PEGylated zinc oxide nanoparticles induce apoptosis in pancreatic cancer cells through reactive oxygen species

Abstract

In this study, the authors have successfully prepared the polyethylene glycol (PEG)‐coated zinc oxide nanoparticles (ZNPs) and studied its effect in pancreatic cancer cells. The authors have observed a nanosized particle with spherical shape. In this study, the authors have demonstrated the cytotoxic effect of ZNP and PZNP in PANC1 cells. To be specific, PZNP was more cytotoxic compared to that of ZNP in PANC1 cancer cells. The authors have further showed that apoptosis is the main mode of cytotoxic activity. It is worth noting that PEGylation of ZNP did not decrease the cell killing activity of zinc particles, whereas it further increases its anticancer effect in the pancreatic cancer cells. The authors have observed a significant upregulation of proapoptotic BAX while expression of antiapoptotic Bcl‐2 was significantly downregulated indicating the potent anticancer effect of zinc nanoparticles. Overall, PEGylation of ZNP could be an effective strategy to improve the stability, while at the same time, its anticancer activity could be enhanced for better therapeutic response.

1 Introduction

Pancreatic carcinoma is one of the leading causes of cancer related in human across the world [1]. Despite the technological development in science and technology, there is no improvement in the outcome of treatment of this carcinoma. The overall 5‐year survival rate is <5% and mortality and morbidity due to this cancer kept increasing [2, 3]. There are several treatment options, however, none is effective in controlling the disease progression and death rate. Chemotherapy is considered as one of the viable option post‐surgeries or when diagnosed at early stages; however, chemotherapeutic drugs often resulted in severe adverse effect compared to its clinical benefits [4]. Therefore, there is a need to develop alternative strategy for the pancreatic cancer treatment.

Nanotechnology allows the creation of materials at a nanoscale which can be modified accordingly for the biological applications. Inorganic nanoparticles are gaining increasing attention for its applications in medicine such as drug delivery or cancer treatment [5, 6]. In this regard, zinc oxide nanoparticles (ZNP) belong to a group of metal oxides, which has been reported to exhibit several biological functions owing to its photo‐oxidising ability. Recently, ZNP has received much attention due to its anticancer property in many cancer cells [7]. The greatest advantage of ZNP is its interaction with the cancer cells. It has been reported that ZNP possess a strong positive charge at normal or physiological conditions which makes it to interact firmly with the negatively charged cell membrane resulting in higher cellular uptake and higher cytotoxic effect in cancer cells [8]. There are several reports in which ZNP has been combined with chemotherapeutic drugs for the enhanced synergistic effect. However, effect of ZNP on the pancreatic cancer cell is not studied in detail, till date [9]. In addition, solubility and stability of ZNP in biological media or conditions is a major concern in cancer therapy [10]. Therefore, attempts have to be made to improve the physicochemical properties of ZNP alongside its biocompatibility has to be improved.

Coating or capping of ZNP with hydrophilic or biocompatible polymers would be one good approach towards this perspective [11]. Surface assembly of polyethylene glycol (PEG) on the nanoparticle is expected to increase the stability, solubility, and biocompatibility characteristics [12]. Furthermore, PEGylation of ZNP also removes the problem of protein adsorption in the blood circulations. There are several studies in which PEGylation of particles improved the systemic performance towards better therapeutic efficacy. For example, PEG‐modified magnetic NP has improved cellular uptake in breast cancer cells [13, 14]. PEG‐surface modification of gadolinium NP has increased the cellular uptake in cancer cells. Overall, surface modification of ZNP with hydrophilic polymers such as PEG will improve its therapeutic effect in pancreatic cancer cells.

