Enhancement of Radio-Thermo-Sensitivity of 5-Iodo-2-Deoxyuridine-Loaded Polymeric-Coated Magnetic Nanoparticles Triggers Apoptosis in U87MG Human Glioblastoma Cancer Cell Line

Abstract

Introduction

With an emphasis on the radioresistant nature of glioblastoma cells, the aim of the present study was to evaluate the radio-thermo-sensitizing effects of PCL-PEG-coated Superparamagnetic iron oxide nanoparticles (SPIONs) as a carrier of 5-iodo-2-deoxyuridine (IUdR) in monolayer culture of U87MG human glioma cell line.

Methods

Following monolayer culture of U87MG cells, nanoparticle uptake was assessed using Prussian blue staining and ICP-OES method. The U87MG cells were treated with an appropriate concentration of free IUdR and PCL-PEG-coated SPIONs (MNPs) loaded with IUdR (IUdR/MNPs) for 24 h, subjected to hyperthermia (water bath and alternating magnetic field (AMF)) at 43 °C, and exposed to X-ray (2 Gy, 6 MV). The combined effects of hyperthermia with or without magnetic nanoparticles on radiosensitivity of the U87MG cells were evaluated using colony formation assay (CFA) and Flowcytometry.

Results

Prussian blue staining and ICP-OES showed that the nanoparticles were able to enter the cells. The results also indicated that IUdR/MNPs combined with X-ray radiation and hyperthermia significantly decreased the colony formation ability of monolayer cells (1.11, 1.41 fold) and increased the percentage of apoptotic (2.47, 4.1 fold) and necrotic cells (12.28, 29.34 fold), when compared to IUdR combined with X-ray and hyperthermia or IUdR/MNPs + X-ray. MTT results revealed that the presence of IUdR/MNPs significantly increased the toxicity of AMF hyperthermia compared to the water bath method.

Conclusions

Our study showed that SPIONs/PCL-PEG, as a carrier of IUdR, can enhance the cytotoxic effects of radiotherapy and hyperthermia and act as a radio-thermo-sensitizing agent.

Graphic Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12195-021-00675-y.

Introduction

Glioblastoma multiforme (GBM) is the most invasive and fatal type of brain malignancy in adults.37 Despite the recent advances in therapy, the prognosis of glioma remains poor, and the median survival rate for GBM patients is less than 2 years. The current therapeutic approach to glioma comprises surgery, followed by radiation therapy and chemotherapy.1 Radiotherapy after surgery is a widely acknowledged approach to patients with glioblastoma, providing a significant increase in patient median survival time.10 Unfortunately, because of the radioresistant nature of GBMs, virtually all patients develop recurrent tumors following their treatment, rendering radiotherapy a mostly palliative modality. Chemotherapeutic agents have no significant effects on GBM due to restrictions such as their non-selective mechanism of action and toxicity, the blood brain barrier (BBB), and the low tolerance of patients.5,39 DNA repair, on the other hand, is also an important mechanism contributing to chemoresistance.18 Accordingly, to improve the response of glioma tumor cells to therapy, a number of strategies have been suggested, e.g., clinical application of radiosensitizers such as 5-iodo-2-deoxyuridine.9 IUdR is a halogenated pyrimidine analogue that is incorporated into the DNA at the S phase, sensitizing the cells to ionizing radiation.6,23 However, the IUdR with a plasma half-life of 5 min is rapidly metabolized in the liver when given in a bolus injection.36 To increase the half-life of the drugs in the circulation, control their release, and overcome the BBB, nanoparticle-based drug delivery systems have been recently considered in hopes of achieving a more effective distribution of therapeutic agents at the tumor site.28,43

In recent years, SPIONs have been appraised for their unique characteristics, particularly their potential to be incorporated into magnetic targeting, magnetic resonance contrast (MRI) and hyperthermia.31,45 A biocompatible polymer can change the surface properties of SPIONs in order to prevent the aggregation and decomposition when exposed to biological systems.11 The amphiphilic diblock poly (caprolactone)-poly (ethylene glycol) copolymers (PCL-PEG) can create a spherical nanostructure with a hydrophobic core and a hydrophilic shell.25 PCL is a hydrophobic polymer with ester links, which can be hydrolyzed during degradation to produce 6-hydroxy hexanoic acid.42 In contrast, PEG is a hydrophilic, non-toxic and non-antigenic material.13

