Research Article www.acsami.org
Continuous Fluorescence Imaging of Intracellular Calcium by Use of Ion-Selective Nanospheres with Adjustable Spectra Chenye Yang, Yu Qin, Dechen Jiang,* and Hong-yuan Chen The State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China S Supporting Information *
ABSTRACT: Continuous fluorescence imaging of intracellular ions in various spectral ranges is important for biological studies. In this paper, fluorescent calcium-selective nanospheres, including calix[4]arenefunctionalized bodipy (CBDP) or 9-(diethylamino)-5-[(2-octyldecyl)imino]benzo[a]phenoxazine (ETH 5350) as the chromoionophore, were prepared to demonstrate intracellular calcium imaging in visible or near-IR regions, respectively. The fluorescence of the nanospheres was controlled by the chromoionophore, and thus the spectral range for detection was adjustable by choosing the proper chromoionophore. The response time of the nanospheres to calcium was typically 1 s, which allowed accurate measurement of intracellular calcium. These nanospheres were loaded into cells through free endocytosis and exhibited fluorescence for 24 h, and their intensity was correlated with the elevation of intracellular calcium upon stimulation. The successful demonstration of calcium imaging by use of ion-selective nanospheres within two spectral ranges in 24 h supported that these nanospheres could be applied for continuous imaging of intracellular ions with adjustable spectra. KEYWORDS: fluorescent calcium-selective nanospheres, intracellular calcium, adjustable spectrum, continuous imaging, chromoionophore
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INTRODUCTION Free ions are involved in many biological processes, so that the continuous detection or imaging of intracellular ions during these processes is of considerable interest to scientists.1,2 Fluorescent probes that emit a spectral response upon binding ions are widely used for detection because of their high selectivity and sensitivity.3−8 When the complexity of biological systems is considered, fluorescent probes with adjustable spectroscopic properties, such as excitation and emission wavelengths, are preferred to avoid the comeasurement of multiple targets. Although the fast development of organic synthesis or genetic encoding offers more fluorescent chemical probes, the design of proper probes with adjustable optical features for biological ion detection is complex and timeconsuming.9 Also, organic probes suffer from drawbacks such as cytotoxicity, dye leakage, and sequestration,10,11 and thus the continuous fluorescence imaging of intracellular ions for days is difficult. Functionalized nanomaterials could improve cellular retention, but the biocompatibility and invasion during delivery of large nanoparticles are problematic, and a delicate design is desired to achieve an adjustable spectrum.9 Therefore, establishing a universal and simple strategy for the preparation of fluorescence probes with adjustable fluorescent features for continuous imaging of intracellular ions is needed. Ion-selective optodes are a new family of optical sensors, composed of ionophore, ion-exchanger, and lipophilic-sensing © XXXX American Chemical Society
components (chromoionophore), such as pH indicators, in a polymeric lipophilic matrix material.12 The sensing process, involving the redistribution of analyte and hydrogen ions between the sensing phase and the sample phase, results in the fluorescence change of pH indicator.9 Selectivity of the optode is determined by the ionophores, so that high specificity is maintained to measure the target ion.10 Recently, Bakker and co-workers9,10,13−16 utilized the precipitation approach to prepare monodisperse ion-selective nanospheres for determination of aqueous ions. In their strategy, all sensing components were dissolved in tetrahydrofuran and mixed with aqueous solution subsequently. Pluronic F-127, a triblock copolymer, was added and functioned as a surfactant to stabilize the nanoparticle. After removal of tetrahydrofuran, the nanospheres were formed without any further purification steps, and they exhibited excellent sensitivity and selectivity to aqueous ions.10 The fluorescence of the nanospheres was controlled by the chromoionophore. Since numerous fluorescent pH indicators could offer abundant spectroscopic properties, the nanospheres should offer appropriate optical features for different biological applications by choosing the proper probes. Meanwhile, Pluronic-based nanospheres have Received: May 6, 2016 Accepted: July 13, 2016
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DOI: 10.1021/acsami.6b05406 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 1. Ion-exchange process of ion-selective nanospheres incorporated with (A) CBDP and (B) ETH 5350.