The ZNP is expected to induce the cytotoxic effect by virtue of apoptosis of cancer cells. Several intracellular signals including reactive oxygen species (ROS) are involved in the induction of apoptosis cascade [15]. The ROS generated in the mitochondria will initiate the DNA damage which will trigger the p53‐mediated signalling pathways and lead to apoptosis and cancer cell death [16]. In this study, we have reported the preparation of bare ZNP and PEGylated ZNP (PZNP) and studied its anticancer effect in pancreatic cancer cells. The particle size distribution and morphology of ZNP and PZNP was evaluated using DLS and TEM. The cytotoxic effect of ZNP and PZNP was studied using MTT assay. Furthermore, apoptosis effect of inorganic particles was studied using Annexin V/PI staining using flow cytometer. Finally, molecular mechanism was evaluated using Western blot analysis.

2 Materials and methods

2.1 Preparation PEG‐coated zinc oxide nanoparticles

The ZNP were prepared from zinc nitrate and sodium hydroxide using wet chemical method. The NaOH was used as a precursor and starch was used as a stabiliser. Briefly, 50 ml of 0.1 M zinc nitrate solution was prepared in water and to this solution 0.2 M NaOH was added in a dropwise manner and vigorously stirred for 2 h. The zinc hydroxide (Zn(OH)₂) was formed as a white precipitate which is centrifuged at 8000 rpm for 10 min. The white precipitate was washed three times with distilled water to get rid of any trace of impurities. The cellulose powder was mixed with zinc hydroxide particles and dried at 100°C. The mixture was thoroughly mixed by grinding in apparatus. The grinded powder was then calcined at 300°C in a muffle furnace for ∼2 h. The ZNPs were formed after the decomposition of zinc hydroxide.

To prepare PEGylated ZNPs (PZNP), ethanolic solutions of ZNO NP was prepared by dissolving the ZNO NP in 10 ml of ethanol. Separately, 50 mM PEG was prepared in water and stirred at 45°C until it becomes transparent and then cooled to room temperature. 50 mM of PEG solution was added to ZNO NP and shaken vigorously. The precipitate was formed which was collected by centrifugation. The final product was collected after repeated washing for three times to remove the unbound PEG polymers. The final PZNP can be dissolved in any solvent according to the use.

2.2 Particle size analysis

The particle size analysis was performed by dynamic light scattering (DLS) method using high‐performance particle sizer (Zetasizer NanoS, Malvern) at room temperature. The samples were adequately diluted and measured in triplicates.

2.3 Morphology analysis

The morphology of particles was analysed by transmission electron microscopy (TEM). Briefly, drop of diluted samples was placed in carbon‐coated copper grids and allowed to dry. The particles were counter stained with phosphotungistic acid and wiped off the excess liquid. The measurement was then performed in TecnaiTM G2 Spirit (FEI, Netherland) at an accelerated voltage of 100 kV.

2.4 Quantification of Zn²⁺ ion release

The release of the Zn²⁺ ion was performed in PBS (pH 7.4) and ABS (pH 5.0) to simulate the alkaline and acidic conditions. The nanoparticle dispersion was placed in a dialysis bag (3500 MW cut‐off) and closed in a conical tube containing 15 ml of release buffer at 37°C. After each time point, aliquots of sample were withdrawn and analysed using flame Atomic Absorption Spectroscopy (Varian 820 ICP‐MS). Analysis was performed in triplicate.

2.5 Cytotoxicity assay

The cytotoxicity effect of particles on pancreatic cancer cell was determined by MTT assay. Earlier, PANC1 cells were cultured in DMEM media supplemented with 10% FBS and 1% antibiotic mixture. The cells at a density of 3 × 10⁵ cells was seeded per well of 96‐well plate and incubated for 24 h. The cells were treated with ZNP and PZNP and incubated for 24 and 48 h, respectively. Next day, supernatant was aspirated and 10 μl of MTT reagent (5 mg/ml, Sigma) was added to the wells followed by incubation for 3 h at 37°C. Then, the solution was added with 100 µl of DMSO to solubilise the formazan crystals. Finally, after 15 min, absorbance of each well was studied using multiwall plate reader Bio‐Tek (Winooski, USA). The concentration versus cell viability plot was drawn in Microsoft excel sheet.

