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. 2022 Oct 8;12(11):313. doi: 10.1007/s13205-022-03377-y

Comparative heating efficiency and cytotoxicity of magnetic silica nanoparticles for magnetic hyperthermia treatment on human breast cancer cells

Melek Acar 1, Kubra Solak 2, Seyda Yildiz 1, Yagmur Unver 1,✉, Ahmet Mavi 2,3,✉
PMCID: PMC9547765  PMID: 36276464

Abstract

Magnetic hyperthermia (MHT) is a promising treatment for a variety of cancers due to its ability to increase the sensitivity of cells to other treatments, such as chemotherapy. Superparamagnetic nanoparticles (MNPs) were used for MHT treatment due to their heat generation ability under an AC magnetic field (AMF). In this study, iron oxide and zinc-doped iron oxide MNPs were produced and modified with silica to obtain eleven different types (MSNP-I to -XI) of magnetic silica nanoparticles (MSNPs). The MSNPs which show the highest heating capacity were selected to investigate their MHT ability on non-tumourigenic MCF-10A and tumourigenic MCF-7 cell lines. The cytotoxicity results indicated that the size, the content of the magnetic core and silica coating thickness were important in the heating capacity of MSNPs under AMF. After MHT treatment, selected MSNPs showed limited cytotoxicity on MCF-10A, but significant cell death on MCF-7.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-022-03377-y.

Keywords: Breast cancer, Magnetic nanoparticles, Magnetic hyperthermia, Iron oxide, Zinc-doped iron oxide

Introduction

Cancer is one of the most serious health problems as it ranks second among causes of death worldwide (WHO 2022). Effective cancer treatments often cannot be achieved with the typical monotherapies, such as chemotherapy or radiotherapy, because of their side effects (Liyanage et al. 2019). Therefore, many strategies have been suggested to fight cancer, such as hyperthermia, gene therapy, photodynamic therapy and a combination of these therapies. Hyperthermia is the process of heating a cell/tissue up to 39–45 °C, to eradicate target/cancer cells while causing minimal injury to surrounding normal tissues. Oncological studies show that multi therapies may cause a more reliable, effective and higher response as compared to monotherapies (Wust et al. 2002).

Magnetic hyperthermia (MHT) is a process where magnetic nanoparticles (MNPs) are heated under an alternating magnetic field (AMF) in order to heat the surroundings cells/tissue. Whilst MHT has been shown to be effective, the challenge still remains of delivering the MNPs to deep-seated tumours (Moroz et al. 2002; Albarqi et al. 2019). MNPs are one of the most interesting nanomaterials due to their wide range of biomedical applications. MNPs are used in cell labelling (Kolosnjaj-Tabi et al. 2013), bioimaging (Liu et al. 2011), targeted drug delivery (Dey et al. 2017; Solak et al. 2021) and hyperthermia (Bañobre-López et al. 2013). Iron oxide MNPs show limited toxicity to normal cells, but high-efficiency in carcinogenic cell destruction, when used in MHT treatments (Kossatz et al. 2014; Martinelli et al. 2019). In addition to iron oxide nanoparticles, bimetallic MNPs, such as zinc-doped iron oxide have also been developed. In addition to the composition of MNPs, their size, shape and surface modifications are equally important for their therapeutic efficiency (Rajan and Sahu 2020). The surface modification of MNPs are very important to help maintain their stability and biocompatibility. Polyethyleneimine (PEI), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyvinyl pyrrolidine (PVP) and silica are the most widely used biocompatible coating materials for MNPs. Of these coatings, mesoporous silica-coated MNPs (MSNPs) are perhaps the most outstanding materials due to their high surface area, bioavailability, ease and cost of production, etc. They are, therefore, suitable for various applications, including drug delivery (Cho et al. 2019; Solak et al. 2021).

Taken together, it is necessary to develop MSNPs that are well dispersed in water, non-cytotoxic and could provide temperature increase in a short time during MHT application. MSNPs get great attention because they offer a porous structure that is ideal for drug delivery, a high surface area for targeted therapies and contain metallic nanoparticles for thermal therapy. Thus, MSNPs might easily be used for multi-therapy applications. In this study, it was aimed to develop a suitable MHT tool based on the magnetic silica nanoparticles that have low toxicity on non-tumourigenic cells, but are highly effective when used for cancer therapy.

