Journal of Cancer Research and Therapeutics

: 2021  |  Volume : 17  |  Issue : 2  |  Page : 393--400

Zinc oxide nanofluids: The influence of modality combinations on prostate cancer DU145 cells

Afsaneh Azhdari1, Razieh Jalal2,  
1 Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
2 Department of Chemistry, Faculty of Science; Department of Research Cell and Molecular Biology, Institute of Biotechnology, Ferdowsi University of Mashhad, Iran

Correspondence Address:
Razieh Jalal
Azadi Square, Ferdowsi University of Mashhad, Mashhad, Razavi Khorasan Province


Aim: The combination of phototherapy and chemotherapy (chemophototherapy), presents a promising multimodal method for comprehensive cancer treatment. The aim of this study is to investigate the influence of low doses of zinc oxide (ZnO) nanofluids and ultraviolet A (UVA) irradiation on the cytotoxicity and cellular uptake of doxorubicin (DOX) on human prostate cancer DU145 cells. Materials and Methods: ZnO nanoparticles were prepared by the solvothermal method and 10% bovine serum albumin was used as the dispersant. The cytotoxic effect of DOX alone and in combination with different concentrations of ZnO nanofluids (0.95-15.6 μg/ml) in the presence and absence of UVA irradiation on DU145 cells was evaluated by -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. DOX residue inside and outside of DU145 cells was explored by fluorescence microscopy and UV-Vis absorption spectroscopy, respectively. The role of ZnO nanofluids and UVA irradiation in DOX-induced apoptosis and cell cycle arrest were evaluated by DAPI staining, comet assay, and flow cytometry. Results: The results revealed that low dose of ZnO nanofluids (0.95 μg/ml) accompanied with irradiation enhanced the cytotoxicity and intracellular delivery of DOX in DU145 cells. The percentage of chromatin fragmentation/condensation and DNA tail of DU145 cells treated simultaneously with DOX and ZnO nanofluids was increased after UVA irradiation, whereas no significant changes in cell cycle progression were observed. Conclusion: The results indicate that ZnO nanofluids in the presence of UVA irradiation could increase DOX efficiency in DU145 cells, suggesting such modality combinations as a promising approach in cancer treatment.

How to cite this article:
Azhdari A, Jalal R. Zinc oxide nanofluids: The influence of modality combinations on prostate cancer DU145 cells.J Can Res Ther 2021;17:393-400

How to cite this URL:
Azhdari A, Jalal R. Zinc oxide nanofluids: The influence of modality combinations on prostate cancer DU145 cells. J Can Res Ther [serial online] 2021 [cited 2021 Oct 28 ];17:393-400
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Full Text


Considerable attention has been paid to semiconductor nanomaterials which can increase the intracellular concentration of drugs and enhance their potential anti-tumor efficiency in the past two decades.[1],[2] Among them, zinc oxide nanoparticles (ZnO NPs) have received much more attention due to their photocatalytic and photo-oxidizing ability against cancer cells. Doxorubicin (DOX) is one of the well-known chemotherapeutic drugs even though its clinical use has unfavorable consequences and its use is limited by its life-threatening side effects.[3] In this study, low concentrations of ZnO NPs were used as vehicles for DOX delivery and photosensitizer. We revealed that ZnO NPs were the cause of enhancement of entrance of DOX into human prostate DU145 cells and ultraviolet A (UVA) irradiation helped increase DOX cytotoxicity in the presence of ZnO NPs.

 Materials and Methods

Materials and reagents

Zn(CH3 COO)2.2H2O was obtained from Bdh. Bovine serum albumin (BSA), trypan blue, low melting point agarose (LMA), and normal melting point agarose (NMA) were purchased from Merck. Trypsin-EDTA, RPMI 1640, and penicillin- streptomycin were bought from Biosera. Methyl green stain was obtained from GhatranShimi. Fetal bovine serum (FBS) was obtained from Fluka. DOX was purchased from Venus remedies limited. 4,6-Diamidino-2-phenylindole (DAPI), propidium iodide (PI), and 3-(4,5-dimethy lthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma.

