Detection of pH Change in Cytoplasm of Live Myocardial Ischemia

Article pubs.acs.org/ac

Detection of pH Change in Cytoplasm of Live Myocardial Ischemia Cells via the ssDNA-SWCNTs Nanoprobes Ru Liu,†,§ Li Liu,‡,§ Jian Liang,‡ Yaling Wang,† Yueteng Wei,† Fuping Gao,† Liang Gao,† and Xueyun Gao*,† †

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China ‡ Affiliated Ruikang Hospital of Guangxi University of TCM, Nanning, Guangxi 530011, China S Supporting Information *

ABSTRACT: Myocardial ischemia is featured by a significant increase in the cytoplasm proton concentration, and such a proton change may be applied as an index for earlier ischemic heart disease diagnostics. But such a pH change in a live heart cell is difficult to monitor as a normal fluorescent probe cannot specifically transport into the cytoplasm of an ischemic cell. This is because the heart cell contains condensed myofibrils which are tight barriers for a normal probe to penetrate. We design fluorescent probes, single-strand DNA wrapped single-wall carbon nanotubes (ssDNA-SWCNTs), where the ssDNA is labeled by the dye molecule hexachloro-6-carboxyfluorescein (HEX). This nanoprobe could transport well into a live heart cell and locate in the cytoplasm to sensitively detect the intracellular pH change of myocardial ischemia. Briefly, protons neutralize the negative charges of nanoprobes in the cytoplasm. This will weaken the stability of nanoprobes and further tune their aggregation. Such aggregations induce the HEX of some nanoprobes condensed together and further result in their fluorescence quenching. The nanoprobes are advantaged in penetrating condensed myofibrils of the heart cell, and their fluorescence intensity is sensitive to the proton concentration change in the live cell cytoplasm. This new method may provide great assistance in earlier cardiopathy diagnosis in the future.

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nanotubes (the ssDNA is labeled by the dye molecule HEX). For nanoprobes, single-wall carbon nanotubes (SWCNTs) help ssDNA to penetrate the live cell membrane. Previous reports revealed that SWCNTs can transport well through the cell membrane and exactly locate in the cell cytoplasm when the SWCNTs’ surfaces are coated with DNA molecules.21,22 The HEX-labeled ssDNA is applied to monitor the pH change in cytoplasm via proton-induced fluorescence change. Our research discloses that the nanoprobe is sensitive to the changes in the cytoplasm pH, which is the main characteristic of the myocardial ischemia process. Previously, many biosensor-based nanomaterials have been reported to detect versatile molecules via a resonance energy transfer mechanism in artificial environment. However, the optical biosensors-based nanomaterials are not widely reported in live cell analysis.23−25 This because a live cell is a more complex environment, and using nanomaterials has potential safety concerns when they are applied in live cell studies. In this report, we first showed that the ssDNA-SWCNT nanoprobe could sensitively and safely detect the protons in live heart cell cytoplasm.

schemic heart disease (IHD) is a major public health problem and the leading cause of death in most industrialized countries.1,2 Myocardial ischemia means that there is reduced cardiac blood perfusion, which will cause serial degrees of damage, such as heart muscle dysfunction, metabolic disorders, and ultrastructural change.3−5 Prolonged myocardial ischemia causes necrosis and contractile dysfunction, which is the molecular pathogenesis of IHD.6−9 Myocardial ischemia is featured by decreasing supply of oxygen, depletion of tissue high energy phosphates, accumulation of ADP and AMP, and lastly, it leads to a significant decrease in the pH of cytoplasm.3,5,10−14 Among these, pH is the main characteristic of the myocardial ischemia process.15 Previous research disclosed that during the ischemia process, the intracellular pH will fall down around 6.0.15−18 As a consequence, a low intracellular pH can be considered as one of the significant indications of myocardial ischemia. Therefore, detecting pH change of the myocardial cell will disclose the myocardial ischemia and assist in early IHD prognosis and diagnosis. However, specific detection of pH change of cytoplasm in a live heart cell has not been achieved yet, as it is very difficult for a traditional organic fluorescent probe to precisely transport into cytoplasm of heart cell.19 Furthermore, the heart cell contains condensed myofibrils which are tight barriers for a normal probe to penetrate.20 In this paper, we design the fluorescent nanoprobes, single-strand DNA wrapped single-wall carbon © 2014 American Chemical Society

