Lasers Med Sci DOI 10.1007/s10103-013-1501-4

ORIGINAL ARTICLE

Photodynamic effects of zinc oxide nanowires in skin cancer and fibroblast Muhammad Fakhar-e-Alam & S. Kishwar & M. Willander

Received: 3 July 2013 / Accepted: 25 November 2013 # Springer-Verlag London 2013

Abstract Cytotoxic effects of zinc oxide (ZnO) nanomaterials, individual and conjugated with a photosensitizer (protoporphyrin IX), were studied in the presence and absence of ultraviolet light exposure (240 nm of light wavelength for a very short time exposure) in cell cultures of human normal and cancerous skin models. Zinc Oxide nanowires (ZnO NWs) were grown on the capillary tip and conjugated with protoporphyrin IX (PpIX). This coated tip was used as tool/pointer for intracellular drug delivery protocol in suggested normal as well as carcinogenic cellular models. After true delivery of optimal drug, the labelled biological model was irradiated with UV-A, which led to a loss of mitochondrial membrane potential, as tested by neutral red assay (NRA). Keyword Protoporphyrin 1X (PpIX) . ZnO nanowires (ZnO NWs)

Introduction Photodynamic therapy (PDT) is a widespread medical technology that is used to treat premalignant and early-stage cancer and reduce tumour size. PDT is a minimally invasive treatment modality that is based on cellular uptake of a photosensitiser, which is excited by light at a specific wavelength of the compound’s absorption peak [1]. The excited photosensitiser will then transfer its energy to molecular

M. Fakhar-e-Alam (*) Department of Physics, GC University, Faisalabad, Pakistan e-mail: [email protected] M. Fakhar-e-Alam : S. Kishwar : M. Willander Department of Science and Technology, Campus Norrköping, Linköping University, 601 74 Norrköping, Sweden

oxygen, which allows the photosensitiser to relax to its ground singlet state and creates an excited singlet oxygen molecule. Singlet oxygen is highly reactive towards biomolecules and might result in cellular damage via oxidation of cellular components, which can lead to apoptosis or necrosis [2]. The therapeutic effect of PDT in vivo is based on the preferential retention of the photosensitiser in the tumour tissue, followed by a restricted illumination at the tumour location [3, 4]. Two principal mechanisms can be considered for PDT-mediated tumour eradication, namely direct damage to the tumour cells and the stroma. The latter has been found to involve microvascular injury and nonspecific immune activation [5, 6]. Nanoparticles (NPs) are photoluminescent semiconductors suggested to have a potent biological cytotoxic effect due to their high quantum yield and size-dependent tuneable wavelength emission over a wide spectrum [7, 8]. Previous experiments have shown that nanoparticulated TiO2 sensitised with ZnPc accumulates in mitochondria, and upon irradiation using wavelengths between 597 and 752 nm, cell toxicity is augmented [9]. A photosensitiser that is in complex with nanowires and activated using UV light results in cell necrosis. This mechanism might involve the production of reactive oxygen species (ROS), which can cause oxidation of biomolecules and finally cell death. In addition, ZnO NWs have a high aspect ratio and are efficient drug carriers [10, 11]. Zinc oxide nanocrystals have a broad range of applications in biomedicine and bio-imaging and can be applied as labelling agents due to their strong photostability and emission properties [12–15]. The drug will be accumulated in an abnormal site; of course, the maximum chances of apoptosis occurred in PDT. Many researchers prove in experimental published results when PDT steps were performed for the treatment of cancerous/malignant tissue model [16]. The neighbouring cells examined for a longer time were found healthy and in normal shape, and their auto-fluorescence was recorded without treatment. In the meanwhile, 20 min is the optimal time