2.6 Apoptosis measurement by flow cytometer

The apoptosis induced by metal nanoparticle is evaluated by Annexin‐V/PI apoptosis assay protocol. The annexin‐V and PI detects the change in phosphatidylserine (PS) in the cancer cells. It is known that during the apoptosis of cancer cells, PS migrate from inner part to outer plasma membrane and Annexin V has a strong affinity towards the PS and thereby detects the apoptosis of cancer cells. Briefly, PANC1 cells were seeded in 12‐well plate at a density of 2 × 10⁵ cells per well and incubated overnight. The cells were treated with the respective formulations and incubated for 24 h. Next day, cells were extracted and centrifuged. The cell pellets were treated with 2 µl of Annexin V and 2 µl of PI each and incubated for 20 min in dark conditions. After that cells were reconstituted with 500 µl of binding buffer as provided by manufacturer. The stained cells were then analysed using the flow cytometer (FACS Calibur; Becton‐Dickinson). Regardless of the cell viability, PS on the cell membrane is detected by Annexin V and considered in the apoptotic phase. Cells positive for PI were considered dead.

2.7 Live dead assay

PANC1 cells were seeded in 12‐well plate at a density of 2 × 10⁵ cells per well and incubated overnight. The cells were treated with the respective formulations and incubated for 24 h. The cells were incubated with calcien AM and ethidium bromide to stain live and dead cells. The respective green and red fluorescence was observed using confocal laser scanning microscope (CLSM).

2.8 Western blot analysis

The effect of ZNP and PZNP on the molecular and cell signalling pathway was determined by Western blot analysis. Briefly, 1 × 10⁶ cells were seeded in the each well of 6‐well plate and incubated with 2 ml of media for 24 h. The cells were treated with ZNP and PZNP at a foxed dose of 10 µg/ml for 48 h. After 48 h, cells were harvested and washed two times with PBS and the cells are lysed. The lysate was quantified for amount of protein in it. Lysates of ZNP‐ and PZNP‐treated cells with equal protein was loaded in a 10% SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% of non‐fat milk for 1 h and then blotted with different antibodies (BAX and Bcl‐2) and incubated overnight at 4°C. The membrane was then incubated with secondary anti‐mouse immunoglobulin antibody which is coupled with horseradish peroxidase. The bands were detected using chemiluminescence (Super Signal West Pico chemiluminescent reagent, Pierce, Rockford, IL).

2.9 Statistical analysis

Data were analysed and the appropriate significance (p  < 0.05) of the differences between mean values was determined by a Student's t ‐test.

3 Results and discussion

3.1 Particle size analysis

In this study, we have coated the ZNP with a hydrophilic PEG that will increase the physicochemical properties and improve its stability in the dispersion medium (Fig. 1).

Fig. 1.

Scheme for the preparation of PEGylated zinc oxide nanoparticles

The particle size of the nanoparticle is an essential factor for the success of the cancer therapy. The ZNP was prepared and then surface modified with PEG. The presence of PEG on the surface expected to change or alter the properties of the zinc. We have evaluated the particle size using DLS analysis. The average particle size of ZNP was 8.12 ± 0.56 nm with a fairly uniform dispersity index (Fig. 2). The particle size increased to 21.8 ± 0.86 nm after the surface PEGylation of the nanoparticles. The increase in the particle size clearly indicates the presence of solid mass on the outer surface. The size is in the nanoscale limit which is very beneficial for the cancer targeting applications. The PEG will induce the steric stability and thereby expected to increase its performance. A small particle size coupled with long circulation ability of PEG would increase the therapeutic efficacy in pancreatic cancers.

Fig. 2.

