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Home > Effects of PEGylated Fe–Fe3O4 core-shell nanoparticles on NIH3T3 and A549 cell lines
Effects of PEGylated Fe–Fe3O4 core-shell nanoparticles on NIH3T3 and A549 cell lines
B.H. Domac a,1, S. AlKhatib b,1, O. Zirhli a,c, N.G. Akdogan d, S¸ .C. O€ çal Dirican e, G. Bulut f, O. Akdogan
1.Introduction
Cancer is considered a major health problem around the world and many attempts have been made to minimize cancer incidence and mortality. Currently, several types of treatments are used for cancer therapy including surgery, radiation, chemotherapy, immunotherapy and targeted therapy, which focus on targeting specific genes and proteins that promote cancer growth and cancer cell survival [1].
Nanotechnology has recently arrived on the scene as a new field of science that opens enticing prospects of several applications, such as the treatment of various diseases including cancer [2]. Nanotechnology provides ways to enhance the efficacy of cancer therapies in targeting only malignant cells without damaging healthy cells [3]. One of the latest advances in nanotechnology is the development of numerous nanoparticle formulations that are engineered to be suitable for cancer research [4].
Nanoparticles (NP) have emerged as novel diagnostic and therapeutic tools for cancer due to their distinctive features in terms of size, shape, and surface characteristics. Organic nanoparticles, such as polymeric micelles, liposomes and dendrimers, and inorganic nanoparticles, such as magnetic NPs, gold NPs, and silica NPs have been used in a drug delivery system as drug carriers. According to the size and shape of nanoparticles, the delivered anti-cancer agents can target the tumor more specifically with fewer side effects and a higher treatment efficacy relies on enhanced permeability and retention (EPR) [5, 6, 7, 8].
Fe3O4 (magnetite) particles are heavily used in cancer diagnosis and treatment due to their biocompatibility and biodegradability. Risk and benefit requirements of magnetite particles make them indispensable for MRI contrast enhancement, targeted drug delivery, and magnetic hyperthermia applications [9, 10]. The latter application requires high coercivity and magnetization.
The size of the nanoparticles directly affects the duration of the NP in the blood circulation. Particles bigger than 200 nm are easily cleared by the reticuloendothelial system, albeit smaller particles are easily cleared by the body through the kidneys. Furthermore, hydrophobicity and negatively charged particles are easily detected and cleared by the mononuclear phagocyte system (MPS). Thereupon, appropriate coating of the particles with synthetic polymers, surfactants, gold, silica, and peptides may be necessary [2].
Particles are generally coated with hydrophilic, uncharged and biocompatible coatings such as poly (ethylene glycol) (PEG) to prevent recognition by the MPS [11, 12, 13]. Thus, particles duration time in the blood circulation is extended. These nanoparticles reach a better performance for most of the aforementioned applications.
The effects of nanoparticles on living cells can vary depending on cell type, activity, type and size of the nanoparticle itself [14]. The aim of this study is to determine the effect of synthesized PEG-coated Fe/Fe3O4 nanoparticles on human non-small cell lung cancer A549 cells and mouse fibroblast NIH3T3 cells and evaluate their potential as anti-cancer agents. It is important to evaluate the toxicity of these nanoparticles by determining the half-maximal inhibitory concentration (IC50) on cultured cells. In parallel to this, cell proliferation assay, a widely applied method in nanotechnology to test the toxicity and effect of the nanoparticles on cell proliferation was performed [15]. Moreover, the possible effect of the nanoparticles on cell motility, which is a preliminary mechanism in cellular motility and the development of metastasis was analyzed through scratch assay [16].
2.Materials and methods
2.1.Nanoparticle synthesis
Nanoparticles were produced via salt reduction, using the Y junction flow tube technique. FeCl2 and NaBH4 were used as the salt and the reducer, respectively. In a typical synthesis route, FeCl2 and NaBH4 are dissolved separately in 50 ml DI water and degassed for 30 min under N2 gas flow to get rid of free dissolved oxygen. The controllable mixture of these chemicals was assured by passing them through the Y junction flow tube (Figure 1). At the junction point, where the intermixing occurs, nanoparticles form and drop into the collection beaker. At this point, the particles get in touch with dissolved capping agents (surfactants, polymers, etc). In this study, PEG (10,000 MW) was used. PEG-coated Fe/ Fe3O4 nanoparticles were cleaned from byproducts (NaCl, etc) of the chemical reaction for four times and the final product was stored in pure EtOH. Two different types of nanoparticles have been synthesized by changing the NaBH4/FeCl2 molar ratio; NP1: 1.67 and NP2: 0.85.
