In situ detection of the Zn²⁺ release process of ZnO NPs in tumour cells by confocal laser scanning fluorescence microscopy

Abstract

The use of zinc oxide (ZnO) nanoparticles (NPs) for cancer is not yet clear for human clinical applications, which is primarily due to the lack of a better understanding of the action mechanisms and cellular consequences of the direct exposure of cells to these NPs. In this work, the authors have selected zinquin ethyl ester, a Zn²⁺ ‐specific fluorescent molecular probe, to efficiently differentiate ZnO NPs and Zn²⁺, and combined with confocal laser scanning microscopy (CLSM) to in situ study the Zn²⁺ release process of ZnO NPs in cancer cell system through detecting the change of Zn²⁺ level over time. During the experiments, the authors have designed the test group ZnO‐2 in addition to assess the influence of a long‐term storage on the characteristics of ZnO NPs in aqueous solution, and the Zn²⁺ release process of ZnO NPs in cancer cell system. After three‐month storage at room temperature, the release process became earlier and faster, which was consistent with previous results of transmission electron microscope, UV‐Vis and PL spectra. It is a good detection method that combination of Zn²⁺ ‐specific fluorescent molecular probe and CLSM, which will be helpful for ZnO NPs using in clinical research.

1 Introduction

Cancer is reported as the second leading cause of death in the United States and accounts for ∼25% of all deaths [1]. Current anticancer chemotherapies frequently fail to produce a complete anticancer response [2], however, nanobiotechnology has been viewed as a more targeted approach potential for providing significant treatment improvements for cancer patients. Zinc oxide (ZnO) is a wide band gap (3.37 eV) semi‐conductor having a high exciton binding energy (60 meV), which leads to efficient excitonic blue and near‐UV emission [3]. Besides the exceptional semi‐conducting, optical, and piezoelectric properties, ZnO is well recognised as a biocompatible and biodegradable material. ZnO is very sensitive to dissolution in acidic conditions, and the unprotected ZnO nanoparticles (NPs) can be decomposed completely at pH 5 in aqueous solution [4]. Meanwhile, Zn is an indispensable trace element involved in a wide number of biological processes, such as metabolism, cell proliferation and differentiation, signal transduction, and control of gene expression [5]. These properties make ZnO NPs excellent candidates for biomedical applications such as drug/DNA delivery [4, 6] and bioimaging [6, 7, 8, 9, 10].

Recently, ZnO NPs have showed a high degree of cancer cell selectivity with the ability to surpass the therapeutic indices of some commonly used chemotherapeutic agents in similar ex vivo studies [11, 12, 13, 14]. Rasmussen et al. [15] systematically reviewed the advantages, approaches and limitations surrounding the use of ZnO NPs for selective destruction of tumour cells and drug delivery applications. Followed, Müller et al. reported that ZnO nanowires were avidly ingested by human monocyte macrophages and showed highly cytotoxicity, due to intracellular release of ionic Zn²⁺, which might be triggered by the lower pH of lysosomes. By comparatively investigating the cytotoxicity of ZnO nanocrystals towards normal human primary cells versus cancer cells, Sasidharan et al. [16] found that the preferential toxicity against cancer cells might be ascribed to the acidic cancer microenvironment which could induce the dissolution of ZnO NCs, and postulated that ZnO NCs would be used to fight against cancer by utilising its own acidic microenvironment. Zhang et al. [4] successfully developed a biodegradable drug delivery system that showing great potential in the treatment of brain cancer based on the pH sensitivity of ZnO NPs.

However, the use of ZnO NPs for cancer is not yet clear for human clinical applications, which is primarily due to the lack of a better understanding of the action mechanisms and cellular consequences of the direct exposure of cells to these NPs.In the past decades, reports about the dissolving behaviour of ZnO NPs in aqueous environments have been published significantly [17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. Meanwhile, Zn²⁺ released from ZnO NPs is at least partially related to their cytotoxic potential [27, 28, 29, 30, 31]. However, the mode of Zn²⁺ cytotoxic remains unclear and, more importantly, it remains uncertain of the dissolving behaviour of ZnO NPs in biological media, as well as the influencing factors, such as the intrinsic physicochemical properties of ZnO NPs, storage conditions and storage time. In this work, we have in situ detected the Zn²⁺ release process of ZnO NPs in cancer cell system and assessed the influence of a long‐term storage on the release process.