Particle size distribution of ZNP and PZNP determined by dynamic light scattering method

3.2 Morphology analysis

The shape of the particle is very important for the anticancer effect. For example, Nair et al. reported that rod‐shaped zinc nanoparticles exerted lower cytotoxicity compared to that of spherical particles in osteoblast cancer cells. In the study, we have observed a perfect spherical shape for ZNP. The particles are uniformly distributed and present as nanosized. The surface PEGylation did increase the size, however, it maintained the spherical shape of the particle. A slight greyish outer shell indicates the presence of PEG‐coated nanoparticles (Fig. 3).

Fig. 3.

Morphology analysis of ZNP and PZNP by TEM

3.3 Release of Zn²⁺ ions from nanoparticles

The release of Zn²⁺ ions from ZNP and PZNP was investigated in physiological and acidic pH conditions. As seen (Fig. 4), continuous release of Zn²⁺ ions from nanoparticles was observed with the prolongation of time. Especially, significantly higher quantity of Zn²⁺ ions was released in the acidic conditions. A higher release of the Zn²⁺ ions in the acidic conditions will provide a beneficial effect in the cancer targeting applications. It has been reported that the Zn²⁺ ions effectively kills the cancer cells by the induction of various intracellular mechanisms. A higher level of Zn²⁺ ions is beneficial for various clinical conditions.

Fig. 4.

Release of Zn²⁺ ions from ZNP and PZNP in the presence of two different pH conditions

3.4 Cytotoxicity assay

The cytotoxic effect of ZNP and PZNP was tested in PANC1 cells, and effect was assessed by MTT assay. As seen, both the ZNP and PZNP killed the cancer cells in a concentration‐dependent manner (Figs. 5 a and b). It should be noted that the ZNP and PZNP were not effective in lower concentrations, whereas higher concentrations of particles were significantly more toxic to the cancer cells. To be specific, PZNP was more cytotoxic compared to that of ZNP in PANC1 cancer cells. For example, at 20 µg/ml, ZNP killed ∼40% of cells, whereas ZNP has killed ∼52% of the cancer cells. It can be seen that free ZnCl₂ was relatively less effect compared to that of ZNP or PZNP. The low efficacy of free ZnCl₂ and relatively higher efficacy of NP's might be attributed to the internalisation efficiency in the cancer cells. The possible reason for the higher cytotoxic effect of PZNP is not known, yet it can be believed that a particle of ∼25 nm size will be retained in the cells, whereas small particles of <10 nm might be expelled from the cancer cells. Overall, cytotoxic effect might be attributed to the release of Zn²⁺ ions form the particles. It should be noted that the anticancer effect increased with the increase in the incubation time of the particles (48 h). It has been reported that the ZNP exhibits its cytotoxic effect by the induction of ROS in the mitochondria which led to the apoptosis of cancer cells and cell death [17, 18].

Fig. 5.

Cytotoxicity analysis of ZNP and PZNP in PANC1 cancer cells. The cytotoxicity was evaluated by MTT assay after 24 and 48 h of respective incubation

3.5 Flow cytometer‐based apoptosis assay & ROS analysis

The apoptosis induced by metal nanoparticle is evaluated by Annexin‐V/PI apoptosis assay protocol. The annexin‐V and PI detects the change in PS in the cancer cells. It is known that during the apoptosis of cancer cells, PS migrate from inner part to outer plasma membrane and Annexin V has a strong affinity towards the PS and thereby detects the apoptosis of cancer cells. Results clearly indicate the high amount of Annexin V positive cells for ZNP and PZNP supporting the fact that apoptosis might be the main mode of action (Fig. 6 a). It is worth noting that PEGylation of ZNP did not decrease the cell killing activity of zinc particles, whereas it further increase its anticancer effect in the pancreatic cancer cells. Approximately 20% of cells were present in early apoptosis and ∼3% in late apoptosis after ZNP treatment. On the contrary, ∼18% of cells were present in early apoptosis phase and ∼10% in late apoptosis phase. In addition, ∼11% of cells were present in necrosis phase indicating the potent cytotoxic effect of PZNP in pancreatic cancer cells. The ROS generation ability was evaluated in these cancer cells. As seen, PZNP exhibited a higher generation of ROS as indicated by the shift in the fluorescence (Fig. 6 b).

