Lasers in Medical Science

, Volume 27, Issue 3, pp 607–614

Sensitivity of A-549 human lung cancer cells to nanoporous zinc oxide conjugated with Photofrin

Authors

  • Muhammad Fakhar-e-Alam
    • Department of Science and TechnologyCampus Norrköping, Linköping University
    • Pakistan Institute of Engineering and Applied Sciences
    • Department of Science and TechnologyCampus Norrköping, Linköping University
    • Department of Electronic EngineeringNED University of Engineering and Technology
  • Zafar Hussain Ibupoto
    • Department of Science and TechnologyCampus Norrköping, Linköping University
  • Khun Kimleang
    • Department of Science and TechnologyCampus Norrköping, Linköping University
  • M. Atif
    • Laser Diagnosis of Cancer, Physics and Astronomy Department, College of ScienceKing Saud University
    • National Institute of Laser and Optronics
  • Muhammad Kashif
    • Nano Biochip Research Group, Institute of Nano Electronic Engineering (INEE)University Malaysia Perlis (UniMAP)
  • Foo Kai Loong
    • Nano Biochip Research Group, Institute of Nano Electronic Engineering (INEE)University Malaysia Perlis (UniMAP)
  • Uda Hashim
    • Nano Biochip Research Group, Institute of Nano Electronic Engineering (INEE)University Malaysia Perlis (UniMAP)
  • Magnus Willander
    • Department of Science and TechnologyCampus Norrköping, Linköping University
Original Article

DOI: 10.1007/s10103-011-0989-8

Cite this article as:
Fakhar-e-Alam, M., Ali, S.M.U., Ibupoto, Z.H. et al. Lasers Med Sci (2012) 27: 607. doi:10.1007/s10103-011-0989-8
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Abstract

In the present study, we demonstrated the use of nanoporous zinc oxide (ZnO NPs) in photodynamic therapy. The ZnO NPs structure possesses a high surface to volume ratio due to its porosity and ZnO NPs can be used as an efficient photosensitizer carrier system. We were able to grow ZnO NPs on the tip of borosilicate glass capillaries (0.5 μm diameter) and conjugated this with Photofrin for efficient intracellular drug delivery. The ZnO NPs on the capillary tip could be excited intracellularly with 240 nm UV light, and the resultant 625 nm red light emitted in the presence of Photofrin activated a chemical reaction that produced reactive oxygen species (ROS). The procedure was tested in A-549 cells and led to cell death within a few minutes. The morphological changes in necrosed cells were examined by microscopy. The viability of control and treated A-549 cells with the optimum dose of UV/visible light was assessed using the MTT assay, and ROS were detected using a fluorescence microscopy procedure.

Keywords

Lung cancer (A-549) cellsMTT assay PhotofrinCell viabilityNanoporous zinc oxide (ZnO NPs)Reactive oxygen species (ROS)Photodynamic therapy (PDT)

Introduction

Activation of a photosensitizer/nanomaterial system by UV light (240 nm) results in tissue necrosis by direct killing or vascular blockade due to singlet oxygen release from the mitochondria [13]. In the field of nanomedicine and nanotechnology numerous nanomaterial metal oxides of variable chemistry and architecture have been introduced for cancer diagnosis and treatment, and involve the use of multifunctional engineering devices [46]. There are many emerging applications involving the use of nanomaterials that make use of the physical, chemical and biological behavior of materials at the nanoscale size [7, 8]. It is considered that nanotechnology will have a positive impact on basic sciences and in particular on clinical applications such as photodynamic therapy (PDT) [9].

PDT is a cancer therapy that is based on the photoinduced generation of highly cytotoxic singlet oxygen (type II reaction), and other reactive oxygen species (ROS) such as the hydroxyl radical, hydroxyl ion, hydrogen peroxide and hypochlorite ion which induce oxidative cell injury that leads to necrotic or apoptotic cell death. It is a nonthermal photochemical process that can be used to treat malignant and nonmalignant diseases. PDT involves the simultaneous presence of light, oxygen and a light-activatable chemical called a photosensitizer. The photosensitizer is administered either systemically or topically and is activated by visible light at a specific wavelength, commonly from a low-power laser, to produce cytotoxic free radicals, namely singlet oxygen, in the presence of molecular oxygen. Singlet oxygen damages cellular organelles which triggers a cascade of events resulting in cell death either by apoptosis or necrosis [1023].