As a typical aquatic exposure system, the aqueous solution of ZnO NPs contains both NPs and dissolved Zn²⁺. In previous work, measurement usually relied on the separation of the solution phase from the NPs via dialysis, filtration, or centrifugation [20, 22, 32, 33, 34, 35, 36, 37]. However, the separation procedures are not suitable for complicated cell system. Meanwhile they are inaccurate, as small NPs may remain in the supernatant of filtrate, and the equilibrium process can yield underestimation of the Zn²⁺ value in the dialysis experiments [21]. Therefore, we need to overcome the difficulty of differentiation between ZnO NPs and Zn²⁺. Fluorescent molecular probes have been proved to be useful tools to sense in vitro and in viv o biological important species such as metal ions and small molecules based on their simplicity and high sensitivity [38]. Several Zn²⁺ ‐specific fluorescent molecular probes have been used to detect the cellular free Zn²⁺, such as Newport Green DCF [16, 32], FluoZin3‐AM [28], and zinquin ethyl ester [4, 30, 31]. Among these fluorescent molecular probes, zinquin ethyl ester is water‐soluble and membrane permeable, and it has higher selectivity, sensitivity and stability. In our study, we have combine dzinquin ethyl ester and confocal laser scanning microscopy (CLSM) to in situ study the Zn²⁺ release process of ZnO NPs in cancer cell system through detecting the change of Zn²⁺ level over time.

2 Experimental

2.1 Materials

Our collaborators supplied ZnO NPs, which were synthesised through a two‐step copolymerisation method [4]. In our work, we designated the pristine synthesised ZnO NPs as ‘ZnO‐1’, after three‐month storage at room temperature, ‘ZnO‐1’ was renamed ‘ZnO‐2’.

Zinquin ethyl ester was obtained from Enzo Life Sciences (Plymouth Meeting, PA) and the sensor was solubilised in DMSO (cell culture grade) at stock concentrations of 25 mM and stored in the dark at 4°C.

2.2 NPs characterisations

Transmission electron microscope (TEM) images were obtained using a JEM‐2010 TEM operating at 200 kV. The UV‐V is absorption data were recorded by a Unico 2802 UV/Vis spectrometer. Photoluminescent (PL) spectra were recorded by a Varian Cary Eclipse fluorescence spectrophotometer.

2.3 Cell cultivation

HeLa cells were routinely cultured at 37°C in flasks containing Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified atmosphere and with 5% CO₂ in a Thermo culturist. Cells were plated in a tissue culture flask with 100% humidity.

2.4 Confocal laser scanning fluorescence microscopy imaging

The HeLa cells were cultured on 35 mm glass‐based culture dishes at a density of 8 × 10⁴ cells/ml containing DMEM with 10%FBS at 37°C and with 5% CO₂ for 24 hours, then ZnO NPs of different concentrations ([Zn] = 0, 4, 8, 16 μg/ml) were introduced into the culture dishes containing the cells for 3 hours at 37°C and with 5% CO₂. After being washed with PBS (phosphate buffered saline, pH 7.4) twice, cells were loaded with 25 μM of the cell‐permeant Zn²⁺ indicator (zinquin ethyl ester) for 30 min at 37°C and with 5% CO₂. Then, cells were washed twice with PBS and embedded in DMEM solution. Fluorescence imaging was performed using a Leica TCS SP5 microscope and a Leica application suite, advanced fluorescence confocal scanning system. A 63 × IMM objective (oil immersion) lens was used.

For the on‐line real time detection, the cells were incubated with 25 μM of zinquin ethyl ester for 30 min at 37°C and with 5% CO₂. Afterwards, cells were rinsed twice by PBS and then embedded in DMEM solution, followed by addition of ZnO NPs at the concentration of [Zn] = 20 μg/ml. Live imaging was started within 5 min after sample addition. Excitation of zinquin ethyl ester was performed with laser at 405 nm, and emission spectra were collected using a wavelength range of 450–500 nm. Confocal software (Leica, Germany) was used to read the fluorescence intensity of each cell, and then calculate the average fluorescence intensity.

3 Results and discussion

3.1 NPs characterisations

ZnO NPs with a diameter of about 3.5 nm, which were synthesised through a two‐step copolymerisation method [4] with the internal hydrophobic polymethacrylate layer and the external hydrophilic polymethacrylamide layer, were supplied by research group of professor Xiong. The aim of present study was to explore whether a long‐time storage could influence the characteristics of ZnO NPs in aqueous solution, and the Zn²⁺ release process of ZnO NPs in cancer cell system. To better explain the influence of a long storage, we choose three months as the time parameter. We comparatively studied the high‐resolution TEM images (including selected area electron diffraction images [SAED]), UV‐Vis absorption spectra and PL spectra of ZnO‐1 and ZnO‐2, to be the research foundation.

