Mesoporous superparamagnetic hydroxyapatite nanocomposite: A multifunctional platform for synergistic targeted chemo-magnetotherapy

Materials Science & Engineering C 101 (2019) 27–41

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Mesoporous superparamagnetic hydroxyapatite nanocomposite: A multifunctional platform for synergistic targeted chemo-magnetotherapy

T

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Azadeh Izadia, Azadeh Meshkinia, , Mohammad H. Entezarib,c a

Biochemical Research Center, Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran Sonochemical Research Center, Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran c Environmental Chemistry Research Center, Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Magnetic hydroxyapatite Drug delivery system Bone cancer Doxorubicin

In the present study, the aim was to develop a magneto-responsive nanocomposite for application in drug delivery by the integration of magnetic nanoparticles into an inorganic architecture, hydroxyapatite. The magnetic mesoporous hydroxyapatite nanocomposites, MMHAPs, were synthesized using a template-free method and fully characterized by XRD, FT-IR, TEM, FE-SEM, VSM, ICP, BET, and UV–Vis spectroscopy. MMHAPs exhibited a rodlike shape with a structure of large mesopores and high surface area. A sample of the nanocomposites with welldefined properties, MMHAP(2), was selected as a carrier for delivery of chemotherapy drug, doxorubicin (Dox). Then, it was coated with polyethylene glycol (P) and folic acid (F), providing aqueous stability and tumor targeting, respectively. The evaluation of drug release profile revealed that the release of drug occurs in a timestaggered manner under low pH conditions, which simulate the internal condition of lysosome. More important, a significant drug release was observed under a static magnetic field (SMF), displaying a magnetically triggered release. According to the toxicity assessment, MMHAP(2) did not show any noticeable toxic effect against the tumor cells (Saos-2) and normal cells (HEK-293) up to 100 μg ml−1 in the presence or absence of SMF. In contrast, the drug-loaded nanocomposite, F.P.D@MMHAP(2), possesses high antitumor efficacy particularly in the presence of SMF. Moreover, it was found that the cellular internalization of F.P.D@MMHAP(2) could be increased by SMF, providing therapeutic efficiency enhancement. The high cytotoxic effect of F.P.D@MMHAP(2) with the help of SMF caused apoptosis in the tumor cells, which was preceded by a disturbance in the intracellular redox state and then caspase activation. Based on the data obtained, F.P.D@MMHAP(2) is a pH- and magneto-responsive platform opening up a new perspective in terms of its exploitation in cancer therapy.

1. Introduction Nanostructure materials today form the basis for a huge variety of pharmaceutical and medical applications, including diagnosis and drug delivery, and they are employed in modern therapeutic strategies against cancer [1]. Among various types of nanomaterials, magnetic nanoparticles have attracted a great deal of attention in biomedicine and clinical applications owing to their prominent advantages such as superparamagnetism, low toxicity, biocompatibility, and biodegradability [2]. Moreover, magnetic nanoparticles can be conducted by an external magnetic field, providing the guided delivery of drugs and biomolecules. In addition, magnetic field can immobilize and separate magnetically tagged biological entities [3–5]. However, in practical applications, some drawbacks limit the exploitation of bare magnetic nanoparticles in biomedical field, namely their poor chemical stability,

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high aggregation propensity, low adsorption drug capacity, poor release rate, and short retention time in the blood stream. To address these grand issues, some effective protection strategies have been taken into account and made the use of these materials feasible. For instance, the grafting of small biomolecules like surfactants [6,7], the encapsulation within polymers such as dextran [8] or chitosan [9], and the coating with inorganic materials such as silica [10] and ceramics [11] not only provide chemical stability and retard the oxidation of nanoparticles but also render the magnetic nanoparticles nontoxic and nonimmunogenic. Among the aforementioned materials, a bioceramic like hydroxyapatite (HAP) could be considered as a proper inorganic surface coating and potentially expand the biological application scope of magnetic nanoparticles, particularly in biomedicine, owing to its excellent biocompatibility, slow biodegradation, unique mechanical stability and great adsorption drug capacity [12,13]. Because of nontoxic nature and

Corresponding author. E-mail address: [email protected] (A. Meshkini).

https://doi.org/10.1016/j.msec.2019.03.066 Received 8 December 2018; Received in revised form 18 March 2019; Accepted 19 March 2019 Available online 23 March 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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embryonic kidney cells (HEK-293), were evaluated in the presence and absence of static magnetic field (SMF).

non-inflammatory properties of HAP, it has a wide variety of potential applications in drug delivery and tissue engineering [14,15]. Recently, an intensive research has been carried out on the synthesis of mesoporous hydroxyapatite owing to its several alternative features such as high surface area, tunable pore size, and high pore volume [16,17]. These features not only enhance the loading capacity and tune the release of several pharmacy molecules, but also increase the bioactivity properties of nanoparticles with respect to bone cells and tissues [12,18,19]. Therefore, mesoporous magnetic hydroxyapatite (MMHAP) would be an excellent candidate in biomedical applications, particularly in targeted drug delivery systems. Thanks to its high drug adsorption capacity, ability to specifically target tumor cells, and to easily recover excess or unused drugs by an external magnetic field. In general, several synthesis routes have been developed to date for the fabrication of magnetic hydroxyapatite nanocomposites [20]; however, the production of mesoporous ones has been studied less extensively. Recently, Aval et al. reported the synthesis of the magnetic hydroxyapatite nanocomposite with a mesoporous structure by employing F127 as a pore-directing agent for an efficient delivery of a chemotherapy drug [21]. In addition, Bharath et al. obtained the mesoporous magnetic hydroxyapatite nanocomposite by the soft template synthesis method, utilizing cetyltrimethylammonium bromide (CTAB) [22]. To the best of our knowledge, the template-free synthesis of the mesoporous magnetic hydroxyapatite nanocomposite has never been reported before. Considering the magnetic properties of magnetic nanoparticles, magnetic fields can be employed for various biomedical purposes. The combination of magnetic fields and magnetic nanoparticles allows one to transform energy into force or heat, affecting cell signaling and destiny [23]. In this line, Bradshaw and coworkers demonstrated that cell-internalized superparamagnetic nanoparticles control the direction and speed of cell migration in the presence of a magnetic field [24]. Precisely, internalized magnetic nanoparticles cause a tensile force inside neurons by an external magnetic field, leading to the neurite and axon elongation in a particular direction [25]. More recently, it has been reported that the combinational effect of a magnetic field and cellular uptake of BSA-coated magnetic nanoparticles lead to a higher intracellular concentration of nanoparticles. The internalized nanoparticles modulate the activity of the intracellular signaling pathways and enabling magnetic stimulation of mesenchymal stem cells to enhance differentiation toward osteogenic outcome [3]. On the other hand, it has been shown that under magnetic field, mechanical forces exerted by magnetic nanoparticles onto the specific cell or organelle disrupt the integrity of membrane and induce apoptotic signaling [26]. In fact, these contradictory results regarding the biological consequences of magnetic nanoparticles and magnetic fields could be attributed to the type, strength, exposure time of a magnetic field, and also physico-chemical properties of magnetic nanoparticles. The aim of the present study was to synthesize template-free mesoporous magnetic hydroxyapatite nanocomposites (MMHAP) through a hydrothermal process for application in drug delivery systems. The synthesized nanoparticles were characterized by XRD, FT-IR, zeta potential, DLS, BET, FE-SEM, TEM, ICP, and VSM techniques. One sample of magnetic hydroxyapatite nanoparticles with well-defined properties was selected as a carrier for the delivery of a standard chemotherapy drug, doxorubicin (Dox), providing magnetic drug targeting under the external magnetic field. The mesoporous nanocomposites were also coated with polyethylene glycol (PEG), making a stable suspension of the drug-loaded nanoparticles in biological media. To improve tumor targeting, PEG-coated nanoparticles were also decorated by folic acid as an essential ingredient in DNA replication. The designed drug delivery system (F.P.D@MMHAP) was also characterized by FT-IR, zeta potential, and UV–Vis spectroscopy. Since the most unique feature of magnetic nanoparticles is their response to the magnetic field, the kinetic of drug release, cellular uptake of the fabricated system, and the antitumor efficacy of the F.P.D@MMHAP nanocomposites on tumor cell line, osteosarcoma cells (Saos-2), and normal cell line, human