Other ways to increase the efficacy of glioma treatment include the use of auxiliary modalities such as hyperthermia. Hyperthermia is regarded as the oldest and most powerful modality to sensitize the cells to radiation.8 It has been reported that hyperthermia might inhibit the repair of double-stranded breaks in DNA, which are thought to be the factors that often lead to cell death by radiation. It has been confirmed that an increase in temperature (43–45 °C) can induce apoptotic death.27,32 One of the most important issues reported with hyperthermia is the imperfect localization of high temperature at the tumor site.3 For this reason, scientists should consider the development of novel hyperthermia systems capable of precisely localizing the incident energy at the tumor site without harming the adjacent normal tissues.45

It is thought that limiting the heat generated in the procedure to an specific target tissue can increase the therapeutic efficacy of this method.14 SPIONs, due to their high thermal capacity (C_Iron = 0.45 J/g °C) and high thermal conductivity, can be effective at generating local hyperthermia under an alternating magnetic field35 and water bath hyperthermia.20 Thus, in the current study, we synthesized a composite type of nanoparticle as IUdR-loaded PCL-PEG-coated iron oxide core nanoparticles (MNPs) to enhance the therapeutic index in the presence of radiation and water bath hyperthermia. We investigated the cytotoxic effects induced by nanoparticles in combination with 2Gy X-ray (6 MV) and water bath hyperthermia (43 °C) on the U87MG human glioblastoma cancer cells in monolayer cultures. Since most studies have confirmed the significant role of radiation24 and hyperthermia40 at inducing apoptosis in cells, we used colony formation assay and Flow cytometry to study the combined effect of hyperthermia and radiation in the presence of IUdR-loaded magnetic nanoparticles on the differentiating potential of the U87MG cell line. Finally, we investigated the role of SPION nanoparticles for heat generation under alternating magnetic field and water bath through thermometry and MTT assay.

Materials and Methods

Cell Culture

Monolayer Culture

Glioblastoma U87MG human cell line was procured from the Pasteur Institute of Iran. The cells were cultured in complete MEM medium, which was supplemented with 10% fetal bovine serum (FBS).

Spheroid 3D Culture

U87MG cells were seeded at density of 5 × 10⁵ into each 100 mm Petri dish containing 10 mL of complete MEM medium and coated with a thin layer of agar (2% agar + MEM medium [2X]). The Petri dishes were incubated at 37 °C, and 5 mL of the medium was replaced with fresh medium twice per week.

Synthesis of Nanoparticles

Synthesis of IUdR/SPIO/PCL-PEG nanoparticles was based on the multi emulsion method.34 SPIONs were synthetized through the standard co-precipitation method. At first, under ultrasonic irradiation, 200 mg of SPIONs was dispersed in 50 mL of pure ethanol, and then 3-mercaptopropyl (6 mL) and water (1 mL) was added into the mixture and the suspension was stirred for 6 h at room temperature. The obtained nanoparticles were separated using a permanent magnet and washed with ethanol (twice) and water (once). Then, in order to attach the two types of polymers (PEG & PCL) to the surface of SPIONs, photo initiating thiolene click reaction was performed under ultra violet (UV) exposure between thiolated SPIONs and PEG-PCL macromeres at a temperature of 25 °C. To prepare IUdR/MNPs, 10 mg of synthesized nanoparticles was dispersed in acetone (2 mL), and afterwards, IUdR (1 mg) was added to the suspension. The suspension was slowly added to 20 mL of deionized water. Finally, the IUdR-loaded nanoparticles were separated by a magnet and washed once with water to remove the unloaded IUdR. Ultimately, the prepared nanoparticles were freeze-dried, and stored at a temperature of 4 °C.

Characterization of Nanoparticles

Particle Size, Zeta Potential and Morphology of Nanoparticles

The hydrodynamic size and surface charge of the MNPs and IUdR/MNPs were determined via dynamic light scattering (DLS) and a zeta sizer (Brookhaven Instruments, Holtsville, USA). The morphological examination of the IUdR/MNPs was performed using transmission electron microscopy (TEM) (Zeiss LEO906, Germany). Furthermore, the concentration of IUdR in the nanoparticles was evaluated using a UV absorption measurement. IUdR-loaded magnetic nanoparticles were dispersed in acetone, then, the solvent was evaporated, and the remainder was dissolved in the phosphate buffer saline (PBS). The IUdR concentration in the PBS solution was determined by UV absorption at a wavelength of 288 nm, and drug loading content (DLC) and encapsulation efficiency (EE) were calculated using the Eqs. (1) and (2), respectively:

$$\text{DLC}\left(\%\right)=\frac{\text{Weight of the drug in nanoparticles}}{\text{Weight of nanoparticles}}\times 100$$ 1

$$\text{EE}\left(\%\right)=\frac{\text{Weight of the drug in nanoparticles}}{\text{Weight of feeding drugs}}\times 100$$ 2