Morphological, structural and magnetic characterization of the nanoparticles was performed by JEOL JEM-ARM200CFEG UHR-TEM, Bruker D2 Phaser XRD, and EZ9 Microsense VSM, respectively.
2.2.Cell culture
NIH3T3 mouse embryonic cells (CRL1658, ATCC) were grown in DMEM medium and A549 human non-small cell lung cancer cells (CCL185, ATCC) were grown in F12–K medium according to the manufacturer's protocol. All media were supplemented with 10% fetal bovine serum. Cells were maintained in 37 ○C humidified atmosphere with 5% CO2.
2.3.Dilution of nanoparticles
Nanoparticles were dissolved in DMSO at 2 mg/ml stock concentration and stored at room temperature.
2.4.Cell proliferation assay
Cellular proliferation was analyzed using WST-1 Reagent (Roche). Cells were seeded at a density of 10,000 cells/well for NIH3T3 and 5,000 cells/well for A549 in a 96-well plate. The next day, both NIH3T3 and A549 cells were treated with nanoparticles at different concentrations in triplicates. After 48h, 10μl of WST-1 reagent was added onto the cells and cells were incubated for 30mins. Absorbance values were measured at 420nm using MultiScan GO Microplate spectrophotometer. The IC50 values were determined by GraphPad Prism Software.
2.5.Scratch assay
The effect of the nanoparticles on cellular motility was assessed using the scratch assay. Cells were plated at a density of 400,000–600,000 cells/well on a 6-well plate in order to form a confluent monolayer the following day. After 24h, a scratch was made on the cell surface using a sterile p-200 pipette tip. The scratched plate was washed with PBS to remove non-adherent cells. The cells were then treated with the nanoparticles at concentrations that were folds of the relevant IC50 values (NIH3T3 cells at 13.75 μg/ml, 27.5 μg/ml, and 41.25 μg/ml concentrations and A549 cells at 0.5 μg/ml, 1 μg/ml, and 2 μg/ml concentrations) for an additional 48h. Images were captured using an Olympus CRKX41 inverted microscope at 24h and 48h of incubation. The distance of migrated cells compared to the baseline distance was measured in pixels. The numerical data obtained was analyzed by GraphPad Prism software
using“paired t-test (p < 0.05)”.
3.Results and discussion
3.1.Structural, morphological and magnetic characterization of nanoparticles
XRD analysis of NP1 and NP2 shows that the nanoparticles are composed of α-Fe and Fe3O4 (Magnetite) phases (Figure 2a). The average particle size of NP1: 21nm and NP2: 13nm has been predicted from Scherer's Eq. (D 0.9 λ/βcos θ, λ: wavelength, β: FWHM, θ: diffraction angle) by using the full width at half maximum of the highest peak in XRD Data. Room temperature magnetic characterization reveals low magnetization and coercivity (Hc) of 350 Oe (Figure 2b). The high coercivity of these particles makes them suitable for hyperthermia ap- plications due to high hysteresis loss [10, 11].
In order to study the morphology of the particles, TEM study was performed. The bright-field TEM image of the particles shows spherical core-shell morphology, where the core is Fe with =20nm diameter, the shell is Fe3O4 with 3.5nm thickness (confirmed by energy dispersive x- ray spectroscopy) (Figure 3). Predicted values from the XRD data match with the size of the nanoparticles. Although PEG coating is not obvious in TEM bright-field images, PEG coating layer on the nanoparticles was realized due to the hydrophilic nature of the synthesized nanoparticles [2].
3.2.Cellular proliferation assay
Nanoparticles were dissolved in sterile DMSO and stored at room temperature. The stock concentration of the nanoparticles was prepared at 2 mg/ml. The final concentration of the DMSO administered on the cells was fixed at 0.1% due to the potential effect of this reagent on cell viability and proliferation.
NIH3T3 and A549 cells were treated with the NP1 at various concentrations (0–100 μg/ml). After incubation with the nanoparticles, 10μl of WST-1 reagent (Roche) was added onto the cells in order to check for the cell viability according to the manufacturer's protocol. The half- maximum inhibitory concentrations (IC50) of nanoparticles was found to be 27.5 μg/ml and 1 μg/ml in NIH3T3 and A549 cells, respectively. The cell viability versus log concentration plots are given in Figure 4.