The mechanism of apoptosis following the use of PDT in different malignant cell lines has been described [15]. Cells of a gastric cell line were incubated with 10 μg/ml Photofrin for 24 h before irradiating with a He-Cd laser (441 nm, 1 J/cm2). The viability of the cells after laser irradiation was analyzed using the methyl-tetrazolium (MTT) assay, and 95% cell death was found. DNA ladder formation and chromatin condensation were also seen within a period of 60 min. Other findings included an increase in caspase-3-like and caspase-9-like activities after laser irradiation for 15 min. Cell death (apoptosis) in the gastric cancer cell line MKN45 60 min after Photofrin-mediated PDT has also been studied. Damage to the mitochondria has been reported to be the first event in apoptosis.

In this study, we demonstrated the effectiveness of nanoporous zinc oxide (ZnO NPs) in PDT using UV light at a wavelength of 240 nm for activation in cells of the A-549 human lung carcinoma cell line. We also sought to minimize cell death due to mechanical stress or trauma. The results showed a significant reduction in cell viability (almost 88%).

Materials and methods

Deposition and conjugation of ZnO NPs

The focus of the work presented here was to develop a microinjection drug delivery technique involving the deposition of ZnO NPs on the tip of borosilicate glass capillaries and its conjugation with Photofrin photosensitizer. ZnO NPs was deposited on sterile borosilicate glass capillaries (Femtotip II, Eppendorf, Hamburg, Germany) with a tip inner diameter of 0.5 μm, an outer diameter of 0.7 μm and a length of 49 mm by applying a low-temperature aqueous chemical growth technique [24, 25]. The morphology and structure of ZnO NPs are shown in the scanning electron microscope (SEM) images in Fig. 1. An atomic force microscopy (AFM) image of a grown ZnO NPs structure is shown in Fig. 2. AFM images were acquired using a Dimension 3100 scanning probe microscope (Digital Instruments) in tapping mode with Si cantilevers. The AFM images clearly showed the porosity of the ZnO NPs structures grown that varied between 200 nm and 600 nm, and thus the therapeutic outcome of PDT using ZnO NPs may be better than that using ZnO nanorods (ZnO-NRs) [2].
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Fig. 1

SEM images of ZnO NPs grown on a Femtotip borosilicate glass capillary using a low-temperature chemical growth method: a typical low-magnification image; b magnified image

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Fig. 2

AFM image of ZnO NPs

The ZnO NPs grown on the capillary glass tip were functionalized by conjugation with a Photofrin layer using a manual process. Photofrin in powder form was purchased from Sigma Aldrich. Its molecular structural formula has been reported previously [26]. Photofrin was dissolved in phosphate-buffered saline (PBS, pH 7.4) to obtain a stock solution (1,000 μg/ml) [1820] which was stored in the dark at 4°C. ZnO NPs-coated tips were dipped five times into the prepared Photofrin solution. After each dip, the tip was allowed to dry at room temperature [2]. A submicrometer glass pipette covered with bare grown ZnO NPs was also used as a reference PDT device to allow separation of the contribution to fluorescence of the ZnO NPs and the ZnO NPs/Photofrin conjugate. The fluorescence spectrum of the ZnO NPs/Photofrin conjugate excited with 240 nm UV light is shown in Fig. 3. Bare and conjugated ZnO NPs devices were used as local PDT intracellular photosensitizer delivery systems in A-549 human lung carcinoma cells. Treated cells were examined under a Nikon microscope attached to a CCD camera.
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Fig. 3

Fluorescence spectrum of ZnO NPs dispersed in PBS excited by exposure to 240 nm UV light