In Vitro Release

The in vitro IUdR release from PCL-PEG nanoparticles was evaluated in phosphate buffer saline (PBS, pH 7.4) at 37 °C stirring using the dialysis method. Five mg of IUdR-loaded nanoparticles was dispersed in a dialysis bag containing 1 mL of PBS. The bag was placed into 50 mL of PBS. At predetermined time intervals, 2 mL of the solution was collected for UV–visible analysis at a wavelength of 288 nm, and replaced with fresh buffer solution.

Cellular Internalization of Nanoparticles

Prussian Blue Staining for Detecting Nanoparticle in Monolayer

Prussian blue staining was used to visualize the accumulation of SPIONs in the cells. The U87MG cells were seeded at a number of 7 × 10⁴ in multiwell plates (6 wells/plate) and incubated with IUdR/MNPs (1 µM) for 24 h. Then, the cells were washed 2–3 times with PBS to be removed of free nanoparticles, and fixed using a solution of 2% formaldehyde for 20–30 min. Finally, the cells were incubated with a 1:1 mixture of 4% (w/v) potassium ferrocyanide trihydrate solution and hydrochloric acid 1.5% (w/v) at the room temperature for 15 min. The results were evaluated using the light microscopy (Bel). To calculate the percentage of U87MG cells with internalized magnetic nanoparticles, 100 cells in each treatment group were counted under light microscopy.41

TEM Imaging for Detecting Nanoparticle in Spheroid 3D Cultures

Ten days after the seeding of the U87MG cells, the spheroids reached 100 microns in diameter and were treated with nanoparticles for 67 h. Then, the spheroids were washed twice with PBS, and fixed with glutaraldehyde (2.5%) at a temperature of 4 °C for 2 h. Afterwards, the spheroids were rinsed three times with PBS (0.1%) at 4 °C for 30 min. Then, the spheroids were stained with osmium tetroxide (1%) within 1 h, and afterwards, were washed three times with PBS. Samples were dehydrated with varying concentrations of acetone (50, 70, 80, 90 and 100%), embedded in pure resin, and the resin sample blocks were trimmed. Ultra-thin sections were prepared and placed on a 200-mesh grid and stained with lead citrate and ammonium acetate. The ultra-localization of nanoparticles in the outer and inner cells of the spheroid was evaluated using a TEM (Zeiss LEO906, Jena, Germany) at 100 kV.

Coupled Plasma Optical Emission Spectrometry (ICP-OES) Analysis

The cellular uptake of MNPs was investigated in U87MG cells via the ICP-OES analysis. Cell lines were cultured at a density of 2 × 10⁶ in T-75 flasks, and incubated for 24 h. Afterwards, the cells were treated with IUdR/MNPs (0.5 mg/mL) for different times (2, 12 and 24 h) with the 0.1 mg/mL concentrations of Fe₃O₄. After treatment, the cells were washed twice with PBS, and then harvested, and counted. The suspension cells were centrifuged, then digested with HNO₃ solution. Next, the samples were diluted to 5 mL with deionized water. The iron concentration of the cells was measured using ICP-OES analysis (VISTA-PRO, Varian, Australia). Finally, the average iron content per cell was calculated.

Irradiation

The U87MG cells (5 × 10⁵) were incubated in T-25 flasks for 24 h. Then, the cells were treated with 1 µM free IUdR (0.35 µg/mL), 8 µg/mL MNPs, and IUdR/MNPs (equivalent to 0.35 μg/mL IUdR) for 24 h. The X-ray irradiation (6 MV, 2 Gy) protocol were performed using a Varian linear accelerator at the Radiotherapy Centre of Asia Hospital. X-ray Irradiation was performed at a distance of 100 cm to the source with a surface field size of 20 cm × 20 cm and a dose rate of 1 centiGray/monitor unit. The flasks were irradiated vertically in a horizontal position at room temperature.

Water Bath Hyperthermia (WBH)

The cells were cultured at a density of 5 × 10⁵ cells per flask in complete MEM culture medium. After 24 h of incubation, glioma cells were treated with 1 µM free IUdR (0.35 µg/mL), 8 µg/mL MNPs, and IUdR/MNPs overnight. Afterwards, hyperthermia was prompted by means of water bath (Memmert, Germany) with ± 0.5 °C accuracy at 43 °C for 60 min. The control cells were exposed to a temperature of 37 °C. All treatment modalities (nanoparticle, radiation and hyperthermia) were performed alone or in combination. After treatment, the cells receiving monotherapy were counted, and assessed for their viability with trypan blue dye. Colony formulation was used to evaluate the effect of treatments. The efficacy of combination therapy was quantified using a set of equations as presented in Table 1.