Received: December 9, 2013 Accepted: February 25, 2014 Published: February 25, 2014 3048

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Cell Viability Assay. Cell viability was measured by the Cell Counting Kit-8 system (CCK-8) according to manufacturer’s instructions (DOJINDO Lab., Japan). H9c2 cells were seeded at 2 × 103 cells per well in 96-well plates and allowed to attach for 24 h. After incubation with different concentrations of nanoprobe for different time points, cells were immediately washed with PBS and then incubated with fresh medium containing 10 μL of CCK-8 reagent for 2 h at 37 °C for the cytotoxicity assay. Then, the absorbance at 450 nm was measured using a microplate reader (SpectraMAX M2, Sunnyvale, California) with a reference at 600 nm. All data were presented as mean percentages ± standard deviation in triplicate compared to the optical density (OD) values of controlled cells. Confocal Laser Scanning Microscopic Observation of Nanoprobes in a Live Cell. Cellular uptake and intracellular fluorescence intensity of the ssDNA-SWCNTs nanoprobes were tracked by confocal laser scanning microscopic (PerkinElmer spinning disc confocal microscope with a Nikon TI-E inverted microscope with a 60× oil immersion lens). H9c2 cells were plated onto the bottom of a glass dish and cultured at 37 °C overnight. After they were incubated with the nanoprobes in fresh medium, the confocal images 3D object tracking was performed using Volocity Quantitation software; this process allows us to exactly observe the location of the nanoprobe in a live cell and to count the fluorescence intensity of nanoprobes therein.

MATERIALS AND METHODS Method to Prepare Nanoprobes. Fluorescently labeled single-strand DNA, d(CGT)10-HEX, where the HEX is hexachloro-6-carboxyfluorescein, is purchased from Invitrogen (molecular weight 9 KDa). Single-wall carbon nanotubes (SWCNTs, purification 90%) synthesized via the HiPCO methods were purchased from Carbon Nanotechnologies, Inc. In a typical method, 10 mL of deionized water containing 1.33 μM DNA-HEX and 0.015 mg SWCNTs were prepared. Then, the resulting solution was placed in an ice-bath. After the mixture was ultrasonicated for 30 min under 40 W microwave output, the ssDNA-SWCNTs solution was centrifuged at 10 000g for 1 h to discard the free-standing SWCNTs. To remove free DNA-HEX, the supernatant solution was transferred to the dialysis film (molecular weight cut off ∼25 KDa, purchased from Fisher) and bathed in deionized water for 48 h. At the end of the process, the ssDNA-SWCNTs nanoprobes were collected and well-dispersed in the solution.26,27 DLS, Zeta Potential, AFM, and HRTEM Characterization of Nanoprobes. Particle size and Zeta potential of the ssDNA-SWCNTs nanoprobes were measured by dynamic light scattering (DLS) using the NicompTM 380/ZLS and analyzed by ZPW388 software. Three millilters of sample solution was put into a vessel. E-field strength was set a 10 V/cm with 100 s running time for the zeta potential. AFM studies of the ssDNA-SWCNTs products are as follows: the samples were deposited on mica, spun at 100 rpm/ minute for 10 min, and dried for 30 min in air, respectively. The morphology of ssDNA-SWCNTs nanoprobes was observed by atomic force microscope (Multimode V, Veeco, America) operating in the tapping mode at a resonance frequency of 242 kHz. HRTEM studies could disclose the fine morphology of ssDNA-SWCNTs. The nanoprobe samples were dropped on the mesh grids and dried in air. High-resolution images were obtained by using Tecnai G2 F20 S-Twin high-resolution transmission electron microscopy in the 200 KV accelerating voltage. Fluorescent Spectra Studies of Nanoprobes. Different amounts of phosphoric acid standard solution (AR grade) containing either K+, Na+, Ca2+, and Mg2+ or not were transferred into the nanoprobe solution to obtain different pH samples. The pH measurements were performed on FE20 pH meter (Mettler Toledo). The nanoprobes’ concentration was fixed at 250 nM. Emission spectra of nanoprobes were detected by spectrofluorometer (Fluorolog-3, HORIBA Jobin Yvon, Inc., France). The excitation wavelength was 514 nm, and emission was 552 nm. Excitation and emission slits were set to 5 nm. All data were obtained in triplicate studies. Cell Culture. H9c2 cells, a clonal line derived from rat heart, were routinely grown in Dulbecco’s modified Eagle medium (DMEM) containing sodium pyruvate and 1000 mg/L glucose (Gibco) supplemented with 10% fetal calf serum (Gibco) in an atmosphere of 10% CO2. The ischemia model cell was carried out as reported previously.28,29 Briefly, the H9c2 cells were incubated in slightly hypotonic Hanks’ balanced saline solution (1.3 mM CaCl2, 5 mM KCl, 0.3 mM KH2PO4, 0.4 mM MgSO4, 0.5 mM MgCl2, 69 mM NaCl, 4 mM NaHCO3, and 0.3 mM Na2HPO4) without glucose or serum for 2 h at 37 °C, and the resultant H9c2 cells are with ischemia status.