2. Materials and methods 2.1. Materials The cell culture medium (RPMI-1640) and fetal bovine serum (FBS) were purchased from Gibco BRL (Life Technology, Paisley, Scotland). Penicillin–streptomycin was purchased from Biosera (France). Saos-2 cell line was obtained from Pasteur Institute of Iran (Tehran, Iran). Methylthiazolyldiphenyl-tetrazolium bromide (MTT), propidium iodide, and genistein were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Calcium chloride (CaCl2), dipotassium hydrogen phosphate (K2HPO4·3H2O), ferric chloride hexahydrate (FeCl3·6H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), poly(ethylene glycol) (PEG 2000), ethidium bromide, acridine orange, and dimethyl sulfoxide (DMSO) were bought from Merck Millipore (Darmstadt, Germany). Cleaved caspase-3 (Asp175) antibody was bought from Cell Signaling Technology (MA, USA). FITC-conjugated secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Hoechst 33342 was obtained from Tocris Cookson (Bristol, UK). 2.2. Synthesis of Fe3O4 nanoparticle Superparamagnetic nanoparticles were synthesized by chemical coprecipitation of Fe2+ and Fe3+ ions. In a 250 ml three-necked roundbottom flask, a mixture of FeCl3·6H2O (1.8 g) and FeSO4·7H2O (0.96 g) were dissolved in 100 ml of deionized water at 80 °C under argon atmosphere. Then ammonia solution (25%, 10 ml) was added quickly to precipitate Fe2+/Fe3+ ions for the synthesis of magnetite (Fe3O4) particles. The solution was mixed rapidly by an overhead stirrer for 45 min and then cooled to room temperature (RT). The Fe3O4 nanoparticles were collected through magnetic separation and washed with deionized water and ethanol three times. The Fe3O4 nanoparticles were then dried under vacuum condition at 50 °C. 2.3. Synthesis of hydroxyapatite (HAP) nanoparticles and MMHAP nanocomposites CaCl2 was dissolved in 30 ml deionized water (0.24 M) and the solution was adjusted to pH 11 using NaOH (1 M). For the synthesis of MMHAP(1) and MMHAP(2), the naked Fe3O4 nanoparticles at concentration of 0.013 and 0.026 mg ml−1, respectively, were added to the above solution. Then, K2HPO4·3H2O (0.150 M) was dissolved in 50 ml deionized water and slowly added dropwise to the above solution mixture, yielding a milky suspension. The resulting mixture was then transferred to a Teflon-lined stainless-steel autoclave and heated at 150 °C for 12 h. The precipitate was then separated by centrifugation (6000 rpm, 15 min) and washed several times with ethanol and deionized water in turn. A gel-like paste was produced and then it was dried in a vacuum oven at 50 °C for 18 h. To obtain pure HAP, the same procedure was carried out without the addition of Fe3O4. 2.4. Characterization of synthesized nanoparticles The crystal phase of the nanoparticles and nanocomposites was determined by x-ray diffraction (XRD) (EXPLORER, GNR, Italy), using CuKa radiation (λ = 1.54 Å) at 40 kV and 30 mA at a step size of 0.040° and step time of 1 s. The Fourier transform infrared (FT-IR) spectra were recorded with a Nicolet Avatar 370 FT-IR Thermo spectrometer. Transmission electron microscopy (TEM) was performed with a Leo 912 AB (120 kV) microscope (Zeiss, Germany). High-resolution TEM was performed by a Zeiss Libra transmission electron microscope. The morphology of fabricated samples was observed using a field emission scanning electron microscope, FE-SEM, (MIRA3TESCAN-XMU) with 28

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Fig. 1. Characterization of Fe3O4 and HAP nanoparticles and MMHAP nanocomposites. (A) XRD patterns of Fe3O4 (a), HAP (b), MMHAP(1) (c), and MMHAP(2). (B) FT-IR spectra of Fe3O4 (a), HAP (b), MMHAP(1) (c), and MMHAP(2) (d). (C) VSM magnetization curves of Fe3O4 nanoparticles and MMHAP nanocomposites. (Inset: visual image of dispersed MMHAP nanocomposites in PBS in the absence and presence of an external magnetic field). (D) Nitrogen adsorption-desorption isotherm of HAP nanoparticles and MMHAP nanocomposites.

solution (1 mg ml−1). The mixture was shaken at 150 rpm at 25 °C. At different time intervals, the samples were centrifuged and the concentration of Dox in the supernatants, Ct, was then determined by a UV–Vis spectrophotometer at a fixed wavelength of 325 nm. The adsorption quality of Dox was defined as follows:

Table 1 Textural parameters of the HAP nanoparticles and MMHAP nanocomposites.

HAP MMHAP(1) MMHAP(2)

BET(m2 g−1)

Dp (nm)

Vp (cm3 g−1)

25.55 56.72 60.79

23.69 20.51 20.43

0.15 0.29 0.31

Qt =

Symbol explanation: BET is specific surface area as described by theory of Brunauer, Emmett, and Teller. Dp and Vp are mean pore diameter and total pore volume, respectively, as defined by the theory of Barrett, Joyner and Halenda.

(C 0 − Ct ) × V m

where Qt is the amount of drug adsorbed on nanocomposite (mg mg−1), C0 and Ct are the initial and residual concentrations of drug at time t, V is the volume of the Dox solution, and m is the mass of nanocomposite (mg).