Materials and methods

Chemicals

Iron acetylacetonate (Fe(acac)3), oleylamine (OAm), oleic acid (OA), benzyl ether (BE), hexane, chloroform, tetraethyl orthosilicate (TEOS), (3-Aminopropyl) triethoxysilane (APTES), ammonium hydroxide solution (25–30%), ethyl acetate (EtOAc), dimethylformamide (DMF) and paraformaldehyde (PFA) were obtained from Sigma-Aldrich. Cetrimonium bromide (CTAB), sodium hydroxide (NaOH), sodium carbonate and sodium bicarbonate were obtained from Merck. 4ʹ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Genaxxon and Oregon Green 488 (Succinimidyl Ester, 6-isomer) was purchased from Thermo-Fisher Scientific.

Synthesis of magnetic nanoparticles (MNPs)

The MNP-I were prepared using a modified protocol from the co-precipitation method (Lin and Haynes 2009; Jiang et al. 2017). First, nitrogen gas was flowed onto deionized water for 10 min. Then, 2.4 g of FeCl3·6H2O, 1.00 g FeCl2·4H2O and 1 mL OA were added to 50 mL of the deionized water with vigorous stirring, under nitrogen atmosphere. The solution was heated to 90 ºC before adding 6 mL ammonium hydroxide (25 wt %) solution. After 2.5 h at 90 ºC, the solution was allowed to cool to room temperature. The MNPs were collected by centrifugation at 10.300g for 10 min and washed with ethanol several times. After re-suspension of MNP-I in hexane, it was stored at + 4 °C under nitrogen gas.

The MNP-II were prepared by the way of the thermal decomposition method (Metin et al. 2014; Solak et al. 2021). Simply, 1.06 g of Fe(acac)3 (3 mmol) was dissolved in 15 mL of OAm and 15 mL of BE in a three-neck flask. The temperature of the resulting mixture was slowly raised to 110 °C under an inert atmosphere. The solution was kept at 110 °C for 1 h, then the temperature was raised to 300 °C. The mixture was left at this temperature for 1 h and after cooling, the MNPs were collected by centrifugation (10.000g, 10 min) and washed three times with ethanol. The resulting MNPs were dispersed in hexane and stored at + 4 °C under an inert atmosphere. After slight alteration of this method, MNP-III was obtained using 10 mL of OAm, 5 mL of OA and 15 mL of BE. Zinc-doped iron oxide MNPs were prepared with the same method with minor modifications. MNP-IV was produced by adding 0.3 g of FeCl2⋅4H2O (1.5 mmol) and 0.091 g of ZnCl2 (0.7 mmol) and MNP-V was produced with 0.273 g of ZnCl2 (2 mmol) addition to the same protocol.

Preparation of core−shell magnetic silica nanoparticles (MSNPs)

MNP-II, MNP-III, MNP-IV and MNP-V were coated with silica following the protocol of Nyalosaso et al. (2016) and called MSNP-I, MSNP-II, MSNP-III and MSNP-IV, respectively. Typically, 0.125 g CTAB was dissolved in 60 g of ultrapure water containing 0.44 mL of NaOH (2 M). This solution was heated to 70 °C under vigorous stirring (750 rpm) and stabilized for 1 h at this temperature. The MNPs solution (5 mg in 0.5 mL) was dispersed using an ultrasonic water bath for 3 h, then gradually injected (10 × 50 µL) into the CTAB solution over 50 min. Following the injections, the reaction was heated to 80 °C and the reaction was left to stabilize under stirring for 1 h at 80 °C. Then, firstly 0.1 mL of TEOS and after 30 min 0.3 mL of TEOS was added to the reaction and it was maintained for 1 h and 30 min at 80 °C. The solution of MSNPs was centrifuged at 5.200g for 10 min and then the supernatant was centrifuged a second time at 23.000g for 20 min to collect the solid. MSNPs were washed with ethanol.

Moreover, MNP-II were modified by following a protocol present in the literature (Kim et al. 2006) and MSNP-V were produced at the end of the reaction. In brief, 0.5 mL of MNP-II in chloroform (15 mg/mL) was added to 5 mL of the water containing 0.1 g of CTAB to obtain a homogeneous oil-in-water micro emulsion. Chloroform evaporation was achieved by heating the solution to 70 °C for 10 min. Then, 0.5 mL of the resulting aqueous solution was diluted with 10 mL of water. Subsequently, 0.2 mL of NH4OH solution, 0.05 mL of TEOS and 0.5 mL of EtOAc were successively added to the diluted MNP-II aqueous solution. The resulting mixture was stirred for 30 s and then aged for 3 h. The MSNP-V was collected by centrifugation and washed with water and ethanol five times.