Fig. 6.

Anticancer effect of ZNP and PZNP on PANC1 cells:

(a) Apoptosis assay of PANC1 cancer cells after treatment with ZNP and PZNP. The apoptosis was evaluated by flow cytometer after Annexin V and PI staining; (b) Reactive oxygen species (ROS) generation evaluation in cancer cells

3.6 Live/dead assay & Hoechst 33342 staining

Live dead assay was performed to further confirm the anticancer effect of ZNP and PZNP in PANC1 cells. Lives cells were stained with Calcein AM and produce green fluorescence while dead cells were stained with ethidium bromide and produce red fluorescence. Consistent with the MTT assay, PZNP killed significantly higher amount of cancer cells compared to that of ZNP. As seen, untreated cells were dense green fluorescence while ZNP treatment reduced the intensity of green fluorescence. PZNP showed remarkable reduction in the green fluorescence indicating the presence of fewer viable cells which is consistent with the MTT assay (Fig. 7 a). The enhanced anticancer effect was the result of enhanced cellular uptake and release of Zn²⁺ ions in the cancer cells. The results corroborated with the Hoechst 33342‐based cell apoptosis. As shown, PZNP exhibited a remarkable apoptosis of cancer cells and cell nuclei were not intact with large apoptotic body formations (Fig. 7 b).

Fig. 7.

Anticancer effect of formulations in terms of Live dead assay and apoptosis assay:

(a) Live dead assay on PANC1 cells after treatment with ZNP and PZNP; (b) Hoechst 33342 staining assay protocol in pancreatic cancer cells

3.7 Molecular pathway analysis

The effect of ZNP and PZNP on the molecular pathways was investigated by Western blot analysis. We have observed a significant upregulation of proapoptotic BAX, while expression of antiapoptotic Bcl‐2 was significantly downregulated indicating the potent anticancer effect of zinc nanoparticles (Fig. 8). The Zn²⁺ released from the zinc nanoparticle will induce ROS in the mitochondria which might damage the DNA, that will further upregulate the p53 expression and induce the cell death [19]. The cleavage of DNA will provide the DNA fragments which is regarded as the important marker for apoptosis of cancer cells. This will initiate the caspase‐3 which will further initiate the entire caspase family that leads to irreversible apoptosis of cancer cells [20, 21].

Fig. 8.

Western blot analysis of cancer cells after treatment with ZNP and PZNP. The BAX and Bcl‐2 was determined

Overall, although Zn is an essential component of biological system, a high intracellular concentration will kill the cancer cells. Zn²⁺ alters the gene expression, reduce the cell metabolism, and induce the apoptosis.

4 Conclusions

In this study, we have successfully prepared the PEG‐coated ZNPs and studied its effect in pancreatic cancer cells. We have observed a nanosized particle with spherical shape. In this study, we have demonstrated the cytotoxic effect of ZNP and PZNP in PANC1 cells. To be specific, PZNP was more cytotoxic compared to that of ZNP in PANC1 cancer cells. We have further showed that apoptosis is the main mode of cytotoxic activity. It is worth noting that PEGylation of ZNP did not decrease the cell killing activity of zinc particles, whereas it further increase its anticancer effect in the pancreatic cancer cells. We have observed a significant upregulation of proapoptotic BAX, while expression of antiapoptotic Bcl‐2 was significantly downregulated indicating the potent anticancer effect of zinc nanoparticles. Overall, PEGylation of ZNP could be an effective strategy to improve the stability while at the same time its anticancer activity could be enhanced for better therapeutic response.