20 kV accelerating voltage. The BET surface area analysis and pore size distribution were measured on a Belsorp mini II system at −120 °C using N2 as the adsorbate. Dynamic light scattering (DLS) and the surface charge of the nanoparticle were analyzed with a laser zetameter (Zeta compact, CAD instrumentation, France). UV–Vis absorption spectra of the samples were recorded by a UV–Vis spectrophotometer (Optizen 322 OUV, MECASYS, North Korea) in quartz cell. Detection of chemical elements in the prepared samples was carried out by an inductively coupled plasma optical emission spectrometer (ICP-OES, Spectro Arcos, Kleve, Germany).

2.6. Preparation of PEG-coated nanoparticles PEG was dissolved in deionized water (100 mg ml−1) and heated at 80 °C for 1 h. Then, naked nanoparticles or drug-loaded nanocomposites (2 mg ml−1) were added to PEG solution and the mixture was stirred for 5 h in the dark. The PEG-coated samples were collected by a centrifuge (8000 rpm, 10 min) and rinsed several times with water. The obtained powder was dried in an oven at 37 °C for 24 h. 2.7. Conjugation of folic acid on the surface of drug-loaded nanocomposites

2.5. Evaluation of Dox adsorption on the surface of MMHAPs For the conjugation of folic acid on the surface of nanocomposites, P.D@MMHAP(2) nanocomposites were added to the folic acid (F)

MMHAP nanocomposites (2 mg) were added to Dox aqueous 29

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Fig. 2. TEM (A) and FE-SEM (B) images of HAP and Fe3O4 nanoparticles and MMHAP nanocomposites.

trypan blue. The human embryonic kidney (HEK-293) cells were obtained from the Research Institute of Biotechnology (Ferdowsi University of Mashhad, Mashhad, Iran). The cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with glucose (4500 mg L−1), FBS (10%, v/v), streptomycin (100 μg ml−1), and penicillin (100 U ml−1). The cells were incubated under 5% CO2 humidified atmosphere at 37 °C.

Table 2 Particle size and surface charge of synthesized nanoparticles and nanocomposites. DLS Zaverage Fe3O4 HAP MMHAP(1) MMHAP(2) a

350 886 630 527

Zeta potential PDI 0.344 0.921 0.494 0.257

−14.49 ± 0.43 −16.48 ± 0.37 −16.52 ± 0.24 −15.32 ± 0.63a

2.10. Cytotoxicity studies using MTT assay MTT was used as an indicator of cell viability on the basis of its mitochondrial-dependent reduction to formazan. The cells were seeded at a density of 2 × 104 cells/well into 96-well tissue culture plates in order to evaluate the cytotoxicity of free Dox, the synthesized nanoparticles, nanocomposites, and drug-loaded nanocomposites. After 24 h, the culture medium was replaced with a medium containing different concentrations of synthesized samples or free Dox and incubated at 37 °C for different times. Then, 10 μl of MTT (5 mg ml−1) was added to each well and the plate was incubated at 37 °C for 4 h. The culture supernatant was removed and the formazan crystals were dissolved using DMSO. The mixture was agitated for 15 min at room temperature. The absorbance was read at 570 nm using an ELISA reader (BioTek, ELX800, USA). The cellular viability was expressed as the percentage of the surviving cells in the treated sample relative to the control samples. For the experiments under magnetic field, after the treatment of the cells with different concentrations of nanoparticles, nanocomposites, or free Dox for 2 h, the cells were subjected to the static magnetic field (0.9 T) for 12 s. Afterward, the cells were incubated at 37 °C for 48 h. The cells exposed solely to the magnetic field with the identical strength were considered as a control in the experiment of magnetic field treating. The viability of cells was examined by the MTT method.

Statistically is significant relative to MMHAP(1).

solution (0.5 mg ml−1 in deionized water, pH 7.8) and stirred for 24 h in the dark at RT. Then, the sample was centrifuged (8000 rpm, 10 min) and the collected nanocomposites were washed three times with deionized water. This material was named F.P.D@MMHAP(2). 2.8. Kinetic of drug release from F.P.D@MMHAP(2) nanocomposite F.P.D@MMHAP(2) nanocomposites (1 mg) were soaked in the release medium (1 ml, PBS, pH 7.4, pH 6.2, or pH 4.5) for the release of drug and shaken at 180 rpm at 37 °C in a shaking bed in the dark. At different times, samples were centrifuged and the release medium was taken out from each vial. The amount of released drug was also determined by a UV–Vis spectrophotometer. In order to evaluate the effect of magnetic field on the release kinetic of drug from nanocomposites, F.P.D@MMHAP(2) nanocomposites (1 mg) were immersed into the release medium (1 ml, PBS, pH 7.4, pH 6.2, and pH 4.5). Then, samples were exposed to magnetic field (0.9 T) for 12 s and then shaken at 180 rpm at 37 °C in a shaking bed in the dark. The amount of drug release was evaluated by a UV–Vis spectrophotometer.

2.11. Perl's Prussian blue staining 2.9. Cell culture For iron detection within the cells, Perl's Prussian blue staining was performed. Saos-2 cells were treated with P@HAP, P@Fe3O4, P@ MMHAP(1) or P@MMHAP(2) at IC50 concentration for 12 h. Then, the cells were stained with an equal volume of 20% hydrochloric acid and 20% potassium ferrocyanide trihydrate for 20 min and then they were washed three times with deionized water. For the cytoplasm staining,

The human osteosarcoma cell line, Saos-2, was cultured in RPMI1640 medium supplemented with FBS (10%, v/v), streptomycin (100 μg ml−1), and penicillin (100 U ml−1). The cells were incubated under 5% CO2 humidified atmosphere at 37 °C. Cell proliferation was assessed using a hemocytometer and the abilities of the cells to exclude 30

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Fig. 3. Cytotoxic effects of Fe3O4 and HAP nanoparticles and MMHAP nanocomposites on the osteosarcoma cells. Saos-2 cells were treated with different concentrations of nanoparticles or nanocomposites for 48 h in the presence and absence of SMF (0.9 T) and then the cells viability was evaluated by MTT assay. The data are the means of three experiments ± SD. *p < 0.05 and statistically is significant.

2.13. Cellular uptake of F.P.DOX@MMHAP nanocomposites and determination of uptake route

Table 3 IC50 values of the synthesized nanoparticles and nanocomposites in the presence and absence of SMF (0.9 T) in osteosarcoma cells (Saos-2) after 48 h of treatment. a

IC50 ± SD (μg ml

Fe3O4 HAP MMHAP(1) MMHAP(2)

−1

After 4 h of the incubation of Saos-2 cells (1 × 104 cells/well) with F.P.D@MMHAP(2), P.D@MMHAP(2) nanocomposites, or free Dox, the cells were harvested and washed with PBS. The cellular uptake of nanocomposites or free drug was qualitatively analyzed by a fluorescence microscope (Olympus, Japan) and quantitatively estimated by a flow cytometer. For the determination of the cellular internalization mechanism, the Saos-2 cells were treated with F.P.D@MMHAP(2), following the treatment of the cells with different endocytosis inhibitors including sucrose (0.22 M), genistein (0.2 μM), or NaN3 (15 μM) for 30 min. After 4 h of incubation, the cells were harvested, washed with PBS, and analyzed by flow cytometry. The cells were treated only with F.P.D@MMHAP(2) served as the control group.