In another coating method, 75 mg of MNP-II and MNP-III were each dispersed in 4 mL of toluene (ultrasonic water bath for 1 h) and mixed with a mixture of 60 mL of ethanol, 20 mL of deionized water and 2.5 mL of ammonia solution, followed by mechanically stirring at 750 rpm and room temperature for 20 min (Toubi et al. 2015). 0.1 mL of TEOS (in 20 mL of ethanol) was added dropwise to the solution within 10 min. After 4 h, the non-porous core/shell NPs were formed (called MSNP-VI and MSNP-VII, respectively) and then they were separated by centrifugation.

Silica coatings of MNP-I and MNP-II were performed, as well. In a typical procedure, 40 mg of MNPs in chloroform (1 mL) were poured into an aqueous CTAB solution (10 mL, 360 mg) and the resulting solution was sonicated using an ultrasonic water bath for 2 h. The MNPs were collected by centrifugation (10.300g, 20 min) and redispersed into 1 mL of water. The centrifugation/redispersion procedure was repeated two times to remove the excess amount of CTAB. Thereafter, the solution (1 mL) was added to a CTAB solution (9 mL, 8.2 mg/mL) and NaOH solution (0.1 mL, 0.1 M) (Lai et al. 2015). The mixture was stirred for 30 min and 50 μL of TEOS was added. The mixture was stirred for an additional 4 h at 55 °C and the synthesized MSNPs (named MSNP-VIII and MSNP-IX, respectively) were collected by centrifugation (20.000g, 30 min) and washed with water three times.

Lastly, 5 mg of the MNP-II or MNP-III in 0.5 mL of chloroform were poured into 25 mL of 0.1 M aqueous CTAB (0.911 g) solution and the resulting solution was stirred vigorously for 30 min (Cho et al. 2019). This resulted in an oil-in-water micro emulsion. Then, the mixture was heated up to 60 °C and kept at that temperature for 10 min under stirring to evaporate the chloroform. The resulting solution was added to a mixture of 50 mL of water and the pH of this mixture was adjusted to pH 11 using 2 M NaOH and the mixture was heated up to 70 °C under stirring. Then, 0.4 mL of TEOS and 2.4 mL of EtOAc were added to the reaction solution in sequence. After the addition of TEOS, the reaction was allowed to continue for 4 h. The magnetic mesoporous silica nanoparticles (MSNP-X and MSNP-XI, respectively) were collected by serial centrifugation. They were centrifuged at 5.000g for 5 min and then, obtained supernatant was centrifuged at 10.304g for 15 min. The MSNPs were washed three times with ethanol to remove the unreacted species.

CTAB removal was performed by shaking in an acetic acid/ethanol mixture (95/5 v/v) for 4 h (Suteewong et al. 2010). To obtain well-dispersed MSNPs in water, PEI solution (5 mg/mL) was added to the suspension (0.5:1, 1:1 and 2:1 (w:w)) of the MSNPs (1 mg/mL). They were shaken for 4 h to allow PEI to graft on to the surface of the MSNPs. Unbound PEI was removed by centrifugation and washed with water. MSNPs were sterilized by washing with ethanol and sterile PBS.

Synthesis of fluorescent-labelled MSNPs

To investigate the cellular uptake of MSNPs, fluorescent labelled MSNPs were prepared using Oregon Green 488 as described in the manufacturer's protocol. Basically, 100 µL of APTES was dissolved in 0.1 M sodium bicarbonate buffer (0.5 mL). 1 mg of Oregon Green 488 was dissolved in 0.2 mL of DMF. The mixture containing Oregon Green 488 was combined with APTES and stirred for 1 h at room temperature under dark conditions. Then, 50 µL of TEOS and 50 µL of Oregon Green 488 containing APTES solution were mixed and 50 µL of this mixture was added to the MNPs that were prepared as described in the silica coating step (in MSNP-IX preparation). After 4 h at 55 °C, the Oregon Green 488 labelled MSNPs were collected by centrifugation (10.000g) and washed three times with water and ethanol.