Preparation and characterization of zinc oxide nanoparticles

Zinc acetate dehydrate (0.001 mol) was dissolved in 90 ml of deionized water and kept at 4°C for 24 h. Then, 830 ml of deionized water was added to it and it was incubated on ice until its temperature reached 0°C. 80 ml of 0.02 M sodium hydroxide was added slowly to this solution under magnetic stirring in such a way that its temperature remains unchanged. The formed transparent Zn(OH)42− solution was kept in a water bath adjusted at 65 °C for 2 h and 3 days at room temperature. The white powder of ZnO NPs was collected by centrifugation at 12000 rpm for 20 min at 4°C. Then, it was washed with deionized water and ethanol several times, and finally dried in a vacuum oven (Heraeus) at 40°C for 10 h.[4] The X-ray diffraction (XRD) pattern of synthesized ZnO NPs was recorded using a Bruker Axs D8 ADVANCE diffractometer (Ettlingen, Germany) with Cu Kα radiation (λ = 0.15406 nm). The Debye–Scherrer equation (Eq. 1) was used to calculate the average crystalline size of ZnO NPs as follows:


where Dhkl is the crystallite size perpendicular to the normal line of (hkl) plane, k is a constant equal to 0.9, λ is the wavelength of the CuKα radiation, βhkl is the full width at half maximum of the (hkl) diffraction peak, and θhkl is the Bragg angle of (hkl) peak.[5]

Preparation of zinc oxide nanofluid

A suspension of 0.5 mg/ml ZnO nanofluid was prepared in 0.5% BSA and dispersed with the aid of a magnetic stirrer for 6 h. BSA was used as a dispersant to enhance the stability of the suspension.[6] The particle size distribution of the prepared ZnO nanofluids was determined by dynamic light scattering (DLS) on a Malvern Zetasizer Nano-ZS instrument (Malvern, UK).

Cell culture

Human prostate cancer DU145 cell line was obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI-1640 medium supplemented with 100 unit ml−1 penicillin, 100 μg/ml streptomycin, and 10% FBS. The cultures were grown at 37°C, 5% CO2, and full humidity. Monolayers with about 80% cell confluence were used for further experiments ensuring that the cells were in the exponential growth phase.

Cytotoxicity assays

DU145 cells were plated in 96-well plates at a density of 1 × 104 cells cm−2 and incubated for 24 h at 37°C in a 5% CO2 humidified environment. Afterward, the cells were treated with ZnO nanofluids-containing different concentrations of ZnO NPs (0.95, 3.9, and 15.6 μg/ml) or DOX (0.05 μM) alone for 48 h. For combination treatment, 24-h cultured DU145 cells were exposed to 0.95 μg/ml of ZnO nanofluid for 3 h and then DOX was added and they were incubated for an additional 48 h. To explore the influence of UVA irradiation, 3-h treated cells with ZnO nanofluids were irradiated for 180 s with a UVA lamp prior to adding DOX. A 36 W UV lamp (Philips, Germany, LT 36W/009) was used with an incident light intensity of 0.36 J cm−2. The controls were cultivated under the same conditions without addition of ZnO nanofluids and DOX both in the absence and presence of UVA irradiation. Cell viability was measured by MTT assay. After each treatment, 20 μl of MTT (5 mg/ml in PBS) was added to each well and incubated in 5% CO2 at 37°C for 4 h. Then, the cells were treated with 150 μl of dimethyl sulfoxide (DMSO) and the optical absorbance at 570 nm was recorded using an ELx800 Absorbance Microplate Reader (BioTek Instruments, Winooski, VT, USA). The cell viability percentage was expressed as shown in Eq. 2:


where (A) represents the light absorbance at 570 nm. Each experiment was repeated at least three times independently.