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RESULTS AND DISCUSSION Characterization of ssDNA-SWCNTs Nanoprobes. In principle, SWCNTs could be wrapped by ssDNA via π−π stacking between the base of ssDNA and the sidewall of SWCNT.30,31 After ice-bath ultrasonication, ssDNA-SWCNTs were well-dispersed in solution. The free SWCNTs and DNA were removed by centrifugation and film dialysis, respectively. The remaining products were the ssDNA-SWCNTs nanoprobes in solution. To characterize the nanoprobe, DLS, AFM, and HRTEM were used. Number distribution of ssDNASWCNTs obtained by DLS was shown in Figure1a. The residual value is 0, which indicates that there is a homogeneous solution and no carbon nanotube aggregation. Also, we can find that the average diameter of the main products is 105 nm ± 23 nm. The morphology of products was studied by AFM (see Figure1b). The image showed that the majority of ssDNASWCNTs are well-dispersed on mica. In order to confirm the AFM observations and provide a clear image about the wrapping between ssDNA and SWCNTs, HRTEM were used. From Figure1c, we could clearly observe the DNA wrapping along the nanotube. This can provide us strong evidence to prove the ssDNA wrapped around SWCNTs. pH-Induced Emission Decrease of ssDNA-SWCNTs Nanoprobes. In order to examine the relation between emission intensity of nanoprobes and the solution pH in vitro, fluorescent emission spectra of nanoprobes were obtained when changing pH values of solution. The nanoprobes’ concentration was fixed at 250 nM. Different amounts of acid solution were transferred into the nanoprobe solution to obtain samples with pH of 7.2, 6.6, 6.0, 5.0, 4.0, respectively. As shown in Figure 2, ssDNA-SWCNTs solution shows higher fluorescence intensity under neutral physiological conditions (pH = 7.2), but the intensity drops intensively under acidic conditions, as the pH changed from 6.6 to 4.0 by adding acid into the sample solution. The above results confirm that the 3049

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Figure 1. Characterization of nanoprobes. (a) Particle size of the ssDNA-SWCNTs in number distribution. (b) AFM height image of dispersed ssDNA-SWCNTs (see nanoprobes pointed by black circle, scale bar ∼200 nm). (c) HRTEM image of individually dispersed ssDNA-SWCNTs (scale bar ∼10 nm).

Figure 2. (a) pH-dependent fluorescence emission spectra of ssDNASWCNTs nanoprobes, the inset shows the linear relation between pH and emission intensity of nanoprobes. Error bars represent variations between three measurements. (b) The relative emission intensity of nanoprobes when they are incubated with different metal ions, respectively; K+ is at 150 mM, Na+ is at 20 mM, Ca2+ and Mg2+ are at 3 mM, representing their concentrations in a live cell.16,17,32 The mixed ions result represents the above ions and a pH value of 6, which could mimic conditions of myocardial ischemia in vitro.