Characterization of nanoparticles

Characterization of MNPs and MSNPs was carried out with the support of experts and the devices in Eastern Anatolia High Technology Application and Research Center (DAYTAM), Atatürk University, Turkey. The powder X-ray diffractograms (XRD) were obtained in a 2θ range of 10°−80° using CuKα rays. TEM (transmission electron microscope) was used for structural analysis and the morphology of NPs was revealed by SEM (scanning electron microscope). Although TEM images were taken, the sample was dropped on the carbon-coated copper grid. Before SEM imaging, samples dried on glass were coated with gold. Organic and inorganic structures and functional groups were determined by FTIR (Fourier transform infrared) spectroscopy. FTIR samples were dispersed in water and dropped into the sample area of the device and the spectra of the samples in the 4000–400 cm−1 range were recorded under vacuum. The dynamic light scattering (DLS) technique was used for hydrodynamic dimension (Rh), zeta potential (ζ-potential) and polydispersity index (PDI) analysis of the obtained structures. These measurements were recorded at 25 °C and pure water was used as the solvent.

Calculating specific absorption rate (SAR) value of MNPs

The heating capacity of MSNPs was investigated by the MHT device. MSNPs were dispersed in water at the same concentrations (5 mg/mL) and the solutions were exposed to an AMF (300 kHz) for the desired amount of time. The specific absorption rate (SAR) is the measure of heat diffused per unit mass of MNPs (Sharma 2021). The SAR values were calculated according to the following formulation (Albarqi et al. 2019):

SAR=(CVs/m)×(dT/dt)

C: the specific heat capacity of the medium (Cwater = 4.185 J g−1 °C−1), Vs: the sample volume, m: the mass of the sample. dT/dt: the slope of the temperature−time graph. Although calculating the slope of the dT/dt curve, the first 60 s was considered when the temperature reached to 30 °C.

Cellular uptake of MSNPs

Cells were cultured as described in previous studies (Solak et al. 2021). To investigate the ideal magnet retention time for the NPs to be applied to the cells, 1 × 105 cells were planted in each of the glass cuts suitable for 12-well cell culture vessels. After 24 h, Oregon Green 488 labelled MSNPs at a concentration of 150 µg/mL were applied by holding the magnet for different durations. After 4 h, the cells were fixed with 4% PFA for 30 min at room temperature and counterstained with DAPI. The cells were visualized with a laser scanning confocal microscope.

Cytotoxicity and proliferation

The cell proliferation and cytotoxicity effect of MSNP-VII, -VIII and -IX were investigated on MCF-7 human breast adenocarcinoma cells and MCF-10A non-tumourigenic mammary epithelial cell line. Cells were incubated at 37 °C, 5% CO2 in an air atmosphere and 90% humidity and seeded on 96 well plates with a density of 10.000 cells/well. After 24 h, the MSNPs were applied to the cells using a magnet for 30 min. The treated cells were incubated for 48 h. MTS Reagent (20 µL/well) and culture media (200 µL/well) were added to each well. After incubation for 1–3 h in standard culture conditions, 200 µL MTS-containing-media was added into a clean well and the absorbance of treated and untreated cells was measured using a plate reader at OD490 nm. DMSO was also used as a positive control (5 µL).

Magnetic hyperthermia (MHT)

The cells were seeded on 25 cm2 culture flasks with a density of 106 cells/flask and incubated for 24 h before the MSNP-VII and -IX exposure at 100 µg/mL concentrations. A magnet placed at the bottom of flasks for 30 min and cells were incubated for 4-, 12- and 24-h. Before MHT application, cells were washed with PBS and trypsinized. The cell pellets were exposed to an AMF (300 kHz) for 30 min. The treated cells were incubated for 24 and 48 h to examine the cell morphology. For therapy studies, cells were seeded in 24-well plates at a density of 50.000 cells/well and incubated for 24 h before exposure to MSNP-IX at different concentrations (0, 50 and 60 µg/mL). A magnet was placed at the bottom of the well for 30 min and the cells were incubated overnight. Before MHT experiments, cells were washed with PBS and trypsinized. Cell pellets were exposed to AMF (300 kHz) at different times (20, 40 and 60 min). Treated cells were incubated for 48 h and cell cytotoxicity was analyzed.

Statistical analysis

All results were analyzed with Origin Pro Lab 8.5 data analysis and graphing software. Statistical significance was considered when p < 0.05 (n > 3). ImageJ software was used for the statistical analysis of confocal images.

