Magnetic mesoporous bioactive glass for synergetic use in bone regeneration, hyperthermia treatment, and controlled drug delivery

A combination of chemotherapy with hyperthermia can produce remarkable success in treating advanced cancers. For this purpose, magnetite (Fe3O4)-doped mesoporous bioactive glass nanoparticles (Fe3O4-MBG NPs) were synthesized by the sol–gel method. Fe3O4-MBG NPs were found to possess spherical morphology with a size of approximately 50 ± 10 nm and a uniform pore size of 9 nm. The surface area (309 m2 g−1) was sufficient for high drug loading capacity and mitomycin C (Mc), an anticancer drug, was entrapped in the Fe3O4-MBG NPs. A variable rate of drug release was observed at different pH values (6.4, 7.4 & 8.4) of the release media. No significant death of normal human fibroblast (NHFB) cells was observed during in vitro analysis and for Mc-Fe3O4-MBG NPs considerable inhibitory effects on the viability of cancer cells (MG-63) were observed. When Fe3O4-MBG NPs were immersed in simulated body fluid (SBF), hydroxycarbonate apatite (HCA) was formed, as confirmed by XRD and FTIR spectra. A negligible value of coercivity and zero remanence confirms that Fe3O4-MBG NPs are superparamagnetic. Fe3O4-MBG NPs showed a hyperthermia effect in an alternating magnetic field (AMF), and a rise of 11.5 °C in temperature during the first 6 min, making it suitable for hyperthermia applications. Fe3O4-MBG NPs expressed excellent biocompatibility and low cytotoxicity, therefore, they are a safe biomaterial for bone tissue regeneration, drug delivery, and hyperthermia treatment.


Introduction
Bone is a self-healing tissue in minor defects but treating larger defects due to trauma, osteoporosis, tumour removal, infection or thinning is still a clinical and socio-economical challenge. Therefore, the need of the hour is a synthetic bone gra that can also overcome the limitations of other bone treatment methods such as autogras and allogras, which suffer from donor site morbidity, weak osteoinductivity, and potential risk of infection. [1][2][3][4] For this purpose, a wide variety of biomaterials were prepared for bone-tissue regeneration and fascinating are those which exhibit multifunctional abilities such as osteoconductivity, osteogenesis, and angiogenesis.
Moreover, sometimes a huge amount of bone is removed due to cancer and it is impossible to harvest such a greater mass of bone; therefore, tissue regeneration is a viable alternative. 5,6 But even the bone regeneration is substantially hindered by infections that were conventionally treated by antibiotic administration and wound drainage. However, these methods were mostly ineffective and resulted in further complications leading to extra surgeries, which cause pain and economic cost. Subsequently, a multifunctional biomaterial was needed to solve all these problems and would represent a valuable solution in preventing post-surgical infections along with bone regeneration. [7][8][9] Therefore nanobioactive glass such as mesoporous bioactive glass (MBG) is one of the most promising biomaterials which possess exceptional osteoinductive behavior and can form a bond with hard and so tissues through hydroxycarbonate apatite (HCA). [10][11][12] It also plays an important role in regenerative medicines, drug carriers, and biosensors. This wide variety of applications of MBG depend upon the surface area, morphology, pore size, stoichiometric ratio, crystallinity, composition, and crystal size distribution. To obtain tailormade properties, different compositions were studied and doped with different ions. Different metallic (Ag, K, Mg, Sr, Cu, and Co) and non-metallic (B) ions impart several biological functions such as stimulation of osteogenesis, angiogenesis, and anti-bacterial activities. [13][14][15] Although MBG is successfully used for bone regeneration and delivery of anti-cancer drugs to the cancerous bones, it does not kill the cancer cells itself. Therefore, in this study, MBG was doped with magnetite (Fe 3 O 4 ) for its synergetic use hyperthermia treatment of cancer cells. Aer implantation in the affected region, it is exposed to the alternating magnetic eld and produces heat. Thus, relatively high temperature is maintained (>43 C) in the region of neoplastic tissue and malignant cells are selectively killed. [16][17][18]  mol%), TEOS was added to a mixture of 5 g of P123, 10 mL of 0.2 N HNO 3 , 50 mL of absolute ethanol and 500 mL of deionized water. The mixture was stirred for 2 h in an inert atmosphere then TEP, Ca(NO 3 ) 2 $4H 2 O, and Na 2 CO 3 were added and stirred with a time interval of 45 min for each component. Then already prepared Fe 3 O 4 NPs were added, and the mixture was stirred for 1 h so that sol is formed. When ammonia solution (25%) was added dropwise a thick gel was formed, which was further stirred for 2 h, aged overnight at room temperature, dried at 100 C in a vacuum oven and nally calcined at 350 C for 4 h to get Fe 3 O 4 -MBG. 19 Characterizations FTIR spectrum was taken by a Nicolet iS10 FTIR spectrometer and XRD analysis was performed by using an X-ray diffractometer (PANalytical, X'Pert Pro, Almelo, Netherlands); having a Cu Ka radiation source operated at 40 kV. The surface area and pore size of Fe 3 O 4 -MBG NPs were measured by using the BJH (ASAP 2010) and BET (Micromeritics Instrument Corp, Gemini V2.0 analyses). Particle size, shape, morphology, and elemental analysis were achieved by using SEM, EDX (Hitachi S3400N), and TEM (JEM-1400 Plus). UV/Vis spectrophotometer (Shimadzu UV-265) was used to determine the concentration of the drug. Zeta potential and the surface charge was determined with the help of Zetasizer ZS (Malvern Instruments, Malvern, UK). The magnetic properties were measured by using the Quantum Design PPMS magnetometer.