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been proved to have low cytotoxicity, and thus these ionselective nanospheres should be expected to be loaded into cells for the imaging of intracellular ions.17 In this paper, nanospheres incorporated with pH indicators emitting different colors of light were prepared and attempts were made to load them into cells for sensing intracellular ions with adjustable spectra. As compared with organic fluorescence probes or genetically encoded ion indicators with multiple colors, the ionselective nanospheres have the advantages of easy preparation, adjustable spectra, and applicability for the analysis of most intracellular ions. The successful utilization of ion-selective nanospheres for intracellular ion sensing could offer an easy and universal strategy for cell analysis. To demonstrate the concept of intracellular imaging by use of ion-selective nanospheres, calcium, an important intercellular messenger,11 was chosen as the model molecule. Current calcium indicators, such as Fluo-3 or Fluo-411,18and Rhod2,19−22 are well suitd for intracellular calcium measurements;, however, the spectra are limited in the visible range. Although the development of new calcium indicators in blue, green, red, and near-IR regions with excellent cellular retention was achieved,23−27 relatively complicated syntheses or genetic encoding was needed. In our work, two fluorescent pH indicators, calix[4]arene-functionalized bodipy (CBDP, λex/em 480/510 nm)28 and 9-(diethylamino)-5-[(2-octyldecyl)imino]benzo[a]phenoxazine (ETH 5350, λex/em 645/669 nm)29 were applied as the sensing components for preparation of calciumselective nanospheres, so that intracellular calcium could be imaged in visible and near-IR regions, respectively. Fluorescence properties, cytotoxicity, probe leakage, and imaging of intracellular calcium were investigated. The achievement of these two calcium-selective nanospheres for intracellular calcium measurement will provide direct evidence to support intracellular ion detection by use of ion-selective nanospheres with adjustable spectra.
EXPERIMENTAL SECTION
Chemicals. Pluronic F-127 (F127), cation-exchanger sodium tetrakis[3.5-bis(trifluoromethylphenyl)borate (NaTFPB), calcium ionophore II (ETH 129), 9-diethylamino-5-[(2-octyldecyl)imino]benzo[a]phenoxazine (chromoionophore III, ETH 5350), bis(2-ethylhexyl) sebacate (DOS), and tetrahydrofuran (THF) were purchased from Sigma−Aldrich (Switzerland). Fluo-3 AM was obtained from Fanbo Biochemicals (Beijing, China). Phosphate-buffered saline (PBS) and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Keygen Biotech (Nanjing, China). Ionomycin was obtained from Beyotime Institute of Biotechnology (Nantong, China). Calix[4]arene-functionalized bodipy (CBDP) was prepared as reported.28 Cell Culture. HeLa cells were purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Science (Shanghai, China). The cells were seeded in Dulbecco’s modified Eagle’s medium (DMEM)/highglucose medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. Preparation of Calcium-Selective Nanospheres. CBDP or ETH 5350 was used as the sensing component for preparation of nanospheres. To prepare CBDP-incorporated nanospheres, 8.0 mg of DOS, 5.0 mg of F127, 1.2 mg of CBDP, and 3.3 mg of calcium ionophore II were dissolved in 3.0 mL of THF to form a homogeneous solution. An aliquot (0.5 mL) of this THF solution was pipetted and injected into 4.5 mL of deionized water on a vortex with a spinning speed of 1000 rpm. Then compressed air was blown on the surface of the mixture for 30 min to remove THF. The solution including the nanospheres was stored for later experiments. For ETH 5350-incorporated nanospheres, 8.0 mg of DOS, 5.0 mg of F127, 1.2 mg of ETH 5350, 1.86 mg of NaTFPB, and 3.9 mg of calcium ionophore II were dissolved in 3.0 mL of THF to form a homogeneous solution. Then the same procedure as described was used to prepare the nanosphere suspension. Response of Nanospheres to Ca2+ in Vitro. Fluorescence emission spectra were recorded on an EnSpire multimode plate reader with 96-well plates (PerkinElmer). The excitation wavelength was fixed at 480 nm for CBDP-incorporated nanospheres and 645 nm for ETH 5350-incorporated nanospheres. All experimental results were B