TEM is used to characterise the particle size and morphology of ZnO NPs (Fig. 1). The TEM images show that the diameter of ZnO‐2 is larger than that of ZnO‐1. The increase of particle diameter might be a result of ‘Ostwald ripening’ [39]. During the synthesis and growth process of conventional ZnO NPs, ‘Ostwald ripening’ is an important growth mechanism, and the process is inevitable. Zhang et al. successfully synthesised ZnO@silica NPs with green fluorescence. After 20 days’ storage at room temperature, the green‐emitting ZnO NPs changed to emit yellow, along with the diameter increasing from 3.8 to 4.4 nm [8], which was consistent with our results. Furthermore, the SAED images show that ZnO‐1 has a perfect wurtzite structure, while a part of crystal structure of ZnO‐2 has been destroyed, which may be the result of the long‐time storage.

Fig. 1.

HRTEM (left‐hand side) and SAED (right‐hand side) images of ZnO‐1 and ZnO‐2

The UV‐Vis spectra of ZnO‐1 and ZnO‐2 are shown in Fig. 2 a, we can find that the position of the characteristic absorption peak around 330 nm of ZnO‐2 appears a considerable red shift, compared with that of ZnO‐1, which means the increase of the particle diameter. According to Meulenkamp's empirical formula (1240/λ_1/2 = a + b /D² ‐c /D) [40], we can calculate that spherical ZnO NPs with a diameter of about 3.5 nm for ZnO‐1 and about 5.6 nm for ZnO‐2.As shown in Fig. 2 b, the maximum excitation wavelength of 330 nm and emission wavelength of 550 nm are the fluorescence characteristics of ZnO NPs, but the fluorescence intensity of ZnO‐2 is much weaker than that of ZnO‐1,which indicates that the luminescent centres of ZnO NPs have been partially destroyed, in accordance to the SAED images (Fig. 1). Those results above suggest that ZnO NPs could probably be destroyed along with a long‐time storage, due to the ‘Ostwald ripening’. Therefore, it is better for the pristine synthesised ZnO NPs to be used, especially in biomedical applications.

Fig. 2.

UV/Vis absorption spectra

a PL spectra (excited by 330 nm; detected at 550 nm)

b ZnO‐1 and ZnO‐2

3.2 Detection of intracellular Zn²⁺ level using Zn²⁺ ‐specific fluorescent probe

Zinquin ethyl ester is a Zn²⁺ ‐specific fluorescent probe that has been used for imaging the Zn²⁺ in live cell system [4, 30, 31]. Prior to detect the Zn²⁺ release process of ZnO NPs in cancer cell system, we studied the sensitivity and efficiency of zinquin ethyl ester for the intracellular Zn²⁺ released from ZnO NPs (ZnO‐1). In this experiment, HeLa cells were exposed to ZnO NPs at different concentrations ([Zn] = 0, 4, 8, 16 μg/ml), and the accumulation of Zn²⁺ in the cells was measured after 3 h of exposure using a fluorescence confocal microscope. The control group ([Zn] = 0 μg/ml) only shows very weak blue fluorescence (Fig. 3), because zinc salts are indispensable in cell culture medium. In the cells treated with ZnO NPs, the blue fluorescence significantly increases along with increasing ZnO NP concentrations, thus confirming the Zn²⁺ release of ZnO NPs. Considering that the ZnO NPs used in this experiment were just synthesised, we ascribed the release to the acidic microenvironment of endosomes, lysosomes, and even of cancer cells [4, 16, 28, 32]. Besides, the average values of fluorescence intensity at different concentrations were read by confocal software, as shown in Fig. 4. Based on the chelating mechanism between zinquin and Zn²⁺, the almost linear correlation indicated that zinquin ethyl ester could relatively quantify the intracellular Zn²⁺ levels efficiently.

Fig. 3.

CLSM images of HeLa cells after 3 h incubation with ZnO NPs at different concentrations (The Zn2+ was stained with fluorescent zinquin ethyl ester (blue))

Fig. 4.

Average fluorescence intensity of HeLa cells after 3 h incubation with ZnO NPs at differernt concentrations

3.3 In situ detection of Zn²⁺ release process of ZnO NPs in cancer cell system

Based on the good performance of zinquin ethyl ester in detecting the intracellular Zn²⁺ released from ZnO NPs (ZnO‐1), we further studied the Zn²⁺ release process of ZnO NPs (ZnO‐1 and ZnO‐2) in situ. For the on‐line real time detection, cells were incubated with 25 μM of zinquin ethyl ester for 30 min at 37°C and with 5% CO₂. Afterwards, cells were rinsed twice with PBS and embedded in DMEM solution. Live imaging was started within 5 min after the addition of ZnO NPs at the concentration of [Zn] = 20 μg/ml.