ssDNA-SWCNTs nanoprobes display pH-dependent fluorescence intensity, and there is linear relation between pH and fluorescence. As this nanoprobe will be used to detect the pH value in the cytoplasm of a live cell, their specificity fluorescence response to proton rather than the rich metal ions in cytoplasm should be checked first. To do this, the normal metal ions in a live cell (e.g., K+, Na+, Ca2+, and Mg2+) are introduced into solution to study their binding competition with that of proton in solution. In Figure 2b, it is clearly shown that all these metals ions could not suppress the fluorescence of nanoprobes even they are with the highest concentration permitted in a live cell. To disclose the mechanism of how nanoprobes display pHdependent fluorescence behavior, AFM, DLS, and Zeta potential observations were carried out. For the nanoprobe, the SWCNT is wrapped by ssDNA, which is labeled by the HEX dye to form a micelle state. Under neutral physiological conditions (pH = 7.2), the nanoprobes were well-dispersed and no aggregation occurred, such as what we see in Figure 1b. But when the pH is down from 7.2 to 4.0, the aggregation of ssDNA-SWCNTs occurred, and more SWCNTs aggregated as the solution pH became more acidic (see Figure 3a). We know that for the ssDNA-SWCNTs, the negative phosphate of the ssDNA, which was wrapped with SWCNTs, provides the electrostatic repulsion between nanoprobes. Thus, the electrostatic repulsion will maintain the stability of the nanoprobe in solution.31 As the system became more acidic, more H+ will neutralize the negative ion of the nanoprobes. This will weaken electrostatic repulsion, which is the main sustaining factor for the stability, and further tune some nanoprobe aggregation. Such aggregation may induce the HEX of the nanoprobe attached in the condensed manner, which will result in

fluorescence quenching.33,34 To confirm our deduction, the DLS and Zeta potential experiments were carried out to provide the positive proof. In Figure 3b, in initial status (pH 7.2), the main DLS peak was at 105 nm, and the average zeta potential is −31 mv, which indicate that the nanoprobes are quite steady in solution at pH 7.2; this is also verified by AFM studies in Figure 1b. When the pH drops to 6.6, which means the system changes from neutral to acidic conditions, the main DLS peak was moved to 118.8 nm, and the zeta potential is down to −25 mv. The DLS peak moving implied that the ssDNA-SWCNTs aggregated (Figure 3a), and the zeta potential decrease indicates some negative charge of nanoprobe is neutralized by the proton. As the system became more acidic from pH 6.6 to pH 4.0, the DLS main peak and zeta potential change further (e.g., more negative), ssDNA are neutralized by the proton and more nanoprobes aggregate (see Figure 3a,b, respectively). Cell Viability and Cellular Location Studies of ssDNASWCNTs Nanoprobes. The aforementioned studies ensure that ssDNA-SWCNTs probes have the potential ability to measure pH in a live cell. Before they are applied in live cells, their toxicity and location in live cells should be clarified first. The H9c2 cell was selected in those studies. The cell viability was assessed by incubating different amounts of the probe with 3050

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mode, and the Z-axis images were disclosed in Figure 4b. It was determined that after they were incubated with H9c2 cells for 6 h, the probes could transport into the live cells and locate in the cytoplasm. Note that the cytoplasm is the right destination of the nanoprobe, as the pH will change significantly when live cells have ischemia status. ssDNA-SWCNTs Probes Measure the pH of a Live Ischemia Cell. The aforementioned studies disclosed that the fluorescence emission of probe changes with the pH and that the probe could transport into live H9c2 cells. This implies that the probe may reflect the pH changes in ischemia cells. In live cells, the ischemia-induced emission decrease of probes is studied as follows. Briefly, the normal live cells with probes localized in the cytoplasm are prepared by incubating normal live cells with 50 nM probes. Consequently, the cells are divided into control (normal) and ischemia model groups (for the ischemia model group, specific culture conditions were used to simulate ischemia; see Cell Culture in Materials and Methods), and the emission intensity of the probes in the live cells is counted at time points of 0 and 2 h in a real-time process. The three-dimensional fluorescent images of the probes located in the cell are disclosed in Figure 5a, where

Figure 3. (a) AFM images of nanoprobes incubated in different pH concentrations, including images of pH = 6.6, pH = 6.0, pH = 5.0, pH = 4.0, respectively. More and more nanoprobes aggregate when the pH became further acidic (see nanoprobe aggregations indicated by the black circle) (b) Particle size and Zeta potential of the ssDNASWCNT. As the pH drops from 6.6 to 4.0, the net charge of the nanoprobe became smaller and the diameter of the particle became larger.