DOI: 10.1021/acsami.6b05406 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces average values from five replicate measurements. The typical noise in fluorescence measurement was ∼500 au. To investigate the reversible response of nanospheres for aqueous calcium, 0.67 and 0.33 μL aliquots of 10 mM calcium chloride were added into 100 μL of nanosphere solution successively to raise the concentration of calcium to 67 μM and 100 μM. In this process, the volume of the solution remained almost constant. After the response of nanospheres to these two concentrations was tested, 50 μL of PBS was added into this 100 μL solution so that the concentration of calcium ion decreased to 67 μM for the detection. Since the fluorescence intensity was detected by multimode plate reader, the volume change did not affect the measurement. MTT Assay. The cytotoxicity of nanospheres was tested by MTT assay. Cells (1 × 104 cells/well) were seeded in a 96-well plate (100 μL/well) overnight. Then nanospheres with different concentrations in the medium were added into the wells for 1 h in sequence. Pure culture medium was used in the control group. Afterward, 50 μL of 5 mg/mL MTT solution was introduced into each well for 4 h in the dark. After removal of the remaining MTT solution, 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was recorded at 550 nm by an EnSpire multimode plate reader. Cell viability was calculated by the following formula:
cell viability (%) =
in Figure S2 (Supporting Information). The average sizes of these two nanospheres were estimated to be 69.1 ± 8.8 and 78.7 ± 8.5 nm by measuring the diameter of the nanospheres and the length of scale bar with a pixel ruler in TEM images, which were consistent with literature results.10 Following the previous analysis,13 the concentrations of ETH 5350-incorporated nanospheres were estimated as 70 μM according to the concentration of NaTFPB. Since no NaTFPB was involved in CBDP-incorporated nanospheres, the total concentration of exchangeable ions was calculated on the basis of CBDP concentration, and the concentration of the nanosphere was estimated to be 70 μM by CBDP concentration as well. The fluorescence of these two nanospheres exhibited similar emission wavelengths as the corresponding dyes in solution, as shown in Figure S3 (Supporting Information). In particular, when excitation wavelength was fixed at 480 nm, CBDP emitted at 510 nm while the CBDP-incorporated nanospheres (50% diluted in PBS solution) emitted at 516 nm. For ETH 5350, the excitation wavelength was fixed at 645 nm, and the dye showed an emission wavelength at 669 nm while the ETH 5350-incorporated nanospheres (50% diluted in PBS solution) emitted at 673 nm. The small discrepancy in emission wavelengths might be caused by the different polar environments surrounding the dyes. Nevertheless, these two nanospheres exhibited fluorescence features in two spectral windows. Response of Nanospheres to Aqueous Calcium. To investigate the response of nanospheres to calcium, solution experiments were carried out with these two nanospheres. The excitation wavelength was fixed at 480 nm for CBDPincorporated nanospheres and 645 nm for ETH 5350incorporated nanospheres. As presented in Figure 2, a gradual
ODtest − ODblank × 100 ODcontrol − ODblank
Imaging of Intracellular Calcium. Cells were plated on confocal dishes 1 day prior to the loading experiments. To load the nanospheres into the cells, the cells were incubated in 10 mM PBS solution (containing 10 μM CaCl2) with 5 μM Fluo-3 AM or the nanospheres (diluted in PBS) for 1 h at 37 °C. For the staining of Fluo-3 AM, the cells were washed with PBS and kept for 20 min at 37 °C to allow de-esterification. Ionomycin (5 μM) was employed to elevate the intracellular level of calcium. Confocal laser scanning microscope (Leica, TCS SP5) equipped with an oil-immersion 63× objective was used to obtain cell images. Fluorescence images were acquired at an excitation wavelength of 488 nm for Fluo-3- and CBDP-incorporated nanospheres and 633 nm for ETH 5350-incorporated nanospheres. Fluorescent intensity of the cells was analyzed by ImageJ software.