Bone tissue regeneration study
For the investigation of bone-forming ability, 500 mg of the Fe 3 O 4 -MBG NPs were immersed in SBF and incubated at 37 C. Aer one week, Fe 3 O 4 -MBG was ltered, washed gently with acetone, and dried at room temperature. 20 The samples were subjected to XRD and FTIR analysis to explore the HCA formation.

Alkaline phosphatase activity and osteocalcin assay
For ALP measurement osteoblast cells were cultured and treated with Fe 3 O 4 -MBG, for 48, 72, and 120 h. The cells were treated according to the previously described protocol and cell lysate was used for the ALP activity, according to the manufacturer kit (ab83369). A standard curve was calculated using pnitrophenol and ALP activity unit was calculated.
For osteogenic assay cells were cultured on Fe 3 O 4 -MBG in DMEM without FBS in 6 well plates. Aer 120 h, culture medium from each well was aspirated and assayed by following a Human Osteocalcin ELISA kit (Biomedical Technologies Inc, Tyne & Wear, UK). 21

Hyperthermia study
The hyperthermia property of Fe 3 O 4 -MBG NPs was studied by using AC applicator DM100 by nB nanoscale Biomagnetics, working at the frequency of 220-260 kHz and magnetic eld amplitude (H o ) up to 23.9 kAm À1 (300 guess). 1 mg mL À1 of Fe 3 O 4 -MBG was suspended in deionized water at room temperature in a glass tube. The alternating magnetic eld of frequency 250 kHz and magnetic eld strength at 6 kAm À1 were applied to the Fe 3 O 4 -MBG solution for 20 min adiabatically by keeping the surrounding temperature at 25 C. The temperature was measured using a ber optic temperature probe. The specic absorption rate (SAR) was measured by the following equation.
In this equation, C (4.18 J g À1 C À1 ) represent specic heat of water, DT is temperature change, t is the change in time, m Fe is the fractional mass of Fe in Fe 3 O 4 -MBG.

Statistical analysis
Statistical analysis performed by using Graphpad Prism. P < 0.05, was regarded as signicant and data is expressed as mean AE SE.