Table 1.

Equations for analyzing the effects of combined therapies.

Effects	Equations
Synergistic	[A + B*] < [A] × [B]/100
Additive	[A + B] = [A] × [B]/100
Sub-additive	[A] × [B]/100 < [A + B] < [A] IF [A] < [B]
Interference	[A] < [A + B] < [B] IF [A] < [B]
Antagonistic	[B] < [A + B] IF [A] < [B]

A and B are therapeutic methods

In Vitro Anti-tumor Efficacy

Colony Formation Assay

Cell viability of control and monotherapy U87MG glioma cells was determined by trypan blue staining. Subsequently, to evaluate the ability of cells to form colonies, cell suspensions from monolayer cultures were plated at predetermined densities per petri dish (SPL) containing 5 mL of complete MEM medium. After 8 days, the colonies were fixed with 2% formaldehyde solution and stained with crystal violet dye (0.5%). The colonies were counted using an inverted phase microscope (A collection of cells of around 100 mm² (~ 50 cells) was counted as 1 colony) and the plating efficiency (PE) was calculated accordingly:

$$\text{plating efficiency}\left(\text{PE}\%\right)=\frac{\text{Number}\text{of}\text{colonies}\text{formed}}{\text{Number}\text{of}\text{cells}\text{seeded}}\times 100$$ 3

Flow Cytometry Assay

The U87MG cells were cultured for evaluation of cell apoptosis and treated with nanoparticles, ionizing radiation and hyperthermia alone or in combination. After 24 h, the treated and control cells at a concentration of 5 × 10⁵ cells/mL were washed with PBS and suspended in 1X binding buffer. Annexin-V FITC (5 μL) was then added to each sample, and the samples were incubated in the dark. After 15 min, Propidium iodide (5 mL) was added to each sample, and fluorescence of the cells was measured using a flow cytometer (BD, San Jose, USA). Finally, the percentage of apoptotic and necrotic cells induced by different treatments in each sample was calculated.

Alternating Magnetic Field Hyperthermia and Thermometry

Magnetic hyperthermia was conducted through an alternating magnetic field (AMF) with a radiofrequency coil (13.56 MHz, 40 A/m, 80 W) in the presence of magnetic nanoparticles. Upon exposure to the AMF, alterations in the cell temperature were monitored with an IR camera (875-2i, Testo, Germany).

MTT Test

The cytotoxicity of the two hyperthermia methods (WB, AMF) tested on the U87MG cells was evaluated with the MTT test. The U87MG cells were cultured in two 96-well plates (8000 cells/well) overnight. Then, the cultured cells in some wells were treated with IUdR/MNPs. After 24 h, the first plates, treated with nanoparticles and untreated, were placed in water bath at a temperature of 43 °C. Meanwhile, the other plates (including nanoparticle-treated and untreated wells) were placed inside the coil (at 43 °C). After induction of hyperthermia, the media were removed, and MTT solution (100 μL) was added to each well. The cells were then incubated for 4 h. Subsequently, the MTT solution was removed, and DMSO (100 μL) solution was added to each well, and shaken for 15 min. The absorbance was measured with an ELISA Reader set to 570 nm.

Statistical Analysis

The results are presented as mean values ± SD (Standard deviation), from at least three independent experiments. For statistical analysis, one-way ANOVA analysis was performed using GraphPad Prism 6. P values less than 0.05 were considered to be statistically significant.

Results

Characterization of Nanoparticles

The morphology of IUdR-loaded PCL-PEG coated SPIONs is shown in Fig. 1a. TEM data showed that the nanoparticles were spherical in shape, with a particle size less than 60 nm. The nanoparticles also had a uniform shape and size, and were well dispersed. Table 2 summarizes the particle size and zeta potential of nanoparticles measured by DLS. With a size of 83.4 to 98 nm, the zeta potential of MNPs and IUdR-loaded MNPs in this study was − 29.2 ± 0.1 and − 17.6 ± 0.05, respectively (Table 2 and Fig. 1b). The DLC and EE of the IUdR/MNPs were 10.43 and 76.47%, respectively. Figure 2 shows the IUdR cumulative in vitro release curve from the MNPs vs. free IUdR, indicating that nanoparticles released more than 47% of the IUdR within 24 h, while about 90% of the drug was released in the first 10 h. It was found that when the drug was loaded onto the nanoparticles, the release curve displayed a sustained release pattern.