)

SMF(−)

SMF(+)

219.05 ± 22.8 285.98 ± 22.2 230.75 ± 4.4 197.85 ± 22.6

179.30 ± 9.7 269.70 ± 24.9 193.55 ± 0.2 144.65 ± 5.3

a IC50: The concentration of compound required for cell growth inhibition by 50%. Data were expressed as the mean of the triplicate.

the cells were incubated with neutral red solution containing neutral red dye (0.03 M), sodium acetate (0.03 M), and acetic acid (0.02 M) for 5 min. The cells were washed two times with deionized water and observed by an inverted light microscope.

2.14. Indirect immunofluorescence For the assessment of the level of activated caspase within the cells, indirect immunofluorescence was performed. Treated and untreated cells were trypsinized and washed with PBS. The cells were incubated with PBS containing 1% FBS and 0.01% Triton X-100 for 10 min at 37 °C. Then, the cells were incubated with a primary antibody for 1 h and they were washed three times with PBS. Afterward, the cells were incubated with a secondary immunoglobulin G fluorescein isothiocyanate-conjugated antibody for 30 min. The cells were then washed again and analyzed by flow cytometry.

2.12. Fluorescence labeling of synthesized nanoparticles and nanocomposites For the assessment of the cellular uptake of the nanoparticles and nanocomposites quantitatively, P@HAP, P@Fe3O4, P@MMHAP(1), and P@MMHAP(2) were labeled with FITC according to the previous report [27]. Then, the amount of cellular uptake of FITC-labeled nanoparticles or FITC-labeled nanocomposites was analyzed by flow cytometry.

2.15. SubG1 analysis by flow cytometry Saos-2 cells (1 × 104 cells/well) were treated with drug-loaded 31

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Fig. 4. Evaluation of cell death induced by Fe3O4 and HAP nanoparticles or MMHAP nanocomposites in Saos-2 cells. (A) Cells were treated with the synthesized samples at IC50 concentration in the presence and absence of SMF (0.9 T) for 48 h and then they were stained with Ao/EtBr. Images were taken by a fluorescence microscope at 200× magnification (scale bar: 200 μm). Normal viable cells possess uniform bright green nuclei. Apoptotic cells show orange area of condensed or fragmented chromatin in their nuclei. The necrotic cells show uniform bright orange nuclei. (B) 300 cells were counted in each experiment for the estimation of the percentage of apoptotic cells. The data are the means of three experiments ± SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

nanocomposites or free Dox at IC50 concentration and then exposed to magnetic field (0.9 T) for 12 s. After 48 h of incubation, the cells were harvested and washed twice with PBS. Then, the cells were suspended in PBS containing 20 μg ml−1 propidium iodide and 20 μg ml−1 RNase (DNase free) for 30 min. The stained cells were analyzed by flow cytometry.

data were analyzed using one-way analysis of variance and Tukey's post hoc test, and p-values ≤0.05 were considered significant.

2.16. Evaluation of intracellular reactive oxygen species (ROS)

2.17. Statistical analysis

3.1.1. XRD analysis The crystallinity and purity of the Fe3O4 nanoparticles after being coated with hydroxyapatite were examined by XRD. As shown in Fig. 1A (a), all of the peaks can be well indexed to a pure cubic crystal structure for magnetite (JCPDS 04-0139807). The XRD pattern of HAP nanoparticles showed the formation of single-phase hydroxyapatite, and the pattern matched well with JCPDS values (01-0853476) (Fig. 1A (b)). Corresponding diffraction patterns were also observed in the XRD pattern of MMHAP nanoparticles; however, some characteristic peaks related to Fe3O4 were well evident (Fig. 1A (c) and (d)). In addition, there was no obvious peak-shifting in the characteristic peaks of HAP, implying that the phase of hydroxyapatite has not been affected during the synthesis of MMHAP nanoparticles.

Data are expressed as mean ± SD of three independent measurements and statistically analyzed using Student's t-test. The experimental

3.1.2. FT-IR analysis FT-IR spectra of Fe3O4, HAP, and MMHAP nanocomposites are

3. Results 3.1. Characterization of HAP, Fe3O4, and MMHAPs

DCFH-DA is a cell-permeable probe which is widely used for detecting of intracellular ROS. It is hydrolyzed to DCFH by intracellular esterases and trapped within the cells. The intracellular ROS oxidize DCFH to a highly fluorescence compound. Thus, the fluorescence intensity is proportional to the amount of intracellular ROS. The treated and untreated cells were harvested and washed twice with PBS. Then, the cells were incubated with DCFHA-DA (10 μM) for 30 min. The cells were washed with PBS and the intracellular ROS was quantified by flow cytometry.

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Fig. 5. Qualitatively and quantitatively evaluation of the cellular uptake of synthesized nanoparticles and nanocomposites. (A) Saos-2 cells were treated with IC50 concentrations of synthesized nanoparticles or nanocomposites in the presence and absence of SMF (0.9 T) for 12 h and then Perl's Prussian blue staining was carried out. (B) Saos-2 cells were treated with FITC-labeled nanoparticles or FITC-labeled nanocomposites and then the amount of uptake was measured by a flow cytometer. The percentage of the cells with green fluorescence was estimated in FL1 channel, reflecting the cellular internalization of nanoparticles or nanocomposites. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

nanocomposites was measured using VSM. The M-H hysteresis curves have been illustrated in Fig. 1C. No remanence and coercivity were observed in any samples, indicating the superparamagnetic property. The saturation magnetization value of naked Fe3O4 nanoparticles was 65.03 emug−1, and it reduced noticeably after being coated with HAP, which is due to a high Ca/Fe ratio and shielding effect of HAP. The MMHAP(1) and MMHAP(2) nanocomposites showed a similar superparamagnetic property with the values of 4.7 and 10.91 emug−1, respectively. Increasing in the amount of Fe3O4 nanoparticles in the nanocomposites augmented their magnetization, facilitating the magnetic control of their spatial distribution in biological media under an external magnetic field.

displayed in Fig. 1B. The FT-IR spectrum of Fe3O4 nanoparticles was established by the absorption bands at 578 and 3397 cm−1 which are attributed to the stretching vibration of FeeO bond and bending vibration of OH groups, respectively (Fig. 1B (a)). The characteristic absorption bands related to the hydroxyapatite appeared at 561, 959, and 1044 cm−1, which are assigned to phosphate groups [28]. In addition, the stretching band at 3497 cm−1 belongs to the vibrational mode of structural hydroxyl groups in hydroxyapatite (Fig. 1B (b)). In the IR spectrum of MMHAP nanocomposites, all characteristic absorption bands related to the functional groups of hydroxyapatite emerged clearly and no significant changes were observed relative to the HAP spectrum, which demonstrated that the incorporation of magnetic nanoparticles does not have any effect on the structure of conventional HAP (Fig. 1B (c) and (d)).