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RESULTS AND DISCUSSION Fluorescence of Calcium-Selective Nanospheres. Our aim is to introduce fluorescent pH indicators (chromoionophores) with different fluorescence properties into ion-selective nanospheres so that the intracellular ion is detected in more spectral windows. As a demonstration, two chromoionophores, CBDP in the visible region and ETH 5350 in the near-IR region, were added into nanospheres to image calcium within two spectral ranges. The chemical structures of CBDP and ETH 5350 are shown in Figure S1 (Supporting Information). When the nanospheres were exposed to the ions, ion exchange between protons and cationic analyte occurred, leading to the change of optical signal, as shown in Figure 1. Typically, the cationic optode included a neutral chromoionophore, a cation exchanger, and an ionophore. For electrically neutral chromoionophores like ETH compounds, they were electrically neutral in the deprotonated form and became positively charged when protonated. Therefore, the cation exchanger was needed for cationic nanospheres. In contrast, CBDP was a charged chromoionophore that was negatively charged in deprotonated form and neutral in protonated form. As a result, cation exchanger was unnecessary in CBDP-incorporated nanospheres. The CBDP- and ETH 5350-incorporated nanospheres were imaged by transmission electron microscopy (TEM) as shown
Figure 2. Fluorescence emission spectra of (A) CBDP-incorporated nanospheres (λex/em = 480/516 nm) and (B) ETH 5350-incorporated nanospheres (λex/em = 645/673 nm) for different concentrations of calcium in PBS solution, pH 7.4. C
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Figure 3. Fluorescence imaging of (A) CBDP-incorporated nanospheres and (B) ETH 5350-incorporated nanospheres inside cells. Bright-field (left), fluorescence (middle), and overlaid images (right) are shown. Excitation wavelengths for CBDP- and ETH 5350-incorporated nanospheres were 488 and 633 nm, respectively. The scale bar was 50 μm.
(Supporting Information), the fluorescence intensities of these two nanospheres were restored when the concentration of calcium was adjusted back to 67 μM. This result revealed that our nanospheres could respond to calcium reversibly, which is important to track the fluctuation of intracellular calcium in continuous monitoring. Cellular Retention of Nanospheres. Cytotoxicity of these nanospheres was studied by MTT assay before they were loaded into the cells for 24 h at 37 °C. CBDP- or ETH 5350incorporated nanospheres with different concentrations from 0 to 70 μM were applied to cells, and an obvious loss in viability was observed for nanospheres at a concentration of 49 μM, as shown in Figure S8 (Supporting Information). When the concentration of the nanospheres decreased to 35 μM, low toxicity was obtained in cells. Therefore, nanospheres at a concentration of 35 μM were used for intracellular calcium imaging. After loading of CBDP nanospheres into the cells for 1 h and washing by PBS, all cells exhibited fluorescence upon excitation at 488 nm, as shown in Figure 3A. Similarly, incubation of cells in medium with ETH 5350 nanospheres for 1 h gave fluorescence upon excitation at 633 nm (Figure 3B). These phenomena suggested that both CBDP- and ETH 5350incorporated nanospheres were loaded into the cells and exhibited fluorescence in the visible and near-IR ranges, respectively. No fluorescence was observed at the cellular nucleus, indicating that nanospheres were distributed in the cellular cytosol. To elucidate the loading mechanism of nanospheres into cells, the loading temperature was fixed at 4 °C and the loading process was repeated. As shown in Figure S9 (Supporting Information), bright fluorescence was observed at the cells, revealing free endocytosis of nanospheres into cells.30 To compare the cellular retention of our nanospheres with that of the widely used calcium probe Fluo-3 AM, the cells were divided into three groups and loaded with Fluo-3 AM, CBDP-