Figure 1.

Characterization of nanoparticles. (a) Transmission electron microscopy image and (b) size distributions of 5-iodo-2-deoxyuridine (IUdR)-loaded Superparamagnetic iron oxide (SPIONs)/PCL-PEG nanoparticles.

Table 2.

Particle size and Zeta potential of nanoparticles.

Type of nanoparticles	Hydrodynamic particle size (nm)	Polydispersity index (PdI)	Zeta potential ± SD
SPIONs/PCL-PEG	83.4 ± 4.8	0.217	− 17.6 ± 0.05
IUdR-loaded SPIONs/PCL-PEG	98 ± 9.3	0.29	− 29.2 ± 0.1

Figure 2.

In vitro cumulative release profile of IUdR from IUdR/SPIONs/PCL-PEG nanoparticles.

Cellular Uptake of Nanoparticles

Since patient-derived GBM cells grow as three-dimensional spheroids, the entry of IUdR/MNPs into U87MG glioma cells was investigated for both monolayer (Figs. 3a and 3b) and spheroid cultures (Figs. 3c and 3d). Staining the U87MG cells with Prussian Blue facilitated a clear differentiation between cells with internalized nanoparticles and controls (Figs. 3a and 3b). The results of Prussian blue staining in the U87MG cells after incubation indicated that SPIONs were transferred inside the cells, while no blue color was observed in the control cells (Fig. 3a). After an incubation period of 24 h, 67 ± 5.1% of U87MG cells proved to be nanoparticle-positive. Also, The TEM imaging results demonstrated that the nanoparticles can pass the cell membrane and enter the spherical cells (Figs. 3c and d). Furthermore, the internalization rate of nanoparticles was quantified by ICP-OES method (Fig. 3e). Fe uptake by the monolayer U87MG cells after different treatment times was equal to 30.6 ± 2.8, 52.3 ± 6.8 and 62.86 ± 5.3 pg/cell for 2, 12, and 24 h, respectively. These findings demonstrated that the uptake of MNPs by the U87MG cells significantly increased with extending the MNPs incubation time (p < 0.05).

Figure 3.

Detection of IUdR-loaded PCL-PEG-coated Superparamagnetic iron oxide (SPIONs) uptake using Prussian blue staining. (a) Control cells (without nanoparticle), (b) U87MG cells were treated with IUdR/SPIONs/PCL-PEG, (c, d) TEM images of U87MG spheroids cells in different magnifications, (e) The amount of Fe taken up by the U87MG cells after 2, 12 and 24 h treatment was measured by ICP-OES test. The white arrows present the distributed nanoparticles the cytoplasm: All experiments were repeated six times. N nuclear, NM nuclear membrane and NP nanoparticles.

Cytotoxic Effects of IUdR/IUdR-Loaded PCL-PEG-Coated SPIONs in Combination with Hyperthermia and Ionizing Radiation

Colony Formation Assay

To investigate the combined effects of hyperthermia and IUdR or IUdR-loaded MNPs on cell viability, U87MG cells were treated with 1 µM IUdR (0.35 µg/mL), 1 µM IUdR released from nanoparticles, SPIONs/PCL-PEG (8 µg/mL), X-ray (2 Gy), and water bath hyperthermia (43 °C for 60 min). The effect of 1 µM IUdR or IUdR-loaded MNPs, hyperthermia, and X-ray radiation on the PE of U87MG glioblastoma cell lines in monolayer culture is shown in Fig. 4a. As can be observed, IUdR, hyperthermia, and MNPs alone had no significant effect on CFA of the U87MG cells in comparison with the control group (p > 0.05, Table 1S). In contrast, IUdR released from IUdR/MNPs or radiation significantly decreased the plating efficiency of monolayer cells (p < 0.05, Supplementary Table 1S). Figure 4b shows the effect of IUdR/IUdR-loaded nanoparticles on the radiosensitivity of glioblastoma cells. As can be inferred, a decline in PE due to the combination effect of IUdR/IUdR-loaded nanoparticles and radiation is significantly more than that of each treatment alone (p < 0.001, Supplementary Table 2S). These results suggested that IUdR might be an effective radiosensitizer. Based on the equations in Table 1, IUdR and IUdR-loaded nanoparticles had synergistic effects in combination with radiation. In contrast, the effect of MNPs in combination with radiation was sub-additive (the calculations are presented in Table 3). As the Fig. 4b also shows, plating efficiency of MNPs plus X-ray was not significantly higher than that of MNPs (P > 0.05, Supplementary Table 2S). However, nanoparticles carrying IUdR radiosensitizer + X-ray significantly reduced colony formation ability against nanoparticles or IUdR + X-ray (p < 0.05, Table 2S). This suggests that the loading of IUdR onto nanoparticles can effectively help them enter the U87MG cells.