Fluorescence microscopy

To investigate the influence of ZnO nanofluids on cellular uptake of DOX, DU145 cells were seeded on glass coverslips inside 6-well plates at a density of 9.5 × 104 cells/well and incubated at 37°C in a 5% CO2 humidified environment for 24 h. Then, the cells were treated with different concentrations of ZnO nanofluids (0.95, 3.9, and 15.6 μg/ml) for 3 h and then DOX (10 μM) was added. After 6 h, the fluorescence intensity of DOX inside the cells was assessed by an inverted fluorescence microscope (Olympus BX51, Tokyo, Japan).[7] Untreated cells, DOX alone and ZnO nanofluids alone treated cells were taken as controls. To explore the influence of UVA irradiation, 3-h treated cells with ZnO nanofluids were irradiated for 180 s with the UVA lamp before adding DOX.

Ultraviolet-visible absorption spectroscopy

Cell suspensions (8 × 105 cells) were prepared in PBS (0.1 M, pH 7.2) containing DOX (10 μM) alone and combined with ZnO nanofluids containing different concentrations of ZnO NPs (0.95, 3.9, and 15.6 μg/ml) in a final volume of 2 ml for each well of 6-well plates. After 6 h, the cell medium was collected and centrifuged at 4000 rpm for 5 min. The UV spectrum of their supernatants was obtained in the 350–600 nm region by UV-VIS spectrophotometer (Agilent 8453, Waldbronn, Germany).[7]

4,6-diamidino-2-phenylindole staining

DAPI staining is a method that shows DNA destruction including chromatin fragmentation and condensation. DU145 cells were seeded in a 6-well plate at a density of 9.5 × 104 cells per well. The procedure of cell treatment and UVA irradiation was similar to MTT assay. 48 h after UVA exposure, the media and trypsinized cells were collected, centrifuged at 800 rpm for 5 min, and fixed with 300 μl of 4% paraformaldehyde for 8 min at room temperature. Then, the fixed cells were washed twice with 700 μl PBS, permeabilized with 300 μl Triton X-100 for 8 min at ambient temperature, and rinsed twice in PBS. Afterward, the cells were stained with 20 μl of DAPI dye (2 μg/ml) and observed by an inverted fluorescence microscope (Olympus BX51, Tokyo, Japan). The cells without any treatment were considered as negative control. Apoptotic cells were identified by condensation and fragmentation of their nuclei. At least, 100 cells were counted in random fields from three independent experiments of each group. In the quantitative analysis, the percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted ×100.

Comet assay

DNA destruction was analyzed with comet assay. Cells were seeded in a 6-well plate at a density of 9.5 × 104 cells per well. The procedure of cell treatment and UVA irradiation was similar to MTT assay. 48 h after UVA exposure, the media and trypsinized cells were collected and centrifuged at 1000 rpm for 10 min. 10 μl of cell suspension including at least 104 cells was added to 75 μl of 0.5% (w/v) LMA and layered onto microscope slides precoated with 1% (w/v) NMA. The slides were kept in a refrigerator for 10 min and immersed in cold lysis buffer (2 M NaCl, 1 M EDTA, 10 mM Tris-base, 1% Triton X-100; pH 7.5) for 1 h. Then, the slides were kept in denaturing buffer (0.3 M NaOH and 1 mM EDTA; pH 13) and incubated at 4°C for 30 min to allow DNA unwinding. Electrophoresis was conducted in denaturing buffer for 30 min at 300 mA. The slides were then immersed in neutralizing buffer (0.4 M Tris; pH 7.5) for 5 min at room temperature, washed three times with water, and dehydrated with 96% cold ethanol for 5 min. After staining, the slides with ethidium bromide (20 μg/ml), the gels were covered with glass coverslips and viewed using the ×200 objective of an inverted fluorescence microscope (olympus bx51, tokyo, japan). Three slides were prepared for each treatment group and a total of 100 cells from each slide were analyzed by image analysis using Comet Score software. The percentage of DNA tail (tail DNA %) was used as the parameter for DNA damage evaluation. It was defined as the fraction of DNA in the tail divided by the total amount of DNA associated with a cell multiplied by 100.[8],[9],[10]

