objective (excitation at 488 nm for SSNPs and 405 nm for LTB). Intracellular pH Calibration. The HeLa cells were incubated with SSNPs for 2 h at 37 °C, and the growth medium without FBS was removed and replaced with high K+ buffer (30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES, and 20 mM NaOAc) at various pH values (pH = 4−8) in the presence of 10.0 μM of nigericin. After 30 min, the fluorescence images were measured, and the pH calibration curve was constructed with Olympus software (FV10-ASW). Fluorescence Imaging of Intact and Treated Cells. To explore the intracellular pH fluctuations associated with the different stimuli, the SSNPs-loaded HeLa cells treated with a redox substance (1 mM NAC or 0.1 mM H2O2,) or 0.1 mM CQ were incubated for 1 h at 37 °C in PBS (pH 7.4), and then the treated cells were subjected to fluorescence imaging. As a control, the SSNPs-loaded HeLa cells untreated with stimuli were used as intact cells.
Figure 1. (a) SEM image of the SSNPs and (b) the corresponding size distribution by dynamic light scattering. Single nanoparticle fluorescence imaging of the SSNPs from different channels: (c) fluorescence image from the fluorescein channel, (d) fluorescence image from the Rhodamine channel, and (e) a merger of images from the fluorescein channel and the Rhodamine channel.
loaded in the same particle (see section S5 in the Supporting Information). At the same time, the results from the agarose gel electrophoresis (AGE) could demonstrate that the inclusion complex of poly-β-CD/dyes was stable (see section S7 in the Supporting Information). Stability of the SSNPs under various environmental conditions (for example, human serum or human cell lysatecontained biological fluids) was investigated by monitoring its fluorescence ratio (F520/F575) changes (see Figure 2). The results showed that the prepared SSNPs have good photostability in complex environment and long storage time. In addition, according to our previous reports,29 Ad-F and Ad-R could form stable inclusion complexes with poly-β-CD, in which the binding constants of Ad-F or Ad-R with poly-β-CD
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RESULTS AND DISCUSSION The morphology and size of the SSNPs were first investigated by using scanning electron microscopy (SEM) and dynamic light scattering (DLS). SEM image showed that the nanoprobe was spherical in shape with uniform size distribution (∼30 nm) (see Figure 1a), and its average hydrodynamic diameter in water was ∼70 nm by DLS analysis (see Figure 1b). In order to further demonstrate that the formation of the SSNPs was involved with the spatial colocalization of the Ad-F and Ad-R in the same particle, single nanoparticle imaging of the nanoprobes was performed. As shown in Figures 1c−e, spots showed an even distribution, indicating that there was a negligible probability that spots derived from the nanoparticle overlap.30−32 In addition, the fluorescence microscopy images acquired from the green channel and the red channel showed that the nanoprobes were imaged as a combined color due to spatial colocalization of the Ad-F and Ad-R in the same particle (see Figure 1e). As shown in Figures 1c and 1d, ImageJ software analysis determined that the standard deviation of the fluorescence intensity of the green light spot was 10.2%, but that of the red light spot was 5.7%. Thus, we inferred that the standard deviation of the number of dye molecules in the SSNPs were 10.2% for Ad-F and 5.7% for Ad-R, respectively. Furthermore, the existence of FRET between Ad-F and Ad-R also could indicate that the two types of dyes were successfully
Figure 2. (a) Stability of the SSNPs in a pH 7.4 PBS buffer solution, (b) blood serum-contained (15% v/v) solution, (c) HeLa cell lysatecontained solution, and (d) the storage stability of the SSNPs in water. F520 and F575 indicate the fluorescence intensity of the SSNPs at 520 and 575 nm, respectively. These data were obtained by excitation at 488 nm on a microplate reader (Infinite M1000 Pro, Switz). In panels (a), (b), and (c), the corresponding solution was irradiated for different times (0, 10, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600 min) by using a 6 W hand-held UV lamp as the radiation source. In panel (d), the storage times of the SSNPs were 0, 1, 2, 3, 4, 6, 8, and 10 days. The error bars indicate the standard deviations of three experiments. D
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Analytical Chemistry under different pH values are ∼3.5 × 10 3 M −1, the photostability of Ad-F/poly-β-CD and Ad-R/poly-β-CD are higher than the corresponding free dyes. Fluorescence responses of the nanoprobe in vitro have been investigated as shown below. As a critical factor for the pH response of SSNPs, the molar feed ratio of Ad-F to Ad-R was first optimized. As shown in Figure 3a, the ratios of the two
Information). It was found that these species and ionic strength change scarcely affected the ratiometric fluorescent signal of SSNPs. Furthermore, the in vitro cytotoxicity of the SSNPs at varied concentrations was measured using a standard MTT assay. It was found that cell viability was not significantly affected after incubation with SSNPs for 24 h (Figure 4), clearly indicating the good biocompatibility of the SSNPs.