Figure 4.

Plating efficiency of U87MG cells treated with, (a) 5-iodo-2-deoxyuridine (IUdR) (1 µM), Superparamagnetic iron oxide (SPIO)/PCL-PEG (80 µg/mL), IUdR-loaded SPIONs/PCL-PEG (80 µg/mL), radiation (2 Gy, 6MV), hyperthermia (43 °C for 60 min), (b) Groups with or without radiation, (c) Groups with or without hyperthermia and (d) All groups combined with radiation or hyperthermia, or radiation+ hyperthermia, as determined by colony formation ability (CFA) of U87MG cells. (magnetic nanoparticles (MNPs) means SPIONs/PCL-PEG). (e) The optical images of U87MG colonies in control and treated groups. Statistical significance was showed with p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), respectively. (mean ± SD, n = 3).

Table 3.

Examples of studying the effects of combination therapy based on plating efficiency results.

Treatment modality	[A]	[B]	[A + B]	[A] × [B]/100	Effect
A = Hyperthermia B = MNPs	81.6	80.3	53.33	65.52	53.33 < 65.52 ≡ Synergistic
A = Radiation B = MNPs	69.2	80.3	67.66	55.56	55.56 < 67.66 < 69.2 ≡ Sub-Additive If 69.2 < 80.3

In the next step, we studied the role of magnetic nanoparticles in combination with hyperthermia on the thermal sensitivity of U87MG cells (Fig. 4c). As shown, reduction of PE due to the combination effect of IUdR/MNPs and hyperthermia is significantly more than that of each treatment alone (p < 0.001, Supplementary Table 3S). Furthermore, the equations in Table 1 show that, IUdR in combination with hyperthermia exhibited an additive effect, while the MNPs and IUdR/MNPs showed a synergistic effect (Table 3). The significant difference between treatments with/without hyperthermia in combination with magnetic nanoparticles indicated the role of heat on the synergistic effect. Figure 4d shows the effect of hyperthermia, IUdR, and X-ray alone or in combination on U87MG cells. The combination therapy of hyperthermia, IUdR/MNPs, and X-ray significantly reduced the CFA of U87MG glioblastoma cell line compared with any other treatment modalities (p < 0.001, Supplementary Table 4S). These findings indicated that hyperthermia plus X-ray radiation in combination with IUdR, SPIONs/PCL-PEG and IUdR/SPIONs/PCL-PEG had a synergistic effect on U87MG cells.

Cell Apoptosis Assay

In addition to analyzing the colony formation ability of treated U87MG cells with different methods, apoptotic and necrotic cells were also evaluated by Flowcytometry method. The distribution pattern revealed by the flow cytometry assessment of the U87MG cells is shown in 4 regions, including: Q1: green, necrotic cells, Q2: red, late apoptotic cells, Q3: Blue, early apoptotic cells, and Q4: black, live cells (Fig. 5a, more details are given in Supplementary data). As shown in Figs. 5a and 5b, IUdR, nanoparticles, and hyperthermia did not result in significant differences in cell death ratio in comparison with the control group (p > 0.05, Fig. 5b), while in other groups there was a significant difference (p < 0.001), which is in agreement with the results of colony formation assays (Flow cytometry data of the other treatment group are given in Supplementary Fig. 1S.). In addition, the results indicated that, nanoparticles in combination with hyperthermia, IUdR and IUdR loaded onto nanoparticles in combination with radiation exhibited a synergistic effect, indicating the effect of thermal and radio sensitivity of nanoparticles. The synergistic effect was also observed with the combination of hyperthermia and radiation. Based on Flow cytometry results, the highest cell death rate of about 75% belonged to the combined group, IUdR-loaded nanoparticles plus hyperthermia and radiation (p < 0.001, Fig. 5b).

Figure 5.