Results and discussion
The prepared Fe 3 O 4 -MBG NPs were subjected to various characterizations and screening before using them as a drug delivery carrier. The results of BET nitrogen adsorption-desorption analysis for the Fe 3 O 4 -MBG are given in Fig. 1 as type of IVa isotherm, conrming the mesoporous structure. The BJH analysis conrms the narrow pore size distribution at 9.52 nm and BET analysis shows that the surface area is 309 m 2 g À1 . The XRD analysis was performed to evaluate any crystallinity in the Fe 3 O 4 -MBG NPs. Before immersion in SBF diffractogram shows one broad diffraction halo which conrms its amorphous nature. The apatite formation i.e. in vitro test for bone formation was conrmed by immersing the Fe 3 O 4 -MBG NPs in SBF and Xray diffractogram is presented in Fig. 2(a). These peaks were matched with the JCPDS standard for HA 9-432 and HCA 9-272 which conrms the apatite formation. The intensity and sharpness of the peaks at a 2q value of 26 (002), 32 (211), and 46 (222) represent the bone-forming ability of Fe 3 O 4 -MBG, and therefore, it can be used in bone regeneration and repair. 22,23 Immersion of Fe 3 O 4 -MBG in SBF is useful in vitro test for the conrmation of apatite-forming ability. FTIR spectra of Fe 3 O 4 -MBG before and aer mineralization is shown in Fig. 2(b). The peaks at 1043, 796 cm À1 are associated with the Si-O vibration. Aer immersion in SBF for one week, the vibrational bands of carbonated hydroxyapatite were also observed. The vibrational peaks at 1435 and 854 cm À1 are assigned to C-O vibration bands of carbonate. The twin bands at 603 and 575 cm À1 are assigned to P-O vibration bands of a phosphate group, at 1643 and 3421 are assigned to the O-H group. 24,25 The morphology of the Fe 3 O 4 -MBG was analyzed and the SEM and TEM images are shown in Fig. 3, exhibiting spherical and uniform morphology with a particle size around 50 nm.   Fig. 4(b). It suggests that Mc-Fe 3 O 4 -MBG has signicant antiproliferative effects on MG-63 cells. The effects of Fe 3 O 4 -MBG on normal human broblast (NHFB) cell lines were also examined by MTT assay and no signicant effect on cell proliferation was observed at any concentration which suggests that it is non-toxic, biocompatible, and is safer even at higher concentration proposing to be used for biomedical applications and drug delivery. 26,27 It is interpreted that surface area plays an important role in the release of ions and apatite formation. As the surface area of Fe 3 O 4 -MBG is relatively higher, therefore the rate of dissolution is also higher. Biochemical analysis reveals that osteogenic ability and osteoblast differentiation was investigated by ALP activity, which indicates the onset and initial differentiation of osteoblast cells.
Over time, this activity is diminished showing the onset of mineralization which occurs in later stages of osteoblast differentiation. ALP activity works by modulating the phosphate metabolism during the bone-forming process. ALP activity of cultured osteoblast cells on Fe 3 O 4 -MBG is considerably higher at day 1 and 3 but aer that, the increase in value is very less on the 7 th and 14 th day which advocates the process of bone mineralization. 28 To interpret the mature osteoblast phenotype formation, ALP activity alone is less useful, therefore the level of OC is also taken into consideration. [29][30][31] Osteocalcin (OC) is synthesized in the bone by the osteoblasts and its level reects the rate of bone formation. Precisely it is an indicator of the later stage of osteoblastic activity and illustrates the mature lineage of osteocytes. 32 OC level results in osteoblast differentiation and the osteocytes actively produce mineralized bone tissue. As shown in ESI Fig. S1, † the value of OC is considerably higher on the 7 th day and 14 th day and this higher level of OC in response to Fe 3 O 4 -MBG suggest its characteristic bone-forming ability and potential to be used as the material for bone repairing and regeneration. 33 The Fe 3 O 4 -MBG shows an admirable loading efficiency of 93% for mitomycin C, which is quite higher as compared to some previous carriers. Most of the drug was loaded into the inner pores and some are adsorbed on the outer surface. The drug can also form complex with that of metal ions present in the composition. 