Flow cytometry analysis

Staining of cells with PI was used to assess cell cycle arrest in different treatments. DU145 cells were seeded in a 6-well plate at a density of 95,000 cells per well. The procedure of cell treatment and UVA irradiation was similar to MTT assay. 48 h after UVA exposure, media, and trypsinized cells were collected, centrifuged for 6 min at 1200 rpm and 4°C. The cells were washed in PBS and resuspended in 500 μl of PI (100 μg/ml) solution containing 0.1% (v/v) Triton X-100, 0.1% (w/v) sodium citrate, and RNase A at 3.3 μg/ml. After 1 h in the dark and at room temperature, the cell cycle distribution of the cells was measured using a flow cytometer (BD FACSCalibur™, San Jose, CA, USA). FlowJo Software (www. FlowJo. com) was used to analyze the flow cytometry data.

Statistical analysis

The significance of differences between data was specified with one-way ANOVA SPSS statistical software version 16 (SPSS, Inc., Chicago, IL, USA). The results with a value of P < 0.05 were considered significantly different. Data are expressed as the mean ± standard error of the mean of separate experiments. Three independent experiments were performed for each assay.


Zinc oxide nanofluids characterization

The XRD analysis showed the crystalline nature of the synthesized ZnO NPs [Figure 1]a. The (100), (002), (101), (102), (110), (103), (200), (112), and (201) reflections correspond to the wurtzite structure of ZnO.[11] The diffraction peaks are in good agreement with those in the JCPDS card (Joint Committee on Powder Diffraction Standards, Card No. 89-1397). The crystallite size of the synthesized ZnO NPs calculated using the Debye-Scherrer formula was 20.3 nm. The hydrodynamic radius measured by DLS was found to be 58 ± 3 nm [Figure 1]b.{Figure 1}


The MTT assay was employed to explore the cytotoxic effect of DOX in the presence of low concentration of ZnO nanofluids and UVA light on DU145 and human foreskin fibroblasts (HFF) cells. In the absence of UVA, 48 h and exposure of DU145 cells with 0.05 μM DOX reduced cell viability by about 9%. As shown in [Figure 2], the cell viability of DU145 cells was significantly decreased with increase in ZnO nanofluids concentration from 3.9 to 15.6 μg/ml in the absence of irradiation (P < 0.01). UVA irradiation had no impact on DU145 cell viability. Since no significant difference in cell survival was found between the cells treated with 3.9 and 0.95 μg/ml concentrations of ZnO nanofluids in the presence or absence of irradiation, the lowest concentration of ZnO nanofluids was used to explore its influence on DOX cytotoxicity and the following experiments. As evident in [Figure 3], ZnO nanofluids alone and in combination with DOX did not significantly decrease the viability of HFF cells, neither in the absence nor in the presence of UVA (P < 0.05). MTT assay results showed that 0.95 μg/ml ZnO nanofluids increased the cytotoxicity of DOX (0.05 μM) by about 28% upon irradiation (P < 0.05), whereas no dramatic impact was observed in the absence of UVA. In contrast, UVA irradiation had no marked effect on the cytotoxicity of DOX in ZnO nanofluids-treated normal HFF cells [Figure 3], suggesting that ZnO nanofluids in the presence of UVA irradiation enhanced the cytotoxic of DOX in DU145 prostate cancer cells. The different effect of ZnO nanofluids in the presence of UVA irradiation on the cytotoxicity of DOX in cancer and normal cells may be due to their different electrical and chemical properties.[12]{Figure 2}{Figure 3}

In vitro doxorubicin cellular uptake monitoring by inverted fluorescence microscopy and ultraviolet-visible absorption spectroscopy