Results and discussion

MHT can be used as a MNP-mediated thermal therapy for cancer. MNPs can be modified to increase their potential for use in nanomedicine. Silica coating of MNPs enhances the stability of NPs in aqueous solutions due to the electrostatic repulsion of silanol groups present on the surface (Ansari and Malaekeh-Nikouei 2017). In addition, silica shells can minimize unintentional leakage of the drugs that are embedded within the NPs (Ansari and Malaekeh-Nikouei 2017; Patil-Sen et al. 2020). Therefore, various applications of silica NPs have been investigated, especially their convenience for use in multi-therapy approaches. In this study, the usability for MHT treatment of iron oxide and zinc-doped iron oxide MSNPs was investigated (Scheme 1).

Scheme 1.

Scheme 1

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Summary diagram of the research steps designed for this study

First, magnetic core nanoparticles, called MNP-I to -V, were produced by thermal decomposition or co-precipitation methods. Characteristic peaks of magnetite were observed at the XRD results of MNP-I, -II and -III (Supp. Fig. S1 A). The MNPs have a peak of 2θ with each value of 30.18° (220); 35.59° (331); 37.22° (222); 43.1° (400); 53.58° (422); 57.2° (511); 62.77° (440) and 73.96° (533) (Solak et al. 2021). Comparison of the previous XRD results (Solak et al. 2021), to the current work, showed that the synthesis method used here did not affect the XRD peaks of MNPs. The TEM images clearly show that MNP-II have dimensions below 10 nm (Fig. S1 B). The magnetic properties of MNP-II were demonstrated by its movement with a magnet (Fig. S2). Thus, the MNPs produced here have magnetic and crystallite properties.

MNPs were individually coated with a silica shell to form bioavailable magnetic silica nanoparticles (MSNPs). Because MSNPs are intended for use in the effective application of MHT, MSNPs that provide temperature increases within a short timeframe were initially selected. It is well known that the presence of silica influences the magnetic properties of NPs. In many studies conducted to investigate the effect of silica on heat generation, it is seen that silica reduces the heating efficiency of the material. This is due to the reduction in magnetization value because of silica (Villanueva et al. 2010; Ansari and Malaekeh-Nikouei 2017; Patil-Sen et al. 2020). The SiO2 shell thickness hinders heat outflow and thus reduces the heating efficiency (Gonzalez-Fernandez et al. 2009). However, it is also possible to encounter studies with contrary results. The reduced rotational motion of the silica-coated NPs or the well dispersion of the NPs in the SiO2 matrix are suggested as possible causes of increased heating capacity (Yu et al. 2010; Kaman et al. 2011).The graph presented in Fig. 1 A revealed the MHT heating efficiency of various MSNPs at the same concentrations. The SAR values of the MSNPs presented in Fig. 1B were calculated from temperature−time graphs. The SAR of any material used in MHT is important because it is related to the conversion of the material's alternating magnetic radiation into heat. Generally, to increase the magnetic behaviour of NPs, the shape of the NPs is changed or additional ions, such as zinc, manganese, cobalt, nickel, etc., are doped into the material (Jang et al. 2009; Mamiya et al. 2020). In this current study, it was observed that the temperatures of MSNP-VII and -IX solutions were increased from ~ 28 °C to ~ 50 °C in 5 min. Interestingly, it was determined that MSNP-III and MSNP-IV which were produced using zinc-doped MNPs (MNP-IV and MNP-V) did not have higher SAR values compared to iron cores. A previous study had revealed that magnetite MNPs showed a higher magnetic saturation moment than the Zn-doped iron oxide NPs at a certain Zn/Fe ratio (Gordon et al. 2011). Hence, the lower SAR value of MSNP-III and MSNP-IV compared to MSNPs containing iron oxide NPs may be related to the Zn/Fe ratio in the core structures from which MSNP-III and MSNP-IV are produced. The highest SAR values were 95.85 and 84.07 for MSNP-VII and MSNP-IX, respectively. The observed temperature increases indicate that all the synthesized MSNPs can cause heat increments in the presence of AMF, however, some were much more efficient than others. In brief, it was decided to use MSNP-VII, -VIII and -IX in the continuation of the study, as they can provide a rapid temperature increase in a short time for cell studies.

Fig. 1.