34,35 As 93% of drug loading efficiency is very high, therefore it was expected that the drug release will also be higher. The maximum cumulative release of 69.6% was observed for the pH of 6.4 and the lowest release of 42% was observed at a pH of 8.4 as shown in Fig. 5(a). It is hypothesized that there is a strong interaction of the drug with that of glass particles, therefore, the overall release of the drug is lower. Its release rate can be adjusted by changing the pH values. Advantage can be taken from the lower pH of cancer affected body parts and drug is specically released there. This system is an effective drug carrier for curing of tumors and drug release to those parts of the body which are affected by cancer. Maximum release against different pH values is in order 6.4 > 7.4 > 8.4. As the drug delivery is affected by the pH of the release media,  For explaining the surface charges of these NPs, the zeta potential was measured, and the value was found to be À18.3 AE 0.44 mV. This value if helpful as it causes repulsion and longterm stability. [40][41][42] The hyperthermia graph is plotted between time and temperature as shown in Fig. 6(b). Magnetic study shows that Fe 3 O 4 -MBG is superparamagnetic which generates heat due to Brownian and Neel's spin relaxations under the  inuence of the alternating magnetic eld. Fig. 6(b) shows the relationship between time and temperature of the Fe 3 O 4 -MBG solution. Aer keeping the Fe 3 O 4 -MBG solution in an alternating magnetic eld (AMF) for 20 min, the temperature rises from 25 to 43.3 C due to magnetic relaxation loss. Fe 3 O 4 -MBG shows a high heating effect, rise 11.5 C temperature in the rst 6 min which makes it suitable for hyperthermia application. The SAR value of Fe 3 O 4 -MBG is 305.45 W g À1 (Table 1). The high value of SAR, even at a low concentration of magnetite suggests that hyperthermia temperature (>41 C) can easily be achieved within 3 min. 17,18,40 MG-63 cancer cells were kept in an alternating magnetic eld along with Fe 3 O 4 -MGB (25 mg mL À1 ) and the cultured dish was tted in a large size alternating magnetic eld coil with a frequency of 250 kHz for 20 min and cytotoxicity was evaluated by MTT assay. In three separate dishes, these cells were subjected to the control conditions, magnetic eld, and Fe 3 O 4 -MGB (25 mg mL À1 ). MTT assay results in Fig. 6(a) show that most cancer cells were dead at hyperthermia condition whereas, in the absence of AMF, normal cells show high cell viability. These results show that Fe 3 O 4 -MBG is excellent biocompatibility, high hyperthermia temperature, and low cytotoxicity which make it highly effective heat controlled magnetic hyperthermia for cancer treatment. 41,42 Conclusions In this study, a multifunctional magnetic mesoporous bioactive glass was prepared for hyperthermia and controlled drug release of anti-cancer drugs. Magnetite NPs and mesoporous bioactive glass were synthesized to produce (Fe 3 O 4 -MBG) nanoparticles (NPs) of spherical morphology, a uniform pore size of 9 nm, and a surface area of 309 m 2 g À1 . Mitomycin C was loaded to the Fe 3 O 4 -MBG which showed different rate of drug release at different pH values (6.4, 7.4, and 8.4) of the release media. The as-synthesized Fe 3 O 4 -MBG showed no signicant cytotoxicity when subjected to MTT assay by using the NHFB cell line. Aer drug loading, considerable inhibitory effects on the viability of the cancer cells (MG-63) were observed with IC 50 of 12.19 mg mL À1 . Upon immersion in SBF, hydroxycarbonate apatite exhibited the osteogenic ability as supported by XRD and FTIR spectra. Fe 3 O 4 -MBG showed a negligible value of coercivity and zero remanence which conrmed it to be superparamagnetic in behavior. Fe 3 O 4 -MBG showed the heating effect in AMF and rise 11.5 C in temperature in the rst 6 min makes it suitable for hyperthermia application. All the results demonstrated that Fe 3 O 4 -MBG is biocompatible and nontoxic biomaterial which can be used for bone tissue regeneration, targeted drug delivery in chemotherapy, and hyperthermia treatment.

Conflicts of interest
There is no conict of interest to be reported.