Figure 5. (a) Three-dimensional confocal microscope images of nanoprobes in control and ischemia cells. Top to bottom rows refer to control cells and ischemia cells, respectively. Left to right lanes refer to the time-dependent emission of nanoprobe in a live cell from 0 and 2 h, respectively. (b) Relative emission intensity of nanoprobe in control and ischemia cells at 0 and 2 h. F and F0 represent cell fluorescence intensity at different time points of 0 and 2 h. The emission intensity of the nanoprobe is significantly suppressed by the rich proton in ischemia cytoplasm. Error bars represent variations between three measurements.

the H9c2 cells for different time points. The cell viability result is shown in Figure 4a. It is clear that cells treated with 50 nM

the emission intensity of probes located in the cytoplasm can be observed. The corresponding relative emission intensity of the probes in live cells is shown in Figure 5b. Clearly, for the control cells the relative emission intensity of the probes remains stable after 2 h of incubation, where the relative emission intensity of the probes (F/F0) is about 97%. For the control cell, this F/F0 value reveals the stability of their fluorescence for the entire observation, and this means the emission intensity of the probes is not disturbed by the native components in live cells. However, for the ischemia cells, the relative emission intensity of the probes (F/F0) decreases to 70% at 2 h when the cells were with the ischemia condition. The F/F0 of the nanoprobe significantly decreased. As the nanoprobe is sensitive to the pH change rather than the K, Na, Ca, Mg ions in the live cell (see results in Figure 2b), the remarkable pH change of cytoplasm in ischemia cell should result in the fluorescence intensity quenching of the nanoprobe in the live cell. The F/F0 of nanoprobe is down to 67% when the pH changes from 7.2 to 6.0 (see Figure 2a), and the F/F0 of nanoprobe change is down to 70% in the live ischemia cell (Figure 5b), where previously reported cytoplasm of an

Figure 4. (a) Cell viability of H9c2 cells incubated with nanoprobes for different time points, the concentration of nanoprobes is 50 and 100 nM, respectively. The black and red bars represent 24 and 36 h, respectively. (b) Confocal microscope Z-axis images of H9c2 cells incubated with nanoprobes for 6 h. The b1−b6 slices point from top to bottom of 3D images, which illustrate that the nanoprobes transported into the live H9c2 cells and are located in the cytoplasm.

probes have very good morphology and viability ratio (over 95%) even after 36 h of incubation. When the cells are treated with 100 nM nanoprobes, over 90% of the cells remained viable. From the cell viability studies, we get the optimized concentration and time of the nanoprobes for the following cell studies. The transportation and location of the nanoprobes is observed by the confocal microscope using 3D object tracking 3051

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ischemia cell is about pH 6.0 as well.15−18 Obviously, our result is very close to the previous data, and this implies that our nanoprobe could be used to index the pH change of an ischemia cell.

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CONCLUSIONS In conclusion, ssDNA-SWCNTs nanoprobes were designed to detect the pH change of ischemia in live cells in situ. In particular, the fluorescence intensity of the probes is pH dependent, and the nanoprobe possesses proton ion selectivity and cell compatibility. When incubated with live ischemia cells, the relative emission intensity of the probes decreases dramatically to index ischemia-induced pH changes of a live cell. Our results propose an approach to index the pH change of a live heart cell with myocardial disease, which may provide great assistance in cardiopathy diagnosis.

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ASSOCIATED CONTENT

S Supporting Information *

HRTEM characterization of free SWCNT, pH-dependent fluorescence emission spectra of free ssDNA, and transportation of free ssDNA in H9c2 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 86-10-88236456. Present Address §

The authors contributed equally to this work. 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 This work was financially supported by the National Key Basic Research Program of China (2013CB932703) and National Natural Science Foundation of China (21390410, 31271072, 31200751, 81201133, 81101743, 31300827). We also thank Prof. Youyi Zhang at Academy for Advanced Interdisciplinary Studies and Institute of Vascular Medicine of Third Hospital, Ministry of Education Key Lab of Molecular Cardiovascular Sciences, Peking University, for providing us with the H9c2 cells and some clinical information.

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REFERENCES

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