(a) The Annexin-V/fluorescein-5-isothiocyanate (FITC) assay for detecting the apoptosis and necrosis U87MG cells, (b) Percentage of apoptotic and necrotic cells after treatment with 5-iodo-2-deoxyuridine (IUdR) (1 µM), Superparamagnetic iron oxide (SPIO)/PCL-PEG (80 µg/mL), IUdR-loaded SPIONs/PCL-PEG (80 µg/mL), radiation (2 Gy, 6 MV), hyperthermia (43 °C for 60 min) only and in combination. (magnetic nanoparticles (MNPs) means SPIONs/PCL-PEG). (mean ± SD, n = 3).

Heating Profile Under AMF

The thermometry data is shown in Figs. 6a and 6b. The temperature of the untreated cells reached 35 °C after about 30 min of exposure to AMF. When nanoparticles were introduced to the cells, the temperature increased significantly in 30 min and reached 44 °C. This indicated the significant role of SPIONs in generating heat under an alternating magnetic field.

Figure 6.

(a) The profile of temperature variations the U87MG cells treated with AMF (13.56 MHz, 40 A/m, 30 min) in the presence or absence of IUdR/MNPs nanoparticles as a function of time, (b) Thermal image monitored by an IR camera during heating.

In Vitro Cytotoxicity Assay of AMF vs. Water Bath Hyperthermia

As shown in Fig. 7, AMF hyperthermia has a significant inhibitory effect on the growth of U87MG cells compared with water bath hyperthermia. The presence of iron oxide nanoparticles resulted in increased toxicity of both methods, however, the increase in toxicity for magnetic hyperthermia (AMF + MNPs) was more significant compared to the toxicity associated with the water bath method, which was speculated to be due to the mechanism of action of magnetic hyperthermia.

Figure 7.

The toxicity effects of U87MG cells treated with water bath (WB), alternating magnetic field (AMF) hyperthermia in the presence or absence of IUdR/MNPs nanoparticles (Mean ± SD, n = 3).