3.1.4. BET analysis The most important features of mesoporous materials, which make them a promising drug delivery system as compared with nonporous ones, refer to their texture of surface including: i) the high surface area,

3.1.3. VSM analysis The magnetic property of the Fe3O4 nanoparticles and the MMHAP 33

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or macromolecules such as proteins. Hence, textural parameters of bare HAP and MMHAP nanocomposites were determined. The nitrogen adsorption-desorption isotherms of the samples are shown in Fig. 1D, and the corresponding textural parameters are summarized in Table 1 and Fig. SI. 1. The HAP nanoparticles and both of the nanocomposites showed type IV adsorption isotherms, which is characteristic of porous materials with mesopore size distribution. There is a broad hysteresis loop in both of the nanocomposites in the region with higher relative pressure, which is attributed to the large mesopores. Interestingly, the specific area and total pore volume increased in the nanocomposites as compared with those in the HAP nanoparticles. The specific surface area and pore volume registered 25.55 m2 g−1 and 0.15 cm3 g−1, respectively, for HAP, and these values increased to 56.72 m2 g−1 and 0.29 cm3 g−1 for MMHAP(1) and to 60.79 m2 g−1 and 0.31 cm3 g−1 for MMHAP(2). The structure of pores did not significantly alter following magnetite concentration enhancement. Based on these data, it seems that some textural properties of MMHAP nanocomposites can be controlled by varying the concentration of Fe3O4.

Fig. 6. Evaluation of the kinetic of Dox adsorption on the surface of MMHAP nanocomposites as a function of time. The data are the means of three experiments ± SD.

3.1.5. TEM and FE-SEM observations The morphology and elemental analysis of bare Fe3O4, HAP, and nanocomposites were investigated by TEM, HRTEM, SEM micrographs, and energy dispersive spectroscopy (EDS). As it can be seen in Fig. 2A and B, Fe3O4 and HAP nanoparticles were found to be spherical and nanorod-shaped, respectively, having nearly monodispersed and narrow particle size distribution. The average diameter of Fe3O4 was found to be around 11 nm and that of the HAP nanoparticle was calculated to be 32.22 nm in width and 154.34 nm in length. Similarly to the HAP nanoparticles, MMHAP nanocomposites showed nanorodshaped, and magnetic nanoparticles were mainly embedded in the HAP structure (Figs. 2A and SI. 2). Besides, the size of nanocomposites decreased evidently as a function of Fe3O4 concentration. EDS spectrum

which implies a high potential for drug adsorption on the surface of the materials and an impact on their final drug content, ii) the high pore volume, which allows large payload of drug molecules to be entrapped into the pores, consequently preventing drug molecules from degradation in harsh environment during drug administration and inhibiting premature drug release, and iii) the ability to control the rate and period of drug release in targeted tissues [29–32]. Further, as compared with microporous materials, whose pore diameters are smaller than 2 nm, mesoporous materials, with pore diameters ranging from 2 to 50 nm, have received much scientific attention. It is because the pore size of mesoporous materials allows them to host either small molecules

Fig. 7. Characterization of F.P.D@MMHAP(2) nanocomposites. (A) Schematic illustration of the synthetic steps for the preparation of the F.P.D@MMHAP(2) nanocomposites. (B) FT-IR spectra of MMHAP(2) (a), D@MMHAP(2) (b), P.D@MMHAP(2) (c), and F.P.D@MMHAP(2) (d). (C) UV–Vis spectra of MMHAP nanocomposites and surface-modified MMHAP(2) nanocomposites. (D) Zeta potential analyses of MMHAP(2) and surface-modified MMHAP(2) nanocomposites in deionized water at pH 7.2. 34

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Fig. 8. Evaluation of cytotoxic effects of F.P.D@MMHAP(2) on the osteosarcoma cells. Saos-2 cells were treated with different concentrations of free Dox or F.P.D@ MMHAP(2) in the absence (A) and presence (B) of SMF (0.9 T, 12 s) for 48 h and then the cell viability was assessed by MTT assay. (C) Calculated IC50 elicited from MTT assay. The data are the means of three experiments ± SD. *p < 0.05 and statistically is significant.

Fig. 9. Evaluation of drug release from the F.P.D@MMHAP(2) nanocomposites as function of time and pH in the absence (A) and presence (B) of SMF (0.9 T). The data are the means of four experiments ± SD.

−14.49 ± 0.43 mV to lower negative values in MMHAP nanocomposites and reached the values detected for the naked HAP nanoparticles (−16.48 ± 0.37 mV), indicating the coating of the magnetic nanoparticles with HAP.

confirmed the existence of Ca, P, and O elements for HAP and Fe and O elements for the Fe3O4 nanoparticles (Fig. SI. 3). A composition of those elements was also detected in both MMHAP nanocomposites; however, a higher amount of Fe element was found in MMHAP(2) than MMHAP (1). The iron content of the MMHAP nanocomposites is found precisely, using ICP-OES, to be 0.06% and 0.15% per 0.1 g of MMHAP(1) and MMHAP(2), respectively (Table SI. 1). In other words, the percentage of Fe element over total elements was 0.030% and 0.067% for MMHAP(1) and MMHAP(2), respectively.

3.2. In vitro biological studies 3.2.1. Cytotoxic evaluation of PEG-coated nanoparticles and nanocomposites Self-aggregation tendency is a major drawback of magnetic nanoparticles, and it limits their biological application. To circumvent this challenge, magnetic nanoparticles are coated with various types of polymers, tuning the physico-chemical properties of the nanoparticles and subsequently making them stably dispersed [33]. Among different types of biocompatible polymers, polyethylene glycol (PEG) is extensively used in targeted drug delivery owing to its non-toxic, nonimmunogenic, and non-antigenic nature [34,35]. It causes an increase in the stability of the magnetic nanosystem in biological media and improves the efficiency of drug delivery to targeted cells [21]. Hence, in our study, all the synthesized nanoparticles were coated with PEG (P), and the cytotoxicity of the different concentrations of the well-dispersed nanoparticles was then evaluated on the osteosarcoma cells (Saos-2 cells) in the presence and absence of SMF. As evidenced in Fig. 3, a fraction of the surviving cells showed a marked decline, as they were exposed to both the magnetic nanoparticles and SMF. A significant decrease in cell viability was observed, particularly, when the Saos-2 cells were being treated with P@MMHAP(2) and SMF, indicating the