Cell culture conditions

We used A-549 human lung carcinoma cells as the in vitro experimental model. Cells were seeded in 25-cm2 plastic tissue culture flasks (Nunc, Wiesbaden, Germany) in minimum essential medium with Hanks’ salts containing 10% fetal bovine serum and 2 mM l-glutamine and some nonessential amino acids, and were incubated for 24 h to allow attachment to the substrate. Cells were maintained at 37°C in a moist environment as a subconfluent monolayer and were routinely subcultured twice or three times weekly. Cell cultures at 70–80% confluence were harvested using 0.25% trypsin [1822]. After trypsinization, some cells were transferred to a regular Petri dish and incubated again for 24 h to allow attachment to the bottom of the Petri dish at 37°C in a CO2-enriched atmosphere. By mechanical manipulation the capillaries with the conjugated ZnO NPs were inserted gently into the cultured cells.

Incubation of photosensitizer and laser irradiation

The measurements in our experiments were performed in two steps. In the first step A-549 cells were cultured in a 96-well flat-bottomed microtiter plate at a density of about 1 × 105 cells/well. The cells were then incubated with Photofrin at a concentration of 100 μg/ml and were irradiated with light at doses in the range 0–160 J/cm2. In the second step cells were incubated with Photofrin at different concentrations in the range 0–500 μg/ml for 24 h and were then irradiated. A separate 96-well plate was used for each Photofrin concentration. Wells were irradiated through the clear bottom of the plate using a semiconductor diode laser at a wavelength of 635 nm, and 80 J/cm2 was used as the optimal light dose. The laser beam was transmitted to the target by a quartz optical fiber which was set immediately below the bottom of the 6-mm diameter wells to irradiate the cells in the 6-mm diameter wells. Following irradiation, the cell culture medium was removed and cells were washed twice in ice-cold PBS, pH 7.4. The PBS was then replaced with minimum essential medium containing 10% v/v fetal bovine serum and the plates were returned to the incubator for 24 h. After the 24-h incubation, the viability of the light-treated cells was determined.

Cell viability MTT assay

A-549 cell survival was assessed using the MTT assay. This assay is based on the ability of living cells having active mitochondria to convert the tetrazolium compound into an insoluble purple-colored formazan product. The cells were fixed in MTT for 3 h at 37°C. The cells were then washed three times with tap water, and dried in air at room temperature. The color of the cells was dissolved in DMSO. After 15 min of gentle shaking, the plates were read using a Multiskan MCC/340 microplate reader at 540 nm. The percentage of viable cells in the cell population at each concentration of Photofrin was calculated using a standard method [2729] using the following formula:
$$ \% {\text{ Viability = }}\frac{\text{Mean absorbance of ALA treated cells}}{{\text{Mean absorbance of control cells}}} \times \left( {100} \right) $$

Staining of mitochondria

For staining of mitochondria, cells were incubated with MitoTracker Red CMXRos (200 nM, 37°C; Invitrogen) for 30 min in cell culture medium without serum. The labeled cells were then examined and images were obtained using a Nikon Eclipse 400 epifluorescence microscope with a CCD camera and/or a DU-897E camera.

ROS detection

Intracellular ROS production was detected using the nonfluorescent compound CM-H2DCFDA (Invitrogen, Carlsbad, CA). The compound crosses the cell membrane and undergoes deacetylation by esterases producing nonfluorescent CM-H2DCF, which reacts with oxygen species inside the cell to produce the highly fluorescent dye CM-DCF. After inactivation of the cells using ZnO NPs conjugated with different concentration of Photofrin in humidified air containing 5% CO2 at 37°C for 24 h, they were washed gently once in Dulbecco's modified Eagle's medium. Cells loaded with 100 μl of 5 μM CMH2DCFDA were incubated for 30 min at 37°C and protected from light. The cells were then exposed to UV light at a dose of 10 J/cm2 for 2 min, and were visualized for determination of ROS production under an inverted fluorescence microscope attached to a digital camera [30].

Results and discussion

Generally ZnO nanomaterials have two different toxic effects on treated cells. The first effect is chemical toxicity (apoptosis or necrosis) due to the release of toxic ions or the formation of ROS [3033], and the second effect is stress or mechanical trauma caused by the shape and size of the nanostructures as described previously [2].

In our experiments we stained the mitochondria of A-549 cells using MitoTracker red dye (data not shown). Capillaries with the conjugated ZnO NPs at the tip were inserted manually into A-549 cells, and after 30 min of UV irradiation a countable increase in the fluorescence was observed. This was shown by the detection of ROS in multiple PDT steps (data not shown). A relationship between ROS production and cell necrosis was observed, in good agreement with the results reported by Lin et al. [34]. Previous studies have shown that cell toxicity depends on the dose and incubation time. In addition, in studies evaluating the toxicity of ZnO nanoparticles with sizes of 70 and 420 nm in human lung epithelial cells, exposure to the nanoparticles resulted in a reduction in cell viability (82% reduction with the 70-nm nanoparticles, 72% reduction with the 420-nm nanoparticles) via cell membrane leakage, ROS production, reduction in GSH levels, increase in LDH levels, lipid peroxidation and oxidative DNA damage [35, 36].

A steep response pattern has been observed with other metal oxides. Moos et al. found that the toxicity of nanoparticles in RKO cells depended on the time of contact and the concentration of the nanoparticles, but was independent of the amount of Zn in the cell culture medium [37]. In addition, the nanoparticles of ZnO were more cytotoxic than microsized ZnO nanoparticles in the above cell line. The mechanism of cell death included the disruption of mitochondrial function, and robust markers of apoptosis, annexin V staining, loss of mitochondrial potential, and increased generation of superoxide were also observed [37]. The impact of particle size, structure and dimensions of ultrafine nanoparticles on cell toxicity was not addressed in our experimental study, but the correlation of cytotoxicity with particle size in macrophages exposed to silica particles has been investigated [38].

It is a matter of debate whether dietary ultrafine nanoparticle metal oxides, e.g. ZnO, have chronic effects on the colon. The effects of ingestion of large amounts of ZnO have been investigated in humans, and ultrafine nanoparticle metal oxides have been shown to cause corrosive gastroduodenal injury without systemic toxicity. Inhaled ZnO can cause pulmonary toxicity but minimal toxicity to other organs [3945]. Increased consumption of fine and ultrafine particulate matter has been hypothesized to exacerbate inflammatory bowel disease [4649]. However, reducing the intake of nanoparticles has been reported not to affect remission in Crohn's disease [50], so the real picture is still not clear.

ROS fluorescence from a microinjected cell exposed to UV light at the doses and for the irradiation times detailed in the legend visualized under an inverted fluorescence microscope attached to a digital camera (data not shown). Significant ZnO drug toxicity was noted after 30 min of incubation in the studied cells. The cell membrane had almost disappeared, the cytoplasm had a white halo with numerous small needle head-like deposits, and the nucleus was ill-defined. No other cell organelle was visible; apoptosis was almost complete [2].

The basic objective of the present investigation was to minimize cell death due to mechanical stress/trauma, which has been shown in many studies to be almost 25%, and the production of significant quantities of ROS which involves chemical reactions leading to cell death [51]. We employed a microinjection drug delivery technique involving the gentle insertion of a capillary with a conjugate of ZnO NPs and 100 μg/ml Photofrin on the tip into A-549 cells. This resulted in almost complete disappearance of the cell membrane. The cytoplasm showed necrosis with a white halo and the nucleus was ill-defined with small and some larger thick densities in it. Fluorescence induced by Photofrin was evaluated, No other signs of cell necrosis were visualized by microscopy. When a functionalized ZnO NPs capillary tip was gently inserted into A-549 cells manually followed by 30 min of UV irradiation, cells started to show signs of necrosis. Thus, excitation of ZnO NPs by irradiation with UV light at a wavelength of 240 mm and at the optimum dose results in the emission of broad-spectrum white light as shown in Fig. 3, which is able to start photochemical reactions that lead to cell death via mitochondrial damage. In this study, A-549 cells were used to investigate the toxicity of ZnO NPs with and without Photofrin.

The experimental algorithm of the mechanism of PDT is shown schematically in Fig. 4. ZnO-NRs due to its large surface area to volume ratio has attractive features for drug delivery to, for example, tumor sites. In this study we compared the efficacy of PDT using microinjection drug delivery (Fig. 4) and free-standing drug delivery. The figure clearly shows that ZnO conjugated with Photofrin together with a suitable dose of UV light can lead to the emission of broad-spectrum light with multiple peaks, and this emission can excite the photosensitizer (Photofrin) leading to cell necrosis via mitochondrial damage as we showed in this study using A-549 cells.
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Fig. 4

Schematic diagram of the mechanism of PDT

Figure 5 shows the reduction in viability of A-549 cells in relation to the laser light (630 nm) dose applied along with 100 μg/ml of Photofrin. Cell viability reduced sharply with increasing doses up to 60 J/cm2 and thereafter remained constant with higher doses. Maximum uptake of Photofrin was observed in A-549 cells after 12 h (data not shown). Photofrin uptake was almost the same regardless of its concentration, which may have been due to cellular saturation. Increasing absorbance was noted from 7 h to almost 18 h of photosensitizer incubation, and after that a descending trend was observed, certainly because of elimination from cells. A noticeable maximal uptake of Photofrin was observed after 10 h of incubation. The acceptable range of incubation times for a good PDT outcome in the A-549 cells studied could be 10–18 h, with the optimal time being12 h, based upon our data. It is worth noting that no data were previously available for the treatment of lung carcinoma, and this is the novelty of our work.
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Fig. 5

Percentage viability of control and treated A-549 cells in the presence of 100 μg/ml Photofrin and exposed to 640-nm laser light

The aim of our experiments was to determine the feasible and suitable dose of photosensitizer along with diode laser, and our experiments showed a reduction in cell viability of at least 90%. We found a reduction of 92–95% in the viability of the A-549 cells in our experiments. Significant production of ROS was observed with ZnO NPs conjugated with 100 μg/ml of Photofrin, which is to be considered the optimum dose from our current data, being associated with the maximum reduction in cell viability (approximately 92%). A significant increase in ROS production was observed in cells microinjected with ZnO NPs/Photofrin conjugate (data not shown) as compared to nontreated A-549 cells (data not shown). It has been previously shown that production of ROS is dependent on photosensitizer concentration [31, 32]. However, excessive amounts of ROS damage biomolecules, trigger apoptosis and even further induce cell death. ZnO NPs conjugated with 100 μg/ml Photofrin induced a gradual increase in fluorescence in the cells following irradiation with increasing doses (10–20 J/cm2) of UV light. The concentration-dependent enhancement in intracellular ROS levels is consistent with the significant cytotoxicity of nanoporous materials in the presence of UV light, which is in good agreement with our previous results [18, 19, 27, 29]. Currently the generation of ROS and the formation of oxidative stress is the best-developed paradigm to explain the toxic effects of nanomaterials [31].

Conclusion

We successfully demonstrated the cytotoxic effects of bare ZnO NPs and ZnO NPs conjugated with Photofrin in A-549 lung carcinoma cells. We used a microinjection drug delivery technique and a free-standing protocol, and assessed the viability of the labeled cells using the MTT assay. The experimental results showed that reduction in cell viability was related to the production of ROS. Moreover, we also found that the ZnO NPs conjugated with Photofrin under UV (240 nm) light exposure displayed valuable cytotoxic effects as compared to Photofrin alone. Thus, ZnO NPs exhibited synergistic cytotoxicity in A-549 cells under UV exposure, showing the potential of ZnO NPs in photodynamic therapeutic applications.

Acknowledgment

M. Fakhar-e-Alam is grateful for financial support from the Higher Education Commission (HEC), Islamabad, Pakistan.

Copyright information

© Springer-Verlag London Ltd 2011