Fig. 5 in detail records the fluorescence images of the groups of ZnO‐1, ZnO‐2 and control (without ZnO NPs), at 30, 45, 60, 75, 90, 120, 150, 180 and 210 min, respectively. The control group shows weak blue fluorescence, as shown in Fig. 3. The gradually decrease of fluorescence intensity may be the result of the long‐time exposure of laser. While the fluorescence intensity of the test groups (ZnO‐1 and ZnO‐2) both show an increasing tendency, and then decrease slightly. The increasing tendency of the fluorescence intensity indicates that there is increasing amount of Zn²⁺ in the live cells, which was released from ZnO NPs. Followed, the weak reduction may be caused by the long‐time exposure of laser, or by the new combination of Zn²⁺ with some other things in live cell system [24]. However, whether or not the transient fluorescence saturation means complete dissolution of ZnO NPs? We further studied the fluorescence images of ZnO‐1 at 120 min. The fluorescence of ZnO NPs still exists (Fig. 7), which could not mean complete dissolution of ZnO NPs. Both endosomes (pH 5.0–6.0) and lysosomes (pH 4.5–5.0) have an acidic microenvironment, which is distinct from that outside the cells (pH 7.4) [4]. Then we just deduced that the part of ZnO NPs inside the endosomes and lysosomes was completely dissolved.

Fig. 5.

In situ detect the Zn2+ release progress of ZnO NPs. The CLSM images of HeLa cells incubated with ZnO‐1, ZnO‐2 (20 ug/ml) and without ZnO NPs (Control). (The Zn2+ was stained with fluorescent zinquin ethyl ester (blue))

Fig. 7.

CLSM images of HeLa cells incubated with ZnO‐1 (20 ug/ml) at120 min and without ZnO NPs (Control)

The average value of fluorescence intensity at different time points was read by confocal software. The two groups (ZnO‐1 and ZnO‐2) have the similar trend of the fluorescence intensity, but there are two obvious differences (Fig. 6). The fluorescence intensity of ZnO‐2 significantly increases at 45 min, earlier than that of ZnO‐1 (at 60 min or later). Besides, the transient fluorescence saturation of ZnO‐2 (nearly 90 min) happens much earlier than that of ZnO‐1 (nearly 120 min). Those two different performances of ZnO NPs in cancer cell system may be caused by the change of physical and chemical properties during the long‐time storage. The HRTEM and SAED images (Fig. 1) show that the particle diameter increases with worse crystal structure. The PL spectra (Fig. 2 b) show that the fluorescence intensity at the maximum excitation and emission wavelength becomes weaker, with part of luminescent centres destroyed. Therefore, we should consider the factor of storage time during the research of ZnO NPs, especially in biomedicine and pharmaceutical engineering (Fig. 7).

Fig. 6.

Average fluorescence intensity of HeLa cells incubated with ZnO‐1, ZnO‐2 (20 ug/ml) and without ZnO NPs (Control)

4 Conclusions

As a detection method, the combination of Zn²⁺ ‐specific fluorescent molecular probe and CLSM can efficiently differentiate zinc based compounds and Zn²⁺, and in situ detect the Zn²⁺ level in live cell system. We think that this detection method is possible to analyse the Zn²⁺ release from other zinc based compounds such as ZnS, ZnSe, ZnF and so on, which may be helpful for the application research of zinc based compounds in the biomedical and pharmaceutical fields. In our work, we have successfully in situ studied the Zn²⁺ release process of ZnO NPs in cancer cell system through detecting the change of Zn²⁺ level over time. The significant release of Zn²⁺ started at 60 min or later, and then went with a transient saturation. Moreover, the saturation did not mean the complete dissolution of ZnO NPs. It might have had something to do with the acidic microenvironment of endosomes (pH 5.0–6.0) and lysosomes (pH 4.5–5.0), we just deduced that the part of ZnO NPs inside the endosomes and lysosomes was completely dissolved. In addition, we have assessed the influence of storage time on the release process. After three‐month storage at room temperature, the release process became earlier and faster, which might be caused by the change of physical and chemical properties during the long‐time storage. Moreover, the present surface modification methods have already endowed ZnO NPs with good performance in biomedical research. In future research, we just need to make clear the action mechanisms and cellular consequences of the direct exposure of cells to ZnO NPs. Therefore, the combination of Zn²⁺ ‐specific fluorescent molecular probe and CLSM is very helpful for the research of ZnO NPs in clinic.