Discussion

Despite progress in glioma treatment techniques such as surgery, chemotherapy, and radiotherapy, due to the anatomical location of the tumor, and superior resistance to cytotoxic drugs and restriction of therapeutic doses, there has been limited progress in the survival of these patients.7 IUdR is a halogenated pyrimidine whose radiosensitizing effect has been confirmed.12 This drug is widely used for the treatment of tumors with a low proliferation rate of healthy cells surrounding them. Theoretically, glioma can be treated easily using this technique,19 but, intravenous administration of IUdR is associated with several issues issues such as absorption in the gut and bone marrow tissue, as well as a short biological half-life.38 To overcome these challenges, new treatment modalities have been developed based on nanoparticles, so that loading of the drug onto nanoparticles and slow release increases the half-life of the drug in the body.14,37 In this study, PCL-PEG-coated SPIONs were designed and synthetized as a carrier for IUdR. The size of IUdR/SPIONs/PCL-PEG was analyzed by TEM (Fig. 1a), which showed a maximum size of less than 100 nm. Most studies have shown that the best nanoparticles sizes are 10 to 100 nanometers in order to facilitate the entry of nanoparticles into cells, especially brain cells.47 Drug loading and IUdR release by MNPs were investigated for their usability as targeting drug delivery platforms (Fig. 2). Using polymers (PCPP) for IUdR delivery, Yuan et al. showed that the initial rates of IUdR release for all percentages of IUdR loadings were rapid, which subsequently led to a decreased final rate of IUdR release over a duration of 4–5 days.46 The results also showed that the release of IUdR from the SPIONs/PCL-PEG in PBS (pH 7.4) yielded a rate of 45.91% in the first 24 h, which increased to 60% over the next 3 days. Slow drug release from nanoparticles compared to free IUdR is due to biodegradable polymers.38 PCL is the type of slowly degrading polymers that can be completely degraded under physiological conditions (such variables as pH or temperature).16 Surface degradation and bulk degradation of polymers have resulted in rapid primary and secondary drug release. Also, entry of IUdR/MNPs into the U87MG cells and spheroids was confirmed using Prussian blue staining and TEM imaging, respectively (Figs. 3a and 3b). Prussian blue staining method was used to label iron ions in cells in which iron nanoparticles are seen as dark blue spots.29 Taylor et al. showed that after a 24 and 48 h incubation, 59.9 ± 5.6% and 77.4 ± 3.9 of gold nanoparticles (GNPs) were adsorbed by GM7373 cells, respectively, indicating a time-dependent uptake of GNPs.41 The results of the ICP-OES analysis of this study also showed that by increasing the incubation time, the cell uptake of nanoparticles can also increase. In the present study, according to the U87-MG cell doubling time (approximately 24 h)22,32,34 and the incorporation of the IUdR into DNA during the S phase of mitotic cells and specifically targets actively dividing cells, the treatment time of cells with IUdR and IUdR/MNPs was selected as 24 h. On the other hand, Neshastehriz et al. suggested that at an IUdR concentration of 1 µM over the period of doubling time the molecules could be saturated30. This study also demonstrated that 1 µM IUdR led to an increase in the cytotoxic effect of 2Gy X-ray radiations through colony formation assay. Additionally, the increase of cellular damage induced by IUdR plus radiation was significantly higher than IUdR or radiation alone (Figs. 4b and 5b). Furthermore, Figs. 4b and 5b indicates that IUdR in combination with radiation had significant effect on cell damage compared with radiation alone (p < 0.05). The results summarized in Table 3 also display the synergistic effect of the IUdR in combination with radiation. Various researchers have reported the sensitizing effect of the IUdR on radiation.21,38 Furthermore, Figs. 4b and 5b suggests that MNPs in combination with radiation did not have a significant effect on cell damage compared with radiation alone (p > 0.05). The results reported in Table 3 also show the sub-additive effect which indicates an increase in the radiation effect, albeit, not significant. It may have been due to the production of reactive oxygen (ROS), which enhanced in vitro post-irradiation cytotoxicity in the presence of SPIONs nanoparticles.17,33 In thermal therapy, the choice of a suitable system for the specific delivery of heat to the tumor site is a major and challenging issue. Nanoparticles can absorb energy from an external source and increase the effects of hyperthermia by transferring energy to adjacent tissues.9 Based on a study by Laurent et al., iron oxide nanoparticles can be concentrated by a magnetic field in the target tissue and subsequently enhance the drug release and local heat production. They also concluded that the increase in temperature could facilitate the efficacy of hyperthermia.26 The study also indicated that increased cell damage caused by MNPs with hyperthermia (43 °C) was more than each treatment alone mostly because these particles have excellent heat conductivity and induction heating conductivity, can cause hyperthermia effects on the cancerous cells and kill a large number of them (Fig. 4c). Consistent with the findings of the previous studies, our investigation suggests that the major antitumor effect of hyperthermia and also radiation is the induction of apoptosis15 (Figs. 4 and 5). Figure 4 indicates that hyperthermia can increase the effects of radiation on glioma cells as an adjunct therapy. Previous studies have also suggested that by reducing the effective dose of radiation, especially radioresistant tumors, hyperthermia can be a convenient and effective adjuvant treatment.8 Asayesh et al. showed that hyperthermia with brachytherapy significantly improved the survival of patients with GBM. They revealed that 100 nM Gemcitabine in combination with hyperthermia (43 °C for 60 min) and 2 Gy gamma 60 Co inhibited the proliferation rate of the U87MG spheroid cells higher than the radiation alone.4 Figs. 4d and 5b shows that U87MG cells treatment with X-ray exposure (2 Gy, 6 MV) and water bath hyperthermia (43 °C, 1 h) in combination with IUdR/MNPs (1 µM) significantly decreased the CFA and increase the cell death (p < 0.001). On the other hand, a comparison of the two hyperthermia methods (AMF an WB) revealed that iron nanoparticles under an alternating magnetic field had a high potential for generating heat locally (Fig. 7). The magnetic relaxation process prompted generation of heat under AMF. Magnetic relaxation can be explained with two mechanisms, Neel and Brownian relaxations. In this way, MNPs can convert the magnetic energy into thermal energy through flipping motion of the iron oxide magnetic moment in an alternating magnetic field.2,44

Our results showed that IUdR/MNPs enhanced the sensitivity of radiation and heat due to the increased half-life of IUdR, sustained release, radiation damage stability, and the effective transfer of heat produced within nanoparticles to the adjacent region. Furthermore, SPIONs/PCL-PEG nanoparticles can be used simultaneously for magnetic drug targeting and contrast MRI because they can be potentially modified to transfer chemotherapeutic agents, and be magnetically guided to the tumor sites.

Conclusion

Our study indicates that PCL-PEG-coated SPIONs in combination with hyperthermia may function as a thermosensitizer, which can significantly increase the therapeutic effect of hyperthermia with local heat production. On the other hand, with the loading of IUdR as a radiosensitizer onto these nanoparticles, the efficacy of radiation therapy can also be increased by facilitating IUdR entry into the glioma cancer cells. The results demonstrate that PCL-PEG-coated SPIONs can function simultaneously as a radio-thermosensitizer.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This work was supported by Grant No. 25052 from the School of Medicine of Iran University of Medical Sciences (IUMS).