3.1.6. DLS analysis The hydrodynamic size distribution of the synthesized nanoparticles and nanocomposites in aqueous media was determined by DLS. The average size of the Fe3O4 and HAP nanoparticles was 350 and 886 nm, and that of MMHAP(1) and MMHAP(2) was 630 and 527 nm, respectively (Table 2). In addition, the evolution of polydispersity index (PDI) of nanocomposites followed a similar trend to the evolution of particle size. PDI decreased in MMHAP nanocomposites as compared with that in the naked Fe3O4 and HAP nanoparticles. Moreover, as the ratio of Ca/Fe decreased, the PDI value augmented, indicating the effect of Fe3O4 concentration on the improvement of size distribution and dispersion of the MMHAP nanocomposite. 3.1.7. Zeta potential analysis To evaluate the surface charge of the synthesized nanoparticles, the zeta potential was measured in distilled water at pH 7.4. As shown in Table 2, zeta potential values of the Fe3O4 nanoparticles changed from 35

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(caption on next page) 36

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Fig. 10. Evaluation of the cellular uptake of F.P.D@MMHAP(2) in the presence and absence of SMF. (A) Saos-2 cells were treated with F.P.D@MMHAP(2) or free Dox in the presence and absence of SMF (0.9 T) for 4 h and then the cells were stained with Hoechst 33342 for nucleus. All images were taken by a fluorescence microscope (Olympus) at 200× magnification and scale bar represents 200 μm. The fluorescence microscopic images of treated cells with free Dox or F.P.D@MMHAP (2) revealed red and blue fluorescence corresponded to the conjugated folic acid on the surface of nanocomposites and nucleus, respectively. (B) The cellular uptake of drug-loaded nanocomposites or free Dox was quantitatively evaluated in the presence and absence of SMF (0.9 T) by flow cytometry. The percentage of cells with red fluorescence was estimated in FL2 channel, reflecting the cellular internalization of nanocomposites or free Dox. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2.2. Cellular uptake of nanoparticles and nanocomposites under SMF The relevance of the prepared nanoparticles as a successful candidate for biomedical application particularly in drug delivery is dependent on the delivery of nanoparticles into cells. Hence, the cellular uptake efficiency of the synthesized nanoparticles in the presence and absence of SMF was qualitatively verified by Perl's Prussian blue staining. The result indicated the intracellular presence of magnetic nanoparticles inside cells, which can be visualized by blue spots in cells. Interestingly, the cellular uptake efficiency of the magnetic nanoparticles was enhanced with the help of the magnetic field (Fig. 5A). It seems that the effect of SMF on the cellular internalization of MMHAP (2) nanocomposites was much stronger than that on the cellular uptake of the other magnetic nanoparticles. This phenomenon may be related to the smaller size of MMHAP(2) nanocomposites and their lower agglomeration tendency under magnetic fields. The enhancement of the nanoparticles uptake owing to the effect of the magnetic field was also quantified by flow cytometry. The Saos-2 cells were treated with FITC-labeled nanoparticles or FITC-labeled nanocomposites, and then the amount of their internalization was evaluated. The results in Fig. 5B show that the percentage of the fluorescence-labeled cells in the presence of SMF was greater than that of the labeled cells in the absence of SMF. Compatible with the Perl's staining results, the highest cellular uptake efficiency was recorded for the MMHAP(2) nanocomposites in the presence of SMF (72.4%), which was nearly 1.2-fold higher than that for the MMHAP(2) nanocomposites in the absence of SMF.

Fig. 11. Evaluation of the cellular uptake mechanism of F.P.D@MMHAP(2). Saos-2 cells were treated with F.P.D@MMHAP(2) at IC50 concentration for 4 h following treatment of cells with endocytosis inhibitors for 30 min. The data are the means of three experiments ± SD. p < 0.05 statistically is significant.

3.2.3. Drug adsorption onto the surface of MMHAP nanocomposites The drug loading efficiency of the MMHAP nanocomposites was evaluated as a function of time using a chemotherapy drug, doxorubicin. As it is evident in Fig. 6, the time-dependent drug adsorption occurred onto the surface of both nanocomposites; however, MMHAP (2) exhibited higher drug loading capacity than MMHAP(1), which is related to their surface properties. It was supposed that the fewer negative charges and higher surface area in MMHAP(2) than in MMHAP (1) enhance adsorption capacity and provide a favorable surface for more adsorption of Dox. After the nanocomposites were soaked in the Dox solution, the surface of the nanoparticles was saturated by Dox in < 24 h of incubation in 0.5 mg ml−1 of Dox. The equilibrium capacity was calculated to be 0.051 and 0.036 mg mg−1 for MMHAP(2) and MMHAP(1), respectively.

cytotoxic enhancement of P@MMHAP(2) nanocomposites with the aid of the external magnetic field. The IC50 value of P@MMHAP(2) was calculated to be 197.85 ± 22.6 μg ml−1, which had significantly decreased after the magnetic treatment (144.65 ± 5.3 μg ml−1) (Table 3). Moreover, it was found that solo SMF possesses an inhibitory effect on the tumor cell growth and survival (Fig. SI. 4), which is in accordance with the previous reports [36,37]. Since the cell viability decreased in the treated cells with nanoparticles or nanocomposites in the presence and absence of SMF, we were interested to figure out the fate of the treated cells. To identify various types of cell death, the treated cells were stained with Ao/EtBr. As illustrated in Fig. 4A and B, as the cells were treated with Fe3O4, HAP, or both the nanocomposites in the presence of SMF, the percentage of the cells with apoptotic features increased as compared with that of the cells treated with the nanoparticles or nanocomposites alone. However, as predicted from the cytotoxic experiments, a pronounced increase in apoptosis was observed in the cells treated with both P@ MMHAP(2) and SMF as compared with that in the cells treated with the others nanoparticles. Interestingly, an even more precisely evaluation of the results revealed that solo SMF has an anti-proliferative effect without any induction of apoptosis or necrosis (Fig. SI. 4), indicating the “cytostatic” activity of SMF. From these results, it is assumed that SMF not only impacts on the biological activity of magnetic nanoparticles, but also showed a synergistic effect along with the magnetic nanoparticles in the induction of apoptosis, owing to its cytostatic activity.