Figure 4. Cell viability values (%) estimated by MTT assay. HeLa cells were incubated with varied concentrations dyes/poly-β-CD (pink) and dyes (azure) at 37 °C for 24 h. Dyes contained Ad-F and Ad-R with fixed molar ratio of Ad-F to Ad-R (1:4). Cells without any addition were taken as a control, and the viability was set as 100%. The error bars indicated the standard deviations of three experiments.
Figure 3. (a) Plots of F520/F575 versus pH values for SSNPs prepared with different molar feed ratios from 4:1 to 1:19 (Ad-F/Ad-R). F520 and F575 indicate the fluorescence intensity at 520 and 575 nm, respectively. The error bars indicated the standard deviations of three experiments. (b) Fluorescence emission spectra of SSNPs (Ad-F/Ad-R = 1:4) at various pH values. (c) Reversible fluorescence ratio (F520/ F575) changes of SSNPs between pH 4 and pH 9. (d) Antidilution performance of SSNPs under various pH values (diluted with buffer). All data were obtained by excitation at 488 nm.
To demonstrate particle uptake of the SSNPs by cells and to investigate the intracellular distribution of the nanoprobe, colocalization experiments using the nanoprobe and organellespecific fluorescent dyes (LysoTracker Blue) have been performed. Particle uptake was achieved by exposing the cells to the SSNPs for 2 h, presumably by vesicular uptake mechanisms. The blue fluorescence from LysoTracker Blue could overlap with the green or red fluorescence from the nanoprobe, which indicated that the nanoprobe was localized to lysosomes (see section S9 in the Supporting Information). Therefore, we inferred that the nanoprobe could be internalized by cells and used to measure lysosomal pH value in cells. SSNPs have been further used to measure pH in cells. The intracellular pH was set to different values with the H+/K+ ionophore nigericin, which was well used for homogenizing the intracellular pH and culture medium.33 As shown in Figure 5, the fluorescence intensity of the fluorescein unit (green channel) in cells increased with pH value (4−8), whereas that of the Rhodamine unit (red channel) was almost unchanged. The ratio images of green intensity to red intensity, which showed a characteristic pH-dependent signal, demonstrated that the SSNPs could be effectively used to measure intracellular pH with a good linear calibration curve in the pH range from 4.0 to 8.0 (see Figure 6a). To demonstrate the utility of the newly constructed SSNPs, the average pH values of HeLa cells were measured when the cells were exposed to the different stimuli, such as NAC (Nacetylcysteine, a GSH precursor), H2O2, and chloroquine (CQ) (see Figure 6). As shown in the calibration curve in Figure 6a. The pH values for the untreated cells, as well as the NAC-, H2O2-, and CQ-treated cells, were determined to be 5.8 ± 0.1, 5.4 ± 0.2, 6.6 ± 0.2, and 6.9 ± 0.3, respectively. Because of the generation of reduced GSH intracellularly,34 NAC could decrease the intracellular pH to 5.4 ± 0.2, which was in agreement with previous reports that reducing substances
dopants ranging from 4:1 to 1:19 make the pH-responsive sensitivity of the nanoprobe different. The SSNPs prepared with a molar ratio of 1:4 (Ad-F to Ad-R) showed distinct fluorescence ratio (F520/F575) changes at pH = 4−8, which could cover most of the pH ranges for biological applications. The fluorescence response of SSNPs in a wide pH range (pH 4−8) can be explained by the features of pH-sensitive adamantane−fluorescein (Ad-F)/poly-β-CD (pKa = 5.78 ± 0.10) and pH-insensitive adamantane−Rhodamine B (Ad-R)/ poly-β-CD (see section S2 in the Supporting Information). Therefore, the SSNPs with a molar feed ratio of 1:4 (Ad-F to Ad-R) were selected in this work, unless otherwise noted. At the same time, fluorescence emission spectra of the nanoprobe under single-wavelength excitation (at 488 nm) were explored in buffer solution at various pH values. As shown in Figure 3b, the fluorescence (520 