decrease in fluorescence intensity was observed with aqueous calcium from 1 μM to 1.5 mM for CBDP-incorporated nanospheres and from 10 μM to 1 mM for ETH 5350incorporated nanospheres. Response and calibration curves of these two nanospheres to aqueous calcium are shown in Figure S4 (Supporting Information). The observation confirmed the monitoring of calcium fluctuation by use of our nanospheres in different spectral ranges. The detection limits for CBDP- and ETH 5350-incorporated nanospheres were determined at 1 μM and 10 μM, respectively. The difference in the response of calcium for CBDP- and ETH 5350-incorporated nanospheres was attributed to the different pKa values of CBDP and ETH 5350 dyes. As compared with CBDP (pKa 6.5), ETH 5350 (pKa 10) had a higher pKa that needed more calcium in the nanopheres to deprotonate the dye for a detectable response.12 Therefore, CBDP-incorporated nanospheres gave a lower detection limit than ETH 5350incorporated nanospheres. Since cytosolic ligands bound the intracellular free calcium quickly, the increases in cytosolic calcium concentration dissipated rapidly.11 As a result, the quick and reversible responses of nanospheres to calcium were critical for accurate monitoring of intracellular calcium. Figure S5 (Supporting Information) showed that the response time of the nanospheres to calcium was typically 1 s, which was suitable for the following cellular study. The selectivity of CBDP- and ETH 5350-incorporated nanospheres toward 10 μM Ca2+ over other electrolyte ions, including 10 mM K+, Na+, and Mg2+, was tested. As shown in Figure S6 (Supporting Information), in the presence of other electrolyte ions, no significant fluorescence increase of these two nanospheres toward Ca2+ was observed, confirming good selectivity. Meanwhile, the nanospheres were exposed to 67, 100, and 67 μM aqueous calcium successively to investigate the reversibility of the fluorescence response. As shown in Figure S7 D
DOI: 10.1021/acsami.6b05406 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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fluorescence intensity remained in the cells loaded with the two calcium-selective nanospheres even after 24 h. These results manifested the excellent storage property of our nanospheres that provided the possibility of long-time intercellular calcium monitoring. Imaging of Intracellular Calcium by Use of Nanospheres. The typical concentration of intracellular calcium was 100 nM, which could not be detected by our ion-selective nanospheres. Therefore, ionomycin was employed to raise the intracellular level of calcium by allowing direct calcium influx across the cellular plasma membrane.31 For these two calciumselective nanospheres, the elevation of intracellular calcium led to the deprotonation of pH indicator in the nanoparticles. Experimentally, the cells loaded with Fluo-3, CBDP-incorporated nanospheres, or ETH 5350-incorporated nanospheres were stimulated at 0, 4, and 24 h with ionomycin. Fluorescence imaging before and after the stimulation was performed, and fluorescence intensities were measured. As imaged in Figure 5, the fluorescence intensity of the nanospheres declined inside the cells after addition of ionomycin for 10 s. The fluorescence drops at the cells were restored in the following 5 min, as shown in Figure S10 (Supporting Information), which was consistent with the recovery of intracellular calcium after stimulation. All these results supported that our probes could monitor the fluctuation of intracellular calcium in two spectral ranges. Analysis of the fluorescence intensity in Figure 6 illustrates that the intensity decreased about 40−50% for these two nanospheres at 0 h, while an obvious increase in fluorescence intensity occurred for Fluo-3. However, after the cells were
incorporated nanospheres, and ETH 5350-incorporated nanospheres, respectively. After loading of these probes for 1 h, the cells were cultured in fresh medium and the fluorescence at the cells was recorded at 0, 1, 2, 3, and 24 h. As shown in Figure 4,
Figure 4. Normalized fluorescent intensity of cells loaded with CBDP and ETH 5350 nanospheres and Fluo-3 for 1 h at 37 °C, recorded after 0, 1, 2, 3, and 24 h. Fluorescence intensity in the cells at 0 h was defined as 100%. Error bars represent the standard deviation from five cells.