The influence of ZnO nanofluids and irradiation on the uptake of DOX by DU145 cells was evaluated using inverted fluorescence microscopy and UV–Vis spectroscopy. DOX cellular uptake was determined by qualitatively comparing DOX-related fluorescence intensity in cytosol and nuclei. As shown in [Figure 4], the intensity of DOX fluorescence in cytoplasm and cellular nuclei was increased in the presence of ZnO nanofluid under irradiation of UVA light. ZnO nanofluids enhanced the entry of DOX into DU145 cells and UVA irradiation increased ZnO nanofluid-induced DOX uptake in a concentration-dependent manner. The residual DOX in the media of DU145 cells was decreased in the presence of ZnO nanofluids as compared with DOX alone in a concentration-dependent manner and much more absorption of DOX residue outside DU145 cells were observed in the presence of ZnO nanofluids after irradiation [Figure 5]. These results suggest that ZnO nanofluids accompanied with irradiation are effective in DOX entrance and accumulation in DU145 cells. Therefore, they may act as suitable agents in DOX delivery systems.{Figure 4}{Figure 5}

Nuclear staining with 4,6-diamidino-2-phenylindole

Changes in nuclear morphology including chromatin condensation and fragmentation were analyzed by DAPI staining. Normal and apoptotic nuclei were easily distinguished as shown in [Figure 6]. When the cells were treated with DOX in combination with ZnO nanofluids in the absence and presence of UVA light, the typical apoptotic morphology was more apparent than when the cells were treated with each of the agents alone [Table 1]. UVA irradiation caused the highest percentage of apoptotic cells in simultaneously treated cells (P < 0.05).{Figure 6}{Table 1}

Comet assay

DNA damage was quantified by measuring the DNA tail % using Comet Score software [Figure 7]. No significant changes in the DNA tail % of DU145 cells were observed between different treatments in the absence of UVA. As evident in [Figure 7], UVA irradiation caused to increase the tail DNA % of DU145 cells treated simultaneously with DOX (0.05 μM) and ZnO nanofluids (0.95 μg/ml) as compared with DOX-treated alone (P < 0.05). In combined treatment, the extent of DNA damage in DU145 cells was 5.6-fold higher than that of normal HFF cells after irradiation [Figure 7].{Figure 7}

The results indicated that apoptosis might play an important role in killing DU145 cells treated simultaneously with DOX and ZnO nanofluids under UVA irradiation.

Flow cytometry

The influence of UVA irradiation on cell cycle arrest of DU145 cells treated with DOX (0.05 μM) and ZnO nanofluids (0.95 μg/ml), alone or in combination, were investigated by flow cytometry. In the absence of irradiation, exposure of DU145 cells to ZnO nanofluids had no effect on cell cycle distribution whereas DOX treatment alone or in combination with ZnO nanofluids caused the arrest of cells in the G2/M phase [Figure 8]. Irradiation resulted in about 20% increase and decrease in the percentage of cells in the G1-and G2/M-phases, respectively, in combination treatment as compared with no UVA. As evident in [Figure 8], no change in cell cycle progression induced by ZnO nanofluids was observed on UVA irradiation.{Figure 8}


ZnO NP is a semiconductor nanomaterial possessing a lot of interesting biological properties. It has been reported as one of the most promising next-generation PDT photosensitizers.[13],[14],[15],[16] It has been shown that UV irradiation can efficiently induce the formation of ZnO NPs-mediated ROS and enhance the cytotoxicity of chemotherapeutic drugs in the presence of ZnO NPs. ROS can lead to cell membrane destruction and changes in membrane permeability, thereby entering more chemotherapeutic molecules into cancer cells.[7],[17] Several published studies have revealed that ZnO NPs-DOX complexes or DOX-loaded ZnO NPs could have synergistic cytotoxic activity in cancer cells.[18],[19],[20],[21] Considering the possibility of the combined application of ZnO NPs and chemotherapeutic agents, this study was carried out to use a combination of individual ZnO nanofluids and DOX, not a complex of two agents, for enhancing the cytotoxicity and cellular uptake of DOX for the first time.