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rather than in the case that the superficial/faint fluorescence was recorded. The toxicity of a nanomaterial depends partly on its chemical toxicity due to the release of toxic ions and formation of reactive oxygen species. Moreover, the shape and size of the NWs might induce mechanical trauma at the plasma membrane [17]. Similar results were obtained using ZnO nanoparticles [18]. In this article, borosilicate glass capillaries (sterile Femtotip® II with tip inner diameter of 0.5 μm, an outer diameter of 0.7 μm and a length of 49 mm; Eppendorf AG, Hamburg, Germany) were fixed on a flat support in the vacuum chamber of an evaporation system (Satis CR725), so that thin chromium and silver films (with a thickness of 10 and 125 nm, respectively) were uniformly deposited onto the outer surface of the capillary. On the silver-coated capillary glass tip, we grew hexagonal single crystals of ZnO NRs by using a low-temperature method. The morphology and structural information of ZnO NRs were confirmed by scanning electron microscopic (SEM) images, which will be shown in this article. The ZnO NRs are 150–170 nm in diameter and around 1 μm in length. The ZnO NRs on the silver-coated capillary glass tip was conjugated with protoporphryrin IX (PpIX) layer by manual process. ZnO nanowires grown on Femtotip capillary tip were used as pointer. After insertion into the cell model, under an irradiation of 20 J/cm2 of UV light, significant reactive oxygen species were found. In the meanwhile, mitochondria damaging effect was examined by staining the mitochondria after applying MitoTracker Red assay [19, 20].

Materials and methods The growth of ZnO NWs on the tip and the conjugation process Borosilicate glass capillaries (sterile Femtotip® II Eppendorf) with a tip that had an inner diameter less than 0.5 μm (outer diameter of 0.7 μm) and a length of 49 mm were used as substrates to grow ZnO NWs. Thin films of chromium and silver with a thickness of 20 and 120 nm, respectively, were uniformly deposited onto the outer surface of the capillary tips. On the silver-coated capillary glass tip, hexagonal single crystals of ZnO NWs were grown using a low-temperature method [10, 21–30]. A portion of the tips with ZnO NWs was conjugated to Pp1X layer via a manual process, as described [10]. Bare ZnO NWs tips were also used as reference for PDT. Free-standing Zinc Oxide NWs powder was dissolved in phosphate-buffered saline (PBS) via a vortex and ultrasonication process for 20 min to make a 10-mg/mL stock solution, and the working solution was then prepared using different concentrations (0.05–600 μg/mL). The same process was used to dissolve ZnO NWs powder in Pp1X solutions very gently. Intracellular measurements were performed on the stage of a Zeiss Axiovert (Zeiss, Gene, Germany) inverted

microscope, using a mechanical manipulator (Eppendorf) to fix the ZnO NWs and take the intracellular measurements. By free-standing drug delivery, ZnO nanowires were synthesised via a hydrothermal decomposition method under an autogenous pressure condition. All of the raw materials were of analytical reagent grade and used as-received without further purification. In a typical experiment, (NH4)2CO3, Zn (NO3)2·6H2O and NaOH were dissolved in distilled water to prepare a 1.0-M (NH4) CO3 solution, 1.0-M Zn (NO3)·6H2O solution and 1.0-M NaOH solution, respectively. A 1.0-M Zn (NO3)·6H2O solution (100 mL) was dropped slowly into 250 mL of a (NH4)2CO3 solution with vigorous stirring to obtain a precipitate. The precipitate was filtered and repeatedly rinsed with deionised water to remove the residual reactants. Next, it was dispersed in 300 mL of deionised water by stirring for 30 min. A 1.0-M NaOH solution was added to this dispersion to adjust the pH to 9.2. This suspension was transferred into a Teflon-lined stainless steel autoclave with a 500mL capacity and heated to 200 °C for 5 h. After the reaction was completed, the white powder product was filtered via suction and dried in vacuum at 100 °C. The ZnO nanowires were obtained by calcining the powder at 400 °C for 3 h.