the significant drop in fluorescence after 1 h only exhibited the obvious leakage of Fluo-3 from the cells. After 24 h of culturing, no fluorescence was measured, revealing no Fluo-3 retained inside the cells. On the contrary, approximately 50%
Figure 5. Fluorescence imaging of cells loaded with (A) CBDP-incorporated nanospheres and (B) ETH 5350-incorporated nanospheres for 1 h, recorded at 24 h before and after ionomycin stimulation. The scale bar was 50 μm. E
DOI: 10.1021/acsami.6b05406 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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exhibited the feasibility of long-term ion monitoring by use of these nanoparticles. Future work will focus on the introduction of targeting molecules at the nanoparticles so that they could reveal ions in specific subcellular compartments.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b05406. Eleven figures showing characterization of calciumselective nanospheres, cytotoxicity of nanospheres, and additional images (PDF)
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Figure 6. Normalized fluorescent intensity of cells loaded with CBDP and ETH 5350 nanospheres and Fluo-3 for 1 h at 37 °C, recorded at 0, 4, and 24 h before and after stimulation. The fluorescent intensity in the cells before stimulation (for nanospheres) or after stimulation (for Fluo-3) was defined as 100%. Error bars represent the standard deviation from five cells.
AUTHOR INFORMATION
Corresponding Author
*Phone 86-25-83594846; e-mail
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the 973 Program (2012 CB932600 and 2013 CB933800) and the National Natural Science Foundation of China (21327902, 21135003, and 21575060).
cultured with the probes in fresh medium for another 3 h, only a 2-fold increase in fluorescence intensity was observed for Fluo-3 upon ionomycin stimulation. At this time point, the intensity decrease of 40−50% was still obtained for the two nanospheres. This phenomenon might be attributed to leakage of Fluo-3, which eliminated the advantage of Fluo-3 in the imaging of intracellular calcium. Most importantly, after 24 h of culturing, monitoring intercellular calcium became impossible for Fluo-3, while both kinds of nanospheres still gave a 20−30% decrease. These results implied the perspective of applying these nanospheres for continuous ion monitoring in cells. It was noted that our ion-selective nanospheres utilized pH indicators as chromoionophores to measure intracellular ions, and thus the minor fluctuation of intracellular pH would interrupt the measurement. To exclude this possibility, CBDP and ETH 5350 as pH indicators were loaded into the cells, respectively. As shown in Figure S11 (Supporting Information), no significant variation of fluorescence intensity was observed in cells before and after ionomycin stimulation. This result revealed that this stimulation might not induce the alteration of intracellular hydrogen ion, and thus our nanospheres could offer continuous information on intracellular calcium during biological processes.
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REFERENCES
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CONCLUSIONS Calcium-selective nanospheres were prepared and loaded into cells to continuously monitor the fluctuation of intracellular calcium. Two chromoionophores, CBDP and ETH 5350, were incorporated into the nanospheres to achieve the assay of intracellular calcium at visible and near-IR regions, respectively. The success in application of ion-selective nanospheres for imaging of intracellular ions revealed that simply replacing the chromoionophores could regulate the spectral regions of the probe to fulfill the biological study. Moreover, as compared with fluorescent chemical probes that needed complex and time-consuming design to achieve biological ion detection, the achievement of intracellular calcium detection by our ionselective nanospheres should provide a simple and universal strategy to analyze intracellular ions. Additionally, the nanospheres were observed to be retained inside the cells for 24 h and indicated the fluctuation of intracellular calcium, which F
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