In the present study, 48 h exposure of DU145 cells to 0.05 μM DOX had no significant effect on cell viability. This is in agreement with other results showing no cytotoxic effect of DOX at low concentrations.[22],[23],[24],[25] DU145 cell viability was reduced by 49% on exposure to 15.6 μg/ml ZnO nanofluids, whereas no cell viability reduction was observed at low concentrations (3.9 and 0.95 μg/ml). The cytotoxicity results were similar to those of other studies which have shown that ZnO NPs with concentrations lower than about 6 μg/ml have no cytotoxicity effect.[7],[26],[27],[28],[29]

The influence of UV irradiation on the photocatalytic activity of ZnO NPs depends on the concentration and size of the NPs, the type and dose of irradiation, and the cell type.[7],[17],[18],[30]

Here, irradiation with UVA had no significant effect on the cytotoxicity of ZnO nanofluids whereas it increased the cytotoxicity of DOX in ZnO nanofluids-treated DU145 cells, suggesting the enhanced permeability and cellular uptake of DOX after irradiation.

The inverted fluorescence microscope and UV–Vis absorption spectroscopy data showed the role of ZnO nanofluids as a dose-dependent DOX carrier in DU145 cells. Previous studies have shown that the concentration of drugs inside the cells increases in the presence of metal oxide nanoparticles, suggesting that the role of metal oxide nanoparticles in the cellular uptake of drugs.[2],[7],[18],[30],[31],[32] The accumulation of DOX in DU145 cells may be due to the inhibition of P-glycoprotein function by ZnO NPs and/or Zn-DOX complex formation between Zn2+ released from ZnO NPs and the chelating sites of the phenolic oxygen molecules and the quinone on the DOX aromatic moiety.[7],[18],[30] UVA irradiation causes alternations in the cellular balance redox, cell membrane properties such as membrane lipid peroxidation, passive permeability of the lipid bilayer, nuclear transcription factors activity, and gene expression.[33],[34],[35] Herein, DOX cell uptake was more enhanced when the cells were exposed to irradiation. This may be due to plasma membrane damage and increase in membrane permeability due to UVA exposure.

In this study, the major cell population of DU145 cells were found in the G1 phase in control and ZnO NPs-treated cells. Treatment of DU145 cells with DOX (0.05 μM) alone and in combination with ZnO nanofluids resulted in the arrest of cells in the G2M phase. This is in agreement with previous studies that revealed an arrest in the G2M and G1 phases after exposure to low concentrations of DOX and ZnO NPs, respectively.[36],[37],[38] According to previous studies, the effect of ZnO NPs on the cell cycle arrest seems to depend on the size and concentration of ZnO NPs and cell type.[39],[40],[41] To find the influence of UVA light on cell cycle arrest, DU145 cells were treated with DOX (0.05 μM) and ZnO NPs (0.95 μg/ml) in the absence and presence of irradiation both alone and in combination. However, UVA irradiation had no effect on the cell cycle arrest induced by ZnO NPs alone. Rather, it resulted in about 20% decrease in the percentage of cells in the G2/M-phase in combination treatment as compared with no UVA.

To confirm the existence of apoptosis, the morphological changes of the cells and DNA damage were evaluated by DAPI staining and comet assay, respectively. The percentage of apoptotic nuclei (condensed or fragmented chromatin) and DNA damage were increased in combination-treated DU145 cells after irradiation as compared to cells before irradiation (P < 0.05). Previous studies have shown that both ZnO NPs and UV irradiation induce DNA damage and cell death via an apoptotic process.[28],[29],[30],[33],[40],[42] Mitra et al. indicated that the ZnO-FA-DOX-treated MDA-MB-231 cells exhibited that the nuclei underwent apoptosis.[21] Zhang et al. found that DNR-ZnO nanocomposites under UV irradiation kill SMMC-7721 cells by inducing apoptosis rather than necrosis.[30]

In the present study, the influence of UVA irradiation on the cytotoxicity and cellular uptake of DOX in ZnO nanofluids-treated prostate cancer cells was investigated. The results revealed that a low dose of ZnO nanofluid accompanied with irradiation could enhance the anticancer activity and intracellular delivery of DOX in DU145 cells. This observation suggests that UVA irradiation in the presence of ZnO nanofluids can increase the availability and use of DOX in the cellular environment, and thus it can enhance DOX efficiency in DU145 cells.

Financial support and sponsorship

This project was supported by Ferdowsi University of Mashhad, grant number 3/30375.

Conflicts of interest

There are no conflicts of interest.


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