Fig. 1

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Magnetic characterization of NPs by using a hyperthermia system. MSNPs were obtained by different synthesis procedures and B SAR values were calculated from A temperature−time curves recorded under a magnetic field. The data were recorded from the MSNPs aqueous solutions (5 mg/mL)

Characterization studies were carried out for selected MSNPs to determine the highest SAR values. In the TEM images of MNP-II, which were used for the manufacture of MSNP-II, -V, -VI, -IX and -X, it was seen that the size of MNPs was smaller than 10 nm and its size distribution was homogeneous (Fig. S1 B). Obviously, this study focused on MSNP-IX due to its higher SAR value. It was observed that the sizes of MSNP-V and MSNP-IX produced by different silica coating methods using the same core structure were less than 100 nm (Fig. 2A and C). Interestingly, the SAR value of the 500 nm MSNP-VII was higher than 100 nm MSNP-XI despite being produced from the same magnetic core (MNP-III) (Fig. 1B). In fact, considering the TEM images, this phenomenon was an expected result of the multicore nature of MSNP-VII (Fig. 2B and D).

Fig. 2.

Fig. 2

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Characterization results of nanoparticles. A SEM image of MSNP-V and TEM images of B MSNP-VII, C MSNP-IX and D MSNP-XI

Keeping the thickness of silica coating to a minimum is recommended in the literature because the silica coating inhibits heat flow, thus significantly reducing the SAR value and the efficiency of heating the environment of the nanomaterial (Gonzalez-Fernandez et al. 2009; Majeed et al. 2014). In addition, various properties, such as synthesis methods of MNPs, particle−particle interactions and the morphology and size of nanomaterials are extremely important to maximize the SAR value (Sharma 2021).

Furthermore, CTAB, which assists in the transfer of hydrophobic MNPs into aqueous solutions, is potentially cytotoxic to mammalian cells and therefore CTAB extraction was performed before silica coating procedures. Also, polyethyleneimine (PEI), a cationic polymer, was preferred to increase the dispersion of NPs in water or culture media. The silica coating, CTAB removal and the presence of the amine group on the surface were confirmed by FTIR analysis. As seen in Fig. 3A, the −C−H chain of CTAB clearly seen at 2852–2920 cm−1 was absent after CTAB removal. In addition, the transmission signal for −HCH bending vibration that was observed at 1489 cm−1 and could be due to the presence of CTAB in CTAB-containing MSNPs, was not observed after CTAB removal (Jain et al. 2019). The vibration of the − Si−O bond of silica was observed at 1024 cm−1 (Solak et al. 2021). The peaks at 1414 cm−1 and at 1257 cm−1 indicating the amine group and −C−N bending were observed after PEI modification, respectively (Pasini et al. 2020).

Fig. 3.

Fig. 3

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Characterization results of modified nanoparticles. A FTIR and B DLS results of PEI-modified MSNP-IX

The branched structure of high molecular weight PEI may cause an undesirable increase in the hydrodynamic diameter (Rh) of the MSNP-IX. For this reason, different PEI:MSNP-IX ratios were investigated. The DLS results presented in Fig. 3 showed that PEI gave a high positive charge to the MSNPs surface. The high positive charge of MSNPs offers a great advantage for biomedical applications, such as nucleic acid delivery (Yildiz et al. 2021). After PEI modification, ζ-potential values of + 30 and above indicated that MSNP@PEI was well dispersed in water. When the Rh was examined, as expected, the size of MSNP@PEI increased as the PEI ratio increased. Finally, PDI values confirmed that the dispersion of the PEI-modified MSNP-IX in water was extremely advantageous (Fig. 3B). When all the results were evaluated, it was decided to use the structures produced in 1:1 PEI:MSNP ratio in ongoing studies, since they have the highest ζ-potential and good stability.

We showed that MNP-II modified with silica (Fig. 2) and PEI (Fig. 3) might be used for MHT. It clears that the thickness and morphology of MSNPs were changed by both the size and morphology of the core materials and the silica coating method (Fig. 2).

In vitro assays

Nanomaterials with a size of 10–100 nm provide easy penetration to cells and also help to increase the life of MNPs in the physiological environment (Chenthamara et al. 2019). NPs < 10 nm can be completely excreted from the renal system, whereas NPs > 200 nm can also be phagocytic. Both conditions are undesirable for biomedical applications (Hoshyar et al. 2016; Spirou et al. 2018). Therefore, the size of MNPs is an important factor. We prepared MSNP-IX less than 100 nm (Fig. 2) based on the TEM images.