3.2.4. Characterization of F.P.D@MMHAP(2) nanosystem Owing to the mesoporous structure, high surface area, and magnetic property of the MMHAP(2) nanocomposites, they could potentially be employed as a promising drug delivery vehicle in biomedical applications. In order to increase the stability of nanosystem in biological media and improve the efficiency of drug delivery to targeted cells and tissues, the Dox-loaded nanocomposites were functionalized with PEG and folic acid (F), respectively (Fig. 7A). Note that different types of tumor cells overexpress folate receptors, which are exploited in targeted specific-cell delivery of folic acid-decorated nanoparticles containing therapeutic drugs, providing active targeting [38]. FT-IR spectroscopy was performed to investigate the surface functionalization of the MMHAP(2) nanocomposites (Fig. 7B). FT-IR spectrum of the D@ MMHAP(2) nanocomposites showed different bands at 3570 and 3436 cm−1 as compared to the spectrum of the naked nanocomposites 37

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Fig. 12. Manifestation of cytotoxic mechanism of F.P.D@MMHAP(2). (A) Saos-2 cells were treated with drug-loaded nanocomposites in the presence and absence of SMF (0.9 T) for 48 h and then the amount of intracellular ROS levels was measured by flow cytometry, using DCFH-DA. (B) The activated caspase-3 level was determined in treated cells after 48 of incubation by indirect immunofluorescence according the procedure described in the materials and methods section. (C) Cell death induced by F.P.D@MMHAP(2) was evaluated qualitatively in treated cells after 48 h of treatment by Ao/EtBr staining. (D) 300 cells were counted in each experiment for the estimation of the percentage of apoptotic cells. The data are the means of three experiments ± SD. (E) The percentage of apoptotic cells in treated Saos-2 cells was also determined as sub-G1 population in cell cycle analysis by the flow cytometry. The data are the means of two experiments ± SD.

charge of the nanocomposites (−15.32 ± 0.63 mV) increased (−19.68 ± 0.95 mV) as the drug molecules were loaded on the surface of the nanocomposites. Following the coating of the nanoparticles with PEG, the surface charge moved toward positive values (−14.96 ± 1.12 mV). As folic acid was conjugated onto the surface of the nanosystem, an increase in the magnitude of negative charges (−25.95 ± 1.5 mV) occurred. This phenomenon may be related to the unbound carboxylate groups in the folic acid structure, which leaves a negative charge on the surface of the nanocomposites.

assigned to the NeH and OeH stretching vibration, respectively, in the structure of Dox [39]. It indicates the successful adsorption of the drug onto the surface of the nanocomposites. The emergence of strong bands in the spectrum (c) at 2887 and 1114 cm−1 corresponded to CeH symmetric and CeOeC asymmetric stretching vibrations, respectively. Further, two sharp bands at 1467 and 1343 cm−1 were assigned to scissoring and wagging CH2 vibrational modes, respectively [40], demonstrating the surface coating of the drug-loaded nanocomposites with PEG. In spectrum (d), the absorbance IR bands appeared at 3349 and 1682 cm−1, which are respectively attributed to the primary amine and carboxylic acid in the structure of folic acid decorated on the surface of the drug-loaded nanocomposites. The surface functionalization of the MMHAP(2) nanoparticles was further confirmed by UV–Visible spectroscopy (Fig. 7B). In the case of the naked nanocomposites, no characteristic UV–Vis absorbance peak was found. In the spectrum of the Dox-loaded nanocomposites, a distinct peak appeared nearly at 233 nm, which is attributed to Dox; however, the intensity of the peak declined dramatically as PEG was introduced on the surface of the nanoparticles. A prominent peak at 280 nm was observed in the case of F.P.D@MMHAP(2), which is related to the n-π* transition in the structure of folic acid, indicating the effective attachment of folic acid on the surface of the nanosystem. Also, the surface-chemical modifications of the MMHAP(2) nanocomposites were followed by zeta potential measurements. As shown in Fig. 7C, the negative surface

3.2.5. Cytotoxicity of F.P.D@MMHAP(2) under SMF To verify the in vitro cancer cell inhibition induced by drug-loaded MMHAP(2) in a magnetic field, the Saos-2 cells were treated with different concentrations of F.P.D@MMHAP(2) or Dox alone in the presence and absence of SMF. As shown in Fig. 8A and B, an obvious inhibition in the tumor cell viability was observed in the cells treated with F.P.D@MMHAP(2) as compared with that in the Dox-treated cells, both in the presence and absence of SMF. However, the most significant enhancement of the toxic effects emerged in the cells treated simultaneously with the drug-loaded nanocomposites and SMF. As for the Saos2 cells, the IC50 values of free Dox and F.P.D@MMHAP(2) were 2.139 ± 0.098 and 1.831 ± 0.147 μg ml−1, respectively, in the absence of SMF; however, in the presence of SMF, these values reduced significantly to 1.674 ± 0.120 and 0.994 ± 0.299 μg ml−1, 38

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images also disclosed the high fluorescence intensity in the F.P.D@ MMHAP(2)-treated cells as compared with that in P.D@MMHAP(2). To investigate the effect of the magnetic field on the cellular uptake efficiency, the cellular internalization of the F.P.D@MMHAP(2) nanocomposites was surveyed under the influence of SMF. Based on the data presented in Fig. 10A and B, it was found that SMF causes a statistically significant increase in the amount of the F.P.D@MMHAP(2) nanocomposites to be taken up by the cells (MFI: 76.8 a.u.) as compared with that by the untreated cells (MFI: 60.0 a.u.). Therefore, besides folic acid which provided active targeting, SMF brought a convenience to the drug delivery system. In targeted drug delivery, nanomaterials enter cells preferentially through receptor-mediated endocytosis; however, physico-chemical properties, surface reactivity of nanoparticles, cell types, and the differentiation state of cells impact on the endocytosis pathway [45]. To determine the trafficking route of the F.P.D@MMHAP(2) nanocomposites in the Saos-2 cells, endocytosis inhibitors were used. As it is evident in Fig. 11, a significant decrease was observed in the cellular uptake of F.P.D@MMHAP(2) following the inhibition of caveolaemediated endocytosis by genistein. However, the ability of the cells to internalize the drug-loaded nanocomposites decreased dramatically as the clathrin-mediated endocytosis pathway was impeded by sucrose (44%). Hence, it seems that both the clathrin-mediated endocytosis and caveola-mediated endocytosis are involved in the uptake of F.P.D@ MMAHP(2); however, clathrin-mediated endocytosis is the main trafficking route of F.P.D@MMAHP(2). Furthermore, the pretreatment of the cells with NaN3 significantly reduced the cellular uptake of F.P.D@ MMHAP(2) up to 71%, demonstrating the involvement of an energydependent endocytosis pathway in the internalization of the fabricated system.

