Synergistic effect of the combination of triethylene-glycol modified Fe₃O₄ nanoparticles and ultrasound wave on MCF-7 cells

Highlights

• We examined the combination effect of Fe₃O₄ nanoparticles and ultrasound wave on MCF7.

• The combination effect featured significant cytotoxic effects.

• The cytotoxic effect is due to the production of reactive oxygen species.

Abstract

Cancer is a group of disease characterized by uncontrolled growth and spread of abnormal cells in the body. The clinical treatments for cancer include surgery, chemotherapy and radiotherapy. Currently, employing new approaches for treatment has attracted more attentions. One of these approaches is sonodynamic therapy, which is an analogous approach based on the synergistic effect of ultrasound and a chemical component referred to as sonosensitizer. Recent years applications of nanotechnology have witnessed a tremendous expansion of research in medicine especially in treatment of cancers. The combination of sonodynamic therapy and nanotechnology can introduce a new way for cancer therapy. In this study, we used therapeutic ultrasonic waves with intensity of 1 MHz and different concentrations of Fe₃O₄ nanoparticles, as sonosensitizer, to investigate their combination effect on MCF-7 cell line. Briefly, we divided cells into four different groups; control, cells which got in touch with nanoparticles, cells that with exposure to ultrasound waves and cells which were influenced with combination of nanoparticles and ultrasonic waves. Finally, cell viability assay was used for detection of cytotoxicity effects. Experimental results revealed a significant decrease in viability of cells, which were affected by the combined action of ultrasound field and Fe₃O₄ nanoparticles, compared to the separate exposure of Fe₃O₄ nanoparticles or ultrasonic field. The synergic effect of ultrasound waves and Fe ions might be due to the production of toxic free radicals.

Introduction

Cancer is a complex, multifactorial and devastating disease with a leading cause of morbidity and mortality worldwide that has baffled researchers over the years [1], [2]. Use of new approaches for cancer treatment has attracted a lot attention these years.

Combination therapy for the treatment of cancer is also becoming more popular because it generates synergistic anticancer effects. It seems that sonodynamic therapy (SDT) could be considered as a potentially crucial alternative way in comparison to the traditional treatment manner. In this technique, a chemotherapeutic agent known as sonosensitizers are used to increase the influences of ultrasound's preferred effects on cells [3].

Ultrasound is a mechanical wave with properties like: ability to periodic vibrations of particles, short wavelengths and frequencies above 20 kHz [4]. Its irradiation appears to be an appropriate method for damaging malignant cells [5] due to the intrinsic cellular responses. Indeed, it could generate microbubbles, micron sized (1–10 μm) bubbles that fluctuate in response to the incident ultrasonic waves [6], that will collapse under enough intensity to produce the inertial (or stable) cavitation [7], [8].

When ultrasound is used in its therapeutic levels, microbubbles oscillations can lead to an increase in the permeability of microvessels and hence enhance the cellular uptake of different molecules, nanoparticles and therapeutic agents. This event is normally due to sonoporation which is generated by microbubbles oscillating in a stable motion (inertial cavitation) and temporarily open pores in the plasma membrane [6].

Inertial cavities are gas bubbles that expand by mechanical resonance and produce energy through collapsing. Enormous energy release by the expulsion of cavities produce temperature and pressure above 5000 K and 800 atm [7].

This energy could produce reactive oxygen species (ROS) which lead to a severe cytotoxic effect. Indeed, in the present of enough ROS in the cell, a cascade of events will activate inside the cell that eventually result in apoptosis [9], [10], [11]. There are two main apoptotic pathways; the extrinsic (receptor mediated) and the intrinsic (mitochondria mediated). While the extrinsic pathway requires sonosensitizers for interaction with their favorable receptors, the intrinsic pathway of apoptosis could be stimulated by both internal and external triggers, such as ultrasound [12]. Briefly, it could disrupt normal function of mitochondria. Moreover, the hydroxyl radicals, produced by ROS, could cause lipid peroxidation in plasma membrane through misappropriating distribution of electrons [13]. The accumulation of oxidants eventually leads to destruction of cellular proteins, enzymes, lipids, and nucleic acids, consequently the normal processes of the cell disrupt that lead to the improvement of diseases and cell death [14], [15], [16], [17].

To enhance the effect of ultrasonic irradiation, there are enormous pure varieties of sonosensitizers, which could be terminated by cell death through different mechanisms ranging from increasing ROS content to disrupting the vascular networks of malignant cells [13], [18].

The purpose of the present study is to obtain a way to promote the preferential damage of ultrasonic irradiation by using nanoparticles as sonosensitizers.

Nanotechnology is a new emerging frontier of this century which has potential to revolutionize different fields of science. It has a wide range of applications from informational technologies to medicinal applications [19], [20]. In spite of the recent developments in conventional treatment strategies, the effective use of nanotechnology in medicine and pharmaceuticals is a fast growing field that generate a new research field called nanomedicine, which could introduce new suitable approach for cancer diagnosis and treatments because of the properties of nanoscale structures [16], [21], [22], [23].

By reduction in the size of particles until nanoscale range, the ratio of surface to volume increases and therefore different properties of particles such as magnetic, optical and mechanical are changed [24], [25], [26], [27] leading to their applications in different fields of science. For example in medicine, they could be used as a carrier in targeted drug delivery systems to convey therapeutic agents exactly to a certain biological entity [28] or act as therapeutics and interact on a cellular (10–100 nm), subcellular (20–250 nm), protein (3–50 nm) or genetic scale (10–100 nm) [29], [30].

Among the various types of nanoparticles, magnetic nanoparticles (MNPs) especially Fe₃O₄ (magnetite) and Fe₂O₃ (maghemite) have attracted lots of attention particularly those that exhibit superparamagnetic properties (SPIONs), which are desired nanoparticles for use in biomedical applications [31], [32]. For instance they could be used for a specific biological purpose such as cell isolation, drug delivery, diagnostics through magnetic resonance imaging (MRI), cellular imaging and hyperthermia [29], [33], [34], [35].

The toxicity of MNPs in biological environment is dependent on a range of factors related to the properties of the MNP such as size [36], concentration [37], surface properties [38], [39], [40] and structural properties [41], [42].

According to the cellular study, the main cause of toxicity by MNPs is oxidative stress that is primarily formed by the incomplete reduction of oxygen and can impair cell metabolism and increase apoptosis. Therefore, MNPs could be used in cancer therapies to destroy cancer cells [14], [15].

In this study, the combination effect of Fe₃O₄ nanoparticles and therapeutic ultrasound waves in the viability of cancer cells was investigated. To this end, therapeutic ultrasonic waves with intensity of 1 MHz and different concentrations of Fe₃O₄ nanoparticles, as sonosensitizers, were used. Briefly, the cells were assigned to four different groups; control cells; cells which got in touch with nanoparticles; cells with exposure to ultrasound and cells which were influenced with the combination of nanoparticles and ultrasonic waves. Finally, MTT assay has been carried out for detection of cytotoxicity effects.