Magnetic mesoporous silica nanoparticles coated

Microporous and Mesoporous Materials 256 (2018) 1e9

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Magnetic mesoporous silica nanoparticles coated with thermo-responsive copolymer for potential chemo- and magnetic hyperthermia therapy Zhengfang Tian a, 1, Xia Yu b, 1, Zhijun Ruan a, Min Zhu b, Yufang Zhu a, b, *, Nobutaka Hanagata c a

Hubei Key Laboratory of Processing and Application of Catalytic Materials, College of Chemical Engineering, Huanggang Normal University, No. 146, Xingang 2 Road, Huanggang City, Hubei Province, 438000, China School of Materials Science and Engineering, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China c Nanotechnology Innovation Station, National Institute for Materials Science, 1-2-1 Segen, Tsukuba, Ibaraki 305-0047, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2017 Received in revised form 22 July 2017 Accepted 24 July 2017 Available online 26 July 2017

We developed a potential chemo- and magnetic hyperthermia therapeutic platform based on thermoresponsive copolymer coated magnetic mesoporous silica nanoparticles (MMSN@P(NIPAM-co-MAA)). The structure, magnetic heating capacity, drug release behavior, in vitro cytotoxicity, cell uptake, and synergistic therapeutic efficacy of MMSN@P(NIPAM-co-MAA) nanoparticles were investigated. The prepared superparamagnetic MMSN@P(NIPAM-co-MAA) nanoparticles had an average particle size of 255 ± 28 nm. The saturation magnetization was 6.2 emu/g and resulted in heat generation to hyperthermia temperature under an alternating magnetic field within a short period. MMSN@P(NIPAM-coMAA) nanoparticles could load doxorubicin hydrochloride (DOX), and exhibited temperature- and pHresponsive drug release behavior. Importantly, MMSN@P(NIPAM-co-MAA) nanoparticles had low cytotoxicity and were internalized by HeLa cells. The DOX-loaded nanoparticles showed a synergistic effect that combined chemo- and magnetic hyperthermia therapy, resulting in higher efficacy to kill cancer cells. Thus, MMSN@P(NIPAM-co-MAA) nanoparticles have great potential for cancer therapy. © 2017 Elsevier Inc. All rights reserved.

Keywords: Magnetic mesoporous silica Surface modification Thermo-responsive copolymer Hyperthermia Controlled drug release

1. Introduction Magnetic mesoporous silica nanoparticles (MMSNs) are a promising platform for cancer therapy [1e8]. They have attracted much attention because of their potential in chemotherapeutic drug delivery and hyperthermia therapy under an alternating magnetic field (AMF). Gan et al. [3] fabricated a magnetic and reversible pH-responsive drug delivery system by anchoring Fe3O4 nanoparticles on the pore outlet of MSNs via a reversible boronate esters linker. By alternately changing the pH from 3 to 7, these Fe3O4 cap gates could be switched “on” and “off” to release the entrapped drug in a pulsinate manner [3]. Martín-Saavedra et al. [4] synthesized magnetic g-Fe2O3-encapsulated MMSNs that could

* Corresponding author. Hubei Key Laboratory of Processing and Application of Catalytic Materials, College of Chemical Engineering, Huanggang Normal University, No. 146, Xingang 2 Road, Huanggang City, Hubei Province, 438000, China. E-mail address: [email protected] (Y. Zhu). 1 The first two authors contributed equally to this work. http://dx.doi.org/10.1016/j.micromeso.2017.07.053 1387-1811/© 2017 Elsevier Inc. All rights reserved.

conduct magnetic hyperthermia upon exposure to low frequency AMF. MMSNs were efficiently internalized by human A549, Saos-2, and HepG2 cells. Cell viability dropped as a function of the intensity of the heat treatment achieved by MMSNs and AMF exposures [4]. However, chemotherapy alone results in multidrug resistance of tumor cells and magnetic hyperthermia treatment is effective only at localized sites. Thus, to improve their therapeutic efficacy for cancer therapy, much effort has been made to construct MMSNs-based platforms that combine controlled drug delivery and magnetic hyperthermia [9e17]. This might have synergistic therapeutic effects on tumor cells favoring a reduction of the required effective dose of toxic chemotherapeutic drugs. Benyettou et al. [9] reported a doxorubicin (DOX)-loaded- Pluronic F108-coated system (DOX@F108mNPs) that was stable at room temperature and physiological pH. This system released DOX slowly under acidic conditions or in a sudden burst with magnetic heating [9]. Furthermore, treatment of cervical cancer cells (HeLa) with both Dox@F108-mNPs and subsequent AMF-induced hyperthermia resulted in significantly

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Z. Tian et al. / Microporous and Mesoporous Materials 256 (2018) 1e9

enhanced reduction of cell viability than either DOX or DOX@F108mNP treatment alone. Lu et al. [10] prepared magnetic iron oxideloaded hollow mesoporous silica nanocapsules with a particle size of 100 nm. These magnetic nanocapsules generated heat upon exposure to an AMF and remotely triggered drug release, thus exhibiting great potential for synergistic chemotherapy and magnetic hyperthermia [10]. Additionally, our group prepared MMSNs with controllable magnetization. In an AMF, these MMSNs could controllably generate heat to hyperthermia temperature within a short time, while the DOX-loaded MMSNs exhibited pH-controlled drug release behavior [11e14]. These results suggest the contribution of the magnetic property of MMSNs to the hyperthermia capacity, while the functionalization of MMSNs determined the drug release behavior. Therefore, methods to functionalize MMSNs are key issues for the construction of MMSN-based platforms with better therapeutic efficacy. Temperature is an ideal stimulus for controlling drug release because the local body temperature can change with ambient conditions [18]. Diverse chemical materials such as phase-change molecules, DNA molecules, and thermo-responsive polymers possess temperature-responsive property and are exploited to regulate the release of drug molecules from the carriers [19e26]. Aznar et al. [19] and Liu et al. [20] designed temperature-controlled drug release systems based on MSNs using phase-change molecules as caps. However, only limited phase-change molecules could be used for constructing controlled drug release systems because of biosafety requirements and phase-change temperature within the range of body temperature. Chang et al. [21] and Li et al. [22] developed NIR light-triggered nanocarriers for controlled drug release. In these nanocarriers, drugs were loaded into mesoporous silica coated Au nanorods and capped with DNA. Au nanorods functioned as nanoheaters by absorbing the NIR laser. Yu et al. [23] studied the relationship between the chain length of DNA and the critical stimuli temperature. MSN-based drug delivery system was capped with different lengths of single-stranded DNA oligomers and drug release was triggered by gate opening in response to an increase in temperature. Jiao et al. [24] prepared a copolymer of 2(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA) cross-linked by disulfide bonds (P(MEO2MAeseseOEGMA)) to coat hollow MSNs. The drugs loaded in these nanoparticles were released rapidly at a temperature higher than 37 C [24]. Thus, temperature-controlled drug release systems have great potential for enhancing the efficiency of drug delivery. Poly(N-isopropylacrylamide) (PNIPAM) is the most studied thermo-responsive polymer. It shows a sharp phase transition at its lower critical solution temperature (LCST) of about 32 C in aqueous solution [27]. Below its LCST, PNIPAM exists in a hydrophilic state with a highly hydrated and extended “random coil” conformation. As the temperature increases above its LCST, PNIPAM displays hydrophobic nature with an extensively dehydrated and collapsed “globular” conformation [28]. This is a reversible process and can be used to make the polymer behave as an on-off system when the temperature