3.3.Scratch assay
In order to test the potential of NP1 as an anti-cancer agent, scratch assay was performed on NIH3T3 and A549 cells. According to the data obtained on NIH3T3 cells, nanoparticles did not show a significant inhibitory role on cell motility when compared to the non-treated (negative control) cells both at 24 and 48 h of treatment (Figure 5). Cell death due to the toxic effect of NP1 on NIH3T3 cells was observed at
41.25 μg/ml concentration (two folds of the IC50 value) and 48h of treatment. On the contrary, nanoparticles had a significant inhibitory effect on the motility of A549 lung cancer cells when compared to the non-treated cells at 24 and 48 h of treatment (Figure 6).
4.Discussion
Although there have been many studies on effects of Fe3O4 nanoparticles in A549 cells [17, 18, 19, 20], to the best of our knowledge, there is no work related to the effects of Fe/Fe3O4 core-shell particles on these cells. Previous studies show concentration-dependent toxicity of Fe3O4 nanoparticles where a significant effect was observed only for concentrations of 100 μg/ml and higher [17, 18, 19]. Hence, particles are coated to reduce their potential toxic effects, so that they can be used with drugs in nanomedicine [18]. The toxicity of the magnetite nanoparticles is due to the increasing level of lipid peroxidation and decreasing antioxidant enzymes of human lung alveolar epithelial cells (A549), displaying a concentration-dependent toxicity in vitro [17]. Malvindi et. al. proposed that following the intake of nanoparticles by the cells, nanoparticles start to degrade and release Fe ions, which interact with hydrogen peroxide produced by the mitochondria. This process induces the generation of highly reactive hydroxyl radicals and ferric ions (Fe3 ) through the Fenton reaction. Therefore, hydroxyl radicals and ferric ions could damage DNA, leading to cell death [18].
In our study, contradictory to the aforementioned previous studies, it has been found that the synthesized particles are toxic at such low concentrations down to 1 μg/ml. In order to evaluate the cytotoxicity level of NP1 on A549 cells, an additional cytotoxicity experiment with a common chemotherapeutic agent, Paclitaxel was performed on the same cell line. The IC50 value of this drug on A549 cells was found to be 11.1 μg/ml (Figure 7). This data shows that NP1 with an IC50 value of 1 μg/ml is more toxic on A549 cells in comparison to Paclitaxel.
We explain the high toxicity of the nanoparticles via Fenton reaction; following the degradation of Fe3O4, Fe core is exposed which accelerates the release of Fe ions and this leads to cell death. In order to further prove this hypothesis, particles with a thicker Fe3O4 shell was also synthesized (NP2). The IC50 values of this thicker shelled nanoparticles were 29 μg/ ml and 27 μg/ml in NIH3T3 and A549 cells, respectively. The cell viability versus log concentrations are given in Figure 8. Surprisingly, no significant change was observed in NIH3T3 and A549 cell lines after treating them with thicker shelled nanoparticles in scratch assay (data not shown). As stated above, thicker Fe3O4 shell de-accelerates the degradation of the nanoparticles, consecutively slows down the cell death process.
Rosman et al. recently evaluated the effects of Gallic acid (GA) on A549 cells. IC50 values found for GA, Fe3O4-PEG-GA and Doxorubicin were 56 μg/ml, 13 μg/ml and 0.58 μg/ml, respectively [20]. Synthesized NP1 nanoparticles in this study have a lower IC50 value (1 μg/ml) in comparison to GA coated nanoparticles. This data supports the hypothesis that NP1 is a promising nanoparticle that can be used as a potential anti-cancer agent.
5.Conclusions
In summary, Fe/Fe3O4 core/shell nanoparticles with an average size of 20 nm and 13 nm have been synthesized and their performance has been evaluated in vitro. The effect of the nanoparticles on proliferation/ survival and motility of NIH3T3 and A549 cells was assessed by cytotoxicity and scratch assays, respectively. IC50 value of NP1 was found to be 27.5 μg/ml in NIH3T3 and 1 μg/ml in A549 cells. This data indicates that the nanoparticles are more toxic to cancer cells. Comparatively, while the nanoparticles did not have any inhibitory effect on the motility of NIH3T3 cells, they showed a significant inhibitory effect on the motility of A549 lung cancer cells. The nanoparticles were found to be 100 times more toxic on cells when compared to the previous studies. This cellular phenomenon can be explained by the accelerated decomposition of particles due to the Fe core.
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