respectively (Fig. 8C). These findings confirmed that the drug delivery efficiency of MMHAP(2) could be improved with the help of a magnetic field. To examine the tumor-selective activity of F.P.D@MMHAP(2) and free Dox, their cytotoxic effects were evaluated on the normal cells, human embryonic kidney cells (HEK-293) (Fig. SI. 5). Note that most of the biodistribution studies confirm the presence of nanoparticles in a secondary organ, e.g. kidney, located far apart from the point of exposure [41]. Therefore, it is useful to pay great attention to the toxic effect of nanoparticles in kidney cells. Statistically, insignificant growth inhibition was observed when the non-cancer cell line was treated with F.P.D@MMHAP(2) in the presence and absence of SMF, which is probably due to the limited cellular uptake. 3.2.6. Kinetic of drug release from F.P.D@MMHAP(2) under different pHs in the presence and absence of SMF Recently, the design of stimuli-sensitive drug release systems has drawn a great deal of attention as they allow reliable drug release flux for clinical needs. Nanosystems responsive to internal signals like pH variations or intracellular redox changes, or to external signals like temperature variations or UV radiations have already been widely applied, subsequently providing spatio-temporal drug release [42]. To investigate the controlled Dox releasing performance of the F.P.D@ MMHAP(2) nanocomposites under different pH conditions as internal signals, the nanosystems were immersed in the medium with pH 7.4, 6.2, and 4.5, which simulates the cytosol, extracellular microenvironment of tumor cells, and endo/lysosomal compartment, respectively [43]. As shown in Fig. 9A and B, a great amount of Dox release from the nanosystem occurred at acidic pH as compared with that at physiological pH, confirming the stability of the nanoparticles and a low premature drug release in physiological conditions. Moreover, we studied this release behavior in a time-dependent manner (2–72 h) to gain an understanding of the kinetic of the Dox release from the nanosystem. The drug release process was significantly increased after 4 h of incubation with constant kinetic, and the steady state was then achieved after 48 h. In addition to the internal signal, we were interested to examine the effect of the magnetic field as an external signal on the drug release profile. To achieve this goal, the F.P.D@MMHAP(2) nanocomposites were immersed in the medium with different pHs, and then samples were exposed to SMF for 12 s. Interestingly, Dox exhibited a significant drug release in acidic conditions following exposure to SMF as compared with that in similar conditions without SMF, indicating magnetically triggered drug release. Based on these results, it was found that the F.P.D@MMHAP(2) nanosystem is a dual-responsive drug delivery system which provides spatio-temporal control over the release of drug by the environmental pH and a magnetic field.

3.2.8. Cytotoxic mechanism of F.P.D@MMHAP nanocomposites In vitro studies recently revealed that iron oxide nanoparticles disrupt the redox homeostasis inside cancer cells, leading to the selective tumor cell toxicity. A potential mechanism by which iron oxide nanoparticles induce cytotoxicity is the formation of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals, and superoxide anions, which provide oxidative stress [46,47]. In fact, endocytosed iron oxide nanoparticles release iron ions into the cytoplasm of tumor cells where they can participate in the Haber-Weiss chemistry through the Fenton reaction and catalyze the formation of highly reactive hydroxyl radicals. Note that in normal condition, intracellular iron ions are generally bound to specific proteins and only trace amount of free ions are available for Fenton reaction.

Fe3 + + O2˙ → Fe2 + + O2

3.2.7. Cellular uptake of F.P.D@MMHAP(2) nanocomposites in the presence and absence of SMF Owing to the innate fluorescence property of folic acid, the cellular trafficking and internalization of the F.P.D@MMHAP(2) nanocomposites into the cells are traceable by a fluorescence microscope. As illustrated in Fig. 10A, the fluorescence nanocomposites were widely distributed in the cytoplasm of the Saos-2 cells; however, a high fluorescence intensity was observed in the cells treated with the F.P.D@ MMHAP(2) nanocomposites as compared with that in the cells treated with P.D@MMHAP(2) or free Dox. Moreover, the cellular internalization of the drug-loaded nanocomposites was quantitatively determined by flow cytometry. The obtained histograms in Fig. 10B demonstrated that the internalization of the F.P.D@MMHAP(2) nanocomposites into the Saos-2 cells was significantly higher (MFI: 60.0 a.u.) than that of P.D@MMHAP(2) (MFI: 36.8 a.u.). It indicates the critical role of folic acid in the cellular uptake of nanoparticles, which provides active and selective targeting in cancer therapy. Note that Saos-2 cells are the folate receptor positive cells [44], so we assumed that a significant amount of the drug-loaded nanocomposites were probably taken up by the folate receptors. Correspondingly, the fluorescence microscopic

Fe2 + + H2 O2 → Fe3 + + OH– + OH˙ O2˙ − + H2 O2 → O2 + OH– + OH˙ Moreover, the effect of Dox on the intracellular redox state has been well documented [48]. Previous studies on breast and prostate cancer showed that Dox undergoes futile redox cycles to generate elevated superoxide (O2%−) and H2O2, causing massive ROS levels and caspasemediated programmed cell death [49,50]. Based on these facts, it was supposed that the combination of Dox and the released iron ions from F.P.D@MMHAP(2) could produce highly reactive hydroxyl radicals through the Fenton reaction, amplifying oxidative stress and increasing the anti-tumor activity of Dox. To demonstrate the hypothesis, the intracellular ROS levels were monitored in the cells treated with F.P.D@ MMHAP(2) or free Dox in the presence and absence of SMF. As shown in Fig. 12A the intracellular levels of ROS augmented after the cells were treated with either F.P.D@MMHAP(2) or free Dox. Interestingly, the intracellular redox state was more disturbed since the cells were exposed to SMF following treatment with F.P.D@MMHAP(2). Amplifying ROS stress generated by F.P.D@MMHAP(2) in the presence of 39

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SMF caused the activation of apoptosis-involved proteins like caspases. A remarkable increase was observed in the level of activated caspase-3 in the cells treated with both F.P.D@MMHAP(2) and SMF (27%) as compared with the cells treated with free Dox (11.7%) (Fig. 12B). In fact, a high level of caspase activation implies the possibility of a high apoptosis rate. Ao/EtBr staining (Fig. 12B and C) and sub-G1 analyses (Fig. 12D) demonstrated a high percentage of apoptosis in the cells treated with F.P.D@MMHAP(2) in the presence of SMF (37.5%) as compared with that in the cells treated with free Dox (15.5%).

[9]

[10]

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4. Conclusion

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In this study, large mesoporous magnetic hydroxyapatite nanocomposites were successfully synthesized in a template free route. The nanocrystalline pore walls, high surface area, and magnetic property made the MMHAP(2) nanocomposites valuable for application in drug delivery. The designed smart delivery platform, F.P.D@MMHAP(2), displayed spatiotemporal drug release, which was precisely controlled by pH and the magnetic field. Most importantly, it was demonstrated that the cellular internalization of the nanocomposites could be enhanced by SMF. Therefore, the delivery efficiency of the MMHAP(2) nanocomposites increased by decoration of the drug delivery system with folic acid and applying the external magnetic field. Cytotoxic assay disclosed that the magnetic field enhanced the growth inhibition of cancer cell, subsequently exhibiting a synergistic cooperation between the drug-loaded nanocomposites as an “inducer” and the magnetic field as a “sensitizer”. Further, the experiments also revealed that the growth inhibition is preceded by changes in intracellular the redox state, caspase activation and apoptosis. From these results, F.P.D@MMHAP(2) is a potential delivery system which could be used for cancer therapy; however, more detailed investigations are required to establish the in vivo efficacy of this nanosystem.

[13]

[14]

[15]

[16]

[17]

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Acknowledgments [22]

The authors appreciate the financial support for this investigation by the Research Council of Ferdowsi University of Mashhad (Grant No. 3/47459).

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Conflict of interest None.

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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.03.066.

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