In vitro applications, acceleration of cellular uptake of MSNPs is possible with the help of temporary magnets. To observe this situation, Oregon Green 488 labelled MSNP-IX was produced and applied to MCF-10A and MCF-7 cells using magnets for different times. The confocal images showed that the Oregon Green 488 signal recorded around the cell nuclei (DAPI) improved in parallel with the increase in magnet time in both cell lines (Fig. 4A and B). The decrease in DAPI signal was observed in the groups that were kept on the magnet for 60 min, suggesting that prolonged exposure of the cells outside the standard incubator conditions or increased time on a magnetic field may have affected the cells. Taken together, in this study, the magnet application time was suggested as 30 min. Quantitative analysis of confocal images obtained using the ImageJ program confirmed these results (Fig. S3).

Fig. 4.

Fig. 4

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In vitro studies using MSNP-IX. Cellular uptake of fluorescent-labelled MSNP-IX by confocal microscopy belongs to A MCF-10A and B MCF-7 cell lines

Zinc ions have been suggested for anticancer treatment and are selectively toxic to breast cancer cells in vitro (Hanley et al. 2008). A biodegradable mesoporous system is highly beneficial due to its ability to control the release of Zn2+ ions from the silica matrix. In vitro biocompatibility was determined by investigating the cytotoxicity of MSNP-VII, -VIII, -IX and zinc-doped MSNP-VIII. As shown in Fig. 5A and B, in the absence of AMF, MCF-10A cells were affected less by MSNP-VIII treatment than MCF-7 cells, whereas the Zn-doped MSNP-VIII treatment had much more of a dose-dependent response from both cell lines. Consistent with our results, there are some studies indicating that zinc-containing MNPs are dose-dependently toxic to both breast cancer and non-tumourigenic cells (Chen et al. 2020). MTS results (Fig. 5C and D) showed that MSNP-VII and -IX were biocompatible for both MCF-10A and MCF-7 at lower concentrations. Therefore, after cell proliferation and cytotoxicity test, MSNP-VII and MSNP-IX were preferred to perform MHT therapies.

Fig. 5.

Fig. 5

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In vitro cytotoxicity of A MSNP-VIII, B Zn-doped MSNP-VIII, C MSNP-VII and D MSNP-IX in the absence of AMF

In vitro magnetic hyperthermia

The biological mechanisms behind MHT-induced toxicity have not yet been fully elucidated. It has been observed that the effect of MHT depends on various factors including the concentrations, physicochemical properties, intracellular location of the NPs used and even the cell type being investigated. It has been reported that after MHT treatment, intrinsic or extrinsic apoptosis is induced in cells and lysosomal membrane permeability is changed (Domenech et al. 2013; Sanchez et al. 2014; Beola et al. 2020; Pucci et al. 2022).

When considering that non-tumourigenic cells will be more convenient for MHT application due to low sensitivity against heat, MCF-10A cells treated PEI-modified MSNP-VII and MSNP-IX were collected at the 4th, 12th and 24th hours and then MHT was applied to the cells. It was determined that the MSNP@PEI treated cells at 4th and 12th hours were reached over 42 °C in a short time. However, cells incubated for 24th hours could not reach this temperature (Fig. 6A). The reason of this may be that the MSNPs were digested by the cells and the intracellular amount of them caused decreased heating efficiency at 24th hours. Fundamentally, it can say that the maximum concentration of intracellular MSNPs is decreasing by time.

Fig. 6.

Fig. 6

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A Time-dependent temperature graphics under a certain AMF of MSNP-treated cells and B Cell morphologies of the treated cells by MSNPs and 30 min MHT (Scale 50 µm)

It is known that hyperthermia might cause cell necrosis or apoptosis by causing changes in intracellular and extracellular temperature (Kalamida et al. 2015). Actually, MHT specifically causes disruption of cytoskeletal components and impaired cell membrane permeability (James et al. 2010). When we examined the morphology of cells that were exposed to MHT followed MSNP-VII or MSNP-IX treatment, it can be said that MCF-10A cells overcome the applied therapy stress within 48 h. It was also observed that MSNP-VII affected cells more adversely due to the presence of globular cells after 24 h (Fig. 6B). Therefore, in this study, MSNP-IX was suggested as a biocompatible agent for MHT applications.