nm) of fluorescein increased dramatically as the pH increased whereas that (575 nm) of Rhodamine B increased little. Most notably, the fluorescence emission of SSNPs was reversible between pH 4 and pH 9 (Figure 3c), and the SSNPs showed good antidilution performance, because of the ratiometric self-calibration of the probe, avoiding the concentration difference (Figure 3d). To determine the influences of intracellular species on the fluorescence response of SSNPs, fluorescence intensity ratios of the nanoprobes were measured in the presence of essential metal ions, different anions, oxidative-stress-associated redox substances (glutathione (GSH), cysteine (Cys), H2O2) under physiological conditions (PBS buffer, pH 7.4, 37 °C) and different ionic strength (see section S8 in the Supporting E
DOI: 10.1021/ac504458r Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
could be successfully applied to estimation of pH fluctuations associated with different stimuli.
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CONCLUSIONS In summary, a self-assembled supramolecular fluorescent pH nanoprobe has been engineered by linking together fluorescein/rhodamine units via noncovalent interactions with poly-β-CD. First, compared to pH-sensing nanostructures constructed by covalent interactions, this self-assembly method for fabricating a functional nanostructure involves one step, is simple, and is easily tunable, with regard to dyes ratios, to obtain optimal ratiometric pH response. Second, cytotoxicity studies have demonstrated its good biocompatibility. Third, the plot of the fluorescence intensity ratio versus intracellular pH values ranging from 4.0 to 8.0 was linear, which covers most of the pH ranges for biological applications. Fourth, the nanoprobe has been successfully applied for measurements of intracellular pH of intact HeLa cells and estimation of pH fluctuations associated with different stimuli in cells. Importantly, we expect that the self-assembly preparation procedure of the supramolecular nanoprobe shown here can be used for the construction of other sensing nanoprobes by replacing the respective indicator dyes by indicators for other biologically relevant ions and small molecules.
Figure 5. Confocal microscopy images of SSNPs in HeLa cells clamped at (a−d) pH 4, (e−h) pH 5, (i−l) pH 6, (m−p) pH 7, and (q−t) pH 8, respectively. The excitation wavelength was 488 nm and the images were collected in the ranges of 505−545 nm (first row, fluorescein) and 575−615 nm (second row, Rhodamine). The corresponding differential interference contrast images (third row) and the ratio images of green (Igreen) to red (Ired) fluorescence intensity of each cell were obtained from green (505−545 nm) and red (575− 615 nm) channels by Olympus software (FV10-ASW) (fourth row). The bottom-right color strip represents the pseudo-color change with pH. Scale bar = 20 μm.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected] (X. Yang). *E-mail: [email protected] (K. Wang). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the Natural Science Foundation of China (Nos. 21190040, 21175035, and 21375034), National Basic Research Program (No. 2011CB911002), and International Science & Technology Cooperation Program of China (No. 2010DFB30300).
Figure 6. (a) Intracellular pH calibration curve of SSNPs in HeLa cells. R, generated by Olympus software (FV10-ASW), indicates the ratio of green (Igreen) to red (Ired) fluorescence intensity of each cell. The error bars indicated the standard deviations of three experiments. (b) Ratiometric images of green (Igreen) to red (Ired) fluorescence intensity of SSNPs loaded HeLa cells in PBS (pH 7.4). Intact cells, NAC (1.0 mM) treated, H2O2 (100 μM) treated, and CQ (100 μM) treated cells were incubated for 1 h at 37 °C, respectively. Scale bar = 20 μm. (For more details, see section S10 in the Supporting Information.)
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