Cells and culture conditions Melanoma cells (FM55P) were seeded out in 25-cm2 plastic tissue-culture flasks (Nunc, Wiesbaden, Germany) in minimum essential medium (MEM) with Hank’s salts, containing 10 % Fetal Bovine Serum (FBS) and 2 mM of L -glutamine along with some nonessential amino acids and antibiotics (penicillin, streptomycin and neomycin) were incubated for 24 h for proper attachment to the substratum. Cells were maintained at 37 °C in a moist environment as a subconfluent monolayer and were routinely sub-cultured twice or thrice weekly. The cell culture with75–80 % confluence was harvested using 0.25 % trypsin [4]. Human foreskin fibroblasts (AG01518; passages 12–24; Coriell Institute, Camden, NJ, USA) were cultured in Eagle’s minimum essential medium supplemented with 2 mM of glutamine, 50 IU/mL of penicillin-G, 50 μg/mL of streptomycin, and 10 % of Fetal Bovine Serum (all from Gibco, Paisley, UK) [31, 32].

The staining of mitochondria For the staining of the mitochondria, cells were incubated with MitoTracker Red CMXRos (200 nM, 37 °C, Invitrogen) for 25 min in a cell culture medium without serum. Thereafter, fresh culture medium was added, and the cells were examined and photographed in a Zeiss Axiovert (Zeiss, Gene, Germany) inverted microscope using a Nikon photomicroscope.

Lasers Med Sci Fig. 1 a Bare Femtotip model of microinjection drug delivery. b Femtotip model with ZnO nanowires grown

Cell viability Cells were seeded in 24- and 96-well plates and were prepared till desired confluence (about 75 % confluence). After preparation of required cellular quantity, microinjection drug delivery with bare ZnO NWs was performed as reference PDT step. In the next step, ZnO NWs grown on capillary tip conjugated with PpIX with and without UV-A exposure/irradiation phototoxicity were measured. Finally, ZnO NWs conjugated with Pp1X were added to the cultures at concentrations between 0.05 and 600 μg/mL in the cell culture medium with and without serum. Each concentration was assigned one column (six wells) per 24-well plate, and control cells were exposed to a cell culture medium either with or without serum, and solvent controls for the N , N-dimethyl formamide were made. Cells were incubated in humidified air with 5 % CO2 at 37 °C for 24 h. The images were captured using a microscope coupled with a CCD camera. Cell survival was assessed using the NRA. In addition, UV-A irradiation were used for excitation process. Phototoxicity of the said laser was examined, but

Fig. 2 SEM image of a bare Femtotip model and b ZnO nanowires grown model

no significant cell viability loss was recorded. Irradiation of UV-A for 2 min had no effect of cell killing because no significant ROS were collected in the mentioned irradiation effect as experimental results.

Reactive oxygen species detection Intracellular ROS production was detected using the nonfluorescent compound CM-H2DCFDA (2,7dichlorodihydrofluorescein diacetate acetyl ester; Invitrogen Co., USA). This compound crosses the cell membrane and undergoes deacetylation by an esterase, producing the nonfluorescent CM-H2DCF. Cells were seeded in a black 96-well plate and incubated in different concentrations of ZnO NWs and ZnO NWs conjugated with Pp1X in humidified air with 5 % CO2 at 37 °C for 12 h. The cells were also cultured in Petri dishes treated as the above experimental steps, excited using blue light (488 nm) and photographed under an inverted fluorescence microscope with a digital camera [33].

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Results and discussion

Fig. 3 ZnO grown capillary/Femtotip conjugated with PpIX inserted for PDT in foreskin fibroblast

80

% Cell Viability

In the present study, melanoma and foreskin fibroblast cellular model was used for studying on the testing feasibility of cytotoxicity of ZnO nanowires, individual and conjugated with PpIX. The snapshot of bare and ZnO-coated Femtotip made by borosilicate was shown in Fig. 1. In addition, a SEM image of borosilicate Femtotip capillary (bare) is depicted in Fig. 2a, and ZnO grown on the said Femtotip is shown in Fig. 2b. Basically, our group was interested to trace the photodynamic effects of ZnO nanomaterials (ZnO nanowires and nanorods) bare and conjugated with PpIX, which is novelty of our experimental work. It is found that free-standing drug delivery within range of 100–120 nm, ZnO nanomaterial is biocompatible and nontoxic. In the first step, a ZnO NW pointer was inserted into cells for confirmation of cell killing effect of melanoma and foreskin fibroblast as reference of PDT in the absence of any light. No significant cytotoxic effects were counted under the dark, even when the Femtotip was coated with ZnO nanowires used as a reference pointer. In the second step, the coated tip was used as a pointer for intracellular insertion of ZnO NWs in the presence of UV-A illumination (light dose 10 J/cm2). It is examined that no remarkable ill-shaped behaviour was seen under ultraviolet illumination of ZnO nanowires grown on the Femtotip model after insertion into the said suggested cellular model. ZnO grown on capillary/Femtotip conjugated with PpIX is inserted for PDT step as shown in Fig. 3. In the third step, the suggested cells were exposed with and without UV-A light. No significant apoptotic effect was found in the absence of ZnO labelled without UV illumination, as described in Fig. 4. From the said experiment, it is clear that when ZnO nanowires grown on conjugated PpIX Femtotip under UV illumination acted as an experimental model,

100

before UV after UV

60

40

20

0

Control

ZnO

ZnO+ PpIX

Fig. 4 Viability (percentage) before and after UV Irradiation

significant cell apoptosis or necrosis within a few seconds after treatment was recorded. The results were confirmed after measurement of reactive oxygen species (free radicals/singlet oxygen). Intracellular ROS production was detected using the nonfluorescent compound carboxy-H 2 DCFDA (2,7dichlorodihydrofluorescein diacetate acetyl ester). It easily permeates the cell membrane and is not fluorescent. Inside the cell, the acetate groups are hydrolysed. In this way, the substance becomes less prone to leave the cell. This new substance is not fluorescent, but it can react with ROS and produce a highly fluorescent product, as ROS will oxidise this new substance. The resulting product is fluorescent. This product’s fluorescence increases due to reactive oxygen species. Fibroblasts and melanoma cells were incubated with ZnO either with or without conjugation with Pp1X for 21 h before labelled cells were stained with carboxy-H2DCFDA. When analysed using a fluorescence microscope, foreskin fibroblast and melanoma were treated with PpIX-conjugated ZnO NWs grown on a borosilicate Femtotip, under illumination of UV-A, a significant amount of ROS was found in melanoma as compared with foreskin fibroblast. Some researchers published their data that due to access of low-density lipoprotein (LDL) receptors, localization of drug ratio is 3:1 into malignant and normal tissue model, respectively. In consistency of the above statement, marvellous quantity of free radicals/singlet oxygen must be stipulated [25]. The results showed greater toxicity for the ZnO NWs conjugated with PpIX and compared with bare ZnO NWs under irradiation of UV-A. Fibroblasts were cultured on square (1.0×4.0 mm2) coverslips and incubated with ZnO NWs for 24 h. The coverslip was inserted into a cuvette at a 45° angle to the light beam of the fluorometer, and the emission spectra of ZnO NWs were obtained at room temperature. Our results agreed with the fluorescence spectra as in [34]. The fluorescence spectrum shows four peaks of white light.

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The first, second and third green emissions peak at 500, 517 and 575 nm, respectively (data not shown); the nanomaterial is attributed to a recombination of electrons at the conduction band with holes trapped in oxygen-related defects. The fourth peak, which begins at 590 nm is attributed to a defect-related radiative transition in ZnO [21]. In Fig. 5, it is clear that ZnO NWs under irradiation show significant loss of cellular viability in skin cancerous model; in fact, significant reactive oxygen species were produced in malignant model under irradiation of ZnO NWs labelled with melanoma cellular model. But in the absence of UV-A irradiation (10–20 J/cm2 of light dose), no response of photochemical reaction occurred. Some researchers proved in their published data that cell necrosis might be possible due to ZnO NWs morphology via mechanical trauma. Our results agreed with the recent published data [34]. The morphology and structure of the ZnO NWs on the capillary tip were visualised using a scanning electron microscope (Fig. 2b). The ZnO NWs were 100–120 nm in diameter and approximately 1.5 μm in length. ZnO NWs were grown on a capillary tip, conjugated with PpIX and inserted into the cytosol of a fibroblast by using a micromanipulator (data will be shown in a future article). The experimental biological models after treatment were examined by applying microscopy, and viability was tested by NRA [35, 36]. The necrosed and ill-defined shape of melanoma as well as fibroblast was confirmed by selecting staining technique with Mito Tracker Red to visualise the mitochondrial membrane potential. The treatment caused heavy damage to the cell, and the mitochondrial fluorescence disappeared within 20 min with UV-A irradiation in the presence of conjugation of PpIX. However, the

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ZnO NWs without UV Irradiation ZnO NWs with UV Irradiation

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% Cell Viability

95 90 85 80 75 70 65 60 0

100

200

300

400

500

ZnO NWs concentrations (µg/ml)

Fig. 5 Percent cell viability of melanoma in the presence and absence of UV-A irradiation

neighbouring cells that were exposed to UV for 20 min still displayed diffused red fluorescence in their mitochondria. More satisfactory results were obtained in melanoma cells as compared with fibroblast. Some researchers quoted that the localization as well as free radical production amount is significant in malignant cellular model as compared with normal biological model [28, 29]. When a capillary with ZnO NWs on the tip was inserted into the melanoma cells as a result of photochemical reaction, the cells shrunk, and, within minutes, the ill-shaped behaviour was seen. However, the shape of the cell was maintained. Furthermore, when the tip with ZnO NWs was inserted inside the nucleus, the cell began to shrink immediately (not shown). In the literature, when the photosensitiser aminolevulinic acid (ALA) is topically applied to a tumour, the cells will accumulate protoporphyrin 1X via the heme biosynthesis pathway [25–27]. The level of protoporphyrin 1X has been found to be higher in tumour cells due to an altered skin barrier [28, 29] and enzymatic differences [30] compared with normal skin. By directly inserting the ZnO NWscontaining tip inside the cell, we overcome the hindrances to ALA and Pp1X diffusion through the cell membrane. Some researchers have claimed that ZnO is biocompatible and bio-safe [31], but we concluded that within the size of 100– 150 nm, ZnO NWs are biocompatible but, with conjugation of PpIX, exhibit considerable cell toxicity not only for melanoma but also poisonous for fibroblasts when incubated together for 24 h. ZnO nanowires can be used for tumour-selective delivery of chemotherapeutic agents because of the high accumulation/ considerable uptake into tumorous part/malignancy which has been verified by many researchers that the drug uptake ratio in premalignant/malignant to normal healthy cell is 3:1 due to the majority of LDL receptors [37–41].

Conclusion In conclusion, we observed that ZnO NWs constitute a potent photosensitizer when taken up by cells and induce ROS production after UV-A-irradiation. A ZnO NW pointer inserted into cells and irradiated with UV-A caused a loss of mitochondrial membrane potential and significant loss of cell viability. Bare ZnO NWs were nontoxic to fibroblasts as well as for melanoma cells as compared to ZnO NWs conjugated with PpIX in the absence of suitable laser illumination/ irradiation. In addition, it was seen that the toxicity of the suggested cellular model is size-dependent, as the size of the nanomaterial increases above 200 nm, and the cell killing effect of melanoma as well as fibroblast links with shape and size of the suggested nanomaterial. Thus, the biosafety of ZnO NWs for use as drug delivery vehicles is still under debate.

Lasers Med Sci Acknowledgments The authors would like to specially thank the Higher Education Commission (HEC), Pakistan, for the financial support for this research.

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