Hyperthermia treatment changes the function of many structural and enzymatic proteins in cells, which can induce apoptosis (Harmon et al. 1991; Sellins and Cohen 1991). Cells were exposed to MSNP-IX + MHT to determine the effective MSNPs dose at 40 min AMF exposure. As shown in Fig. 7A, MSNP-IX-treated MCF-10A cells were exposed to AMF and retained cell viability at all tested concentrations. In contrast, MCF-7 cells treated with MSNP-IX showed cytotoxic effects when exposed to AMF (Fig. 7B). The results showed us that tumourigenic MCF-7 cells were more affected than non-tumourigenic MCF-10A cells in the presence of AMF with MSNP-IX (Fig. 7). Interestingly, in contrast to MCF-10A, we also noticed the reduced cell viability in MCF-7 cells that were kept under AMF field for only 60 min and lacked MSNP-IX. Even when cancer cells were harvested and seeded after the application of MSNP, a decrease in the cell viability was observed. However, the higher viability observed in the non-cancer cell line under similar conditions may indicate that cancer cells have higher sensitivity even to only MSNPs treatment. The viability rates below 80% in MCF-10A cells were only observed in the groups treated with MHT for 60 min.

Fig. 7.

Fig. 7

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In vitro cytotoxicity of MSNP-IX in the presence of AMF for A MCF-10A and B MCF-7. *p < 0.05

The main causes of cancer-selective cell death are suggested as cytotoxic reactive-oxygen species production and lysosomal membrane permeability. Increased oxidative stress is a treatment approach used against cancer (Cho et al. 2019). When iron oxide NPs are taken into cells, Fe2+ ions can release into the cytoplasm which is a trigger for the Fenton reaction and as a result hydroxyl radicals are formed in the cells (Huang et al. 2013). The hydroxyl radicals are one of the most reactive radicals known. They effectively disrupt cellular functions and therefore can be used to direct cancer cells to apoptosis. On the other hand, the increasing intracellular temperature is one of the explanations for the toxicity of NPs-mediated MHT. Hyperthermic stress causes protein unfolding and aggregation in the cytoplasm that affects cellular activities, such as DNA replication. In addition, hyperthermia also affects the permeability of the plasma membrane and causes changes in the mitochondrial membrane potential, resulting in an increase in calcium along with a change in the redox state of the cell (Roti Roti 2008). When all these stress conditions occur at the same time, cancer cells cannot overcome this situation, while non-tumourigenic cells are less affected by these conditions using stress overcoming pathways.

All these results are once again revealed the cancer-selective effect of MHT therapy. MHT sensitizes cells to other cancer treatments, such as radiotherapy or chemotherapy. In some studies, it was observed that the efficacy of chemotherapeutic drugs increases up to ten times in patients after hyperthermia (Wust et al. 2002).

Conclusion

Different nanomaterials are being developed for various cancer therapies and diagnosis, include mesoporous silica NPs and iron oxide NPs. These MNPs have great potential because of their interesting features, which include response to external magnetic fields, high charge, ease of surface modification to prevent adverse biological interactions and excellent biocompatibility. Thus, MNPs, and in particular MSNPs, offer researchers a great platform to overcome the main challenges in cancer treatment.

In this study, monodispersed and bioavailable MSNPs were produced. The MSNPs can be applied to cells in a very easy way by the use of a magnet. It was also observed that the MSNPs concentration in cells decreased after 12 h. It can also be said that effectiveness of MHT treatment depends on the shape, size and composition of MSNPs. Contemplating the MHT temperature increase rate and cytotoxicity effects, it was determined that the most suitable MSNPs for this application were MSNP-IX. Thus, MSNPs, which can heat up rapidly under magnetic field and remain stable after MHT application and which are not cytotoxic for MCF-10A cells have been synthesized. As demonstrated in the results with MCF-7, these MSNPs are good candidates to be used in MHT treatment for breast cancer.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1576 kb) (1.5MB, docx)

Acknowledgements

The authors kindly acknowledge TÜBİTAK (The Scientific and Technological Research Council of Turkey) (Project number: 118Z150) for the financial supports and DAYTAM (East Anatolia High Technology Application and Research Center, Atatürk University, Erzurum, Turkey) for the equipment support. M.A. and K.S. are also thankful for The Council of Higher Education (CoHE, 100/2000) PhD Scholarship Program, Turkey.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Contributor Information

Yagmur Unver, Email: yunver@atauni.edu.tr.

Ahmet Mavi, Email: amavi@atauni.edu.tr.

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Supplementary Materials

Supplementary file1 (DOCX 1576 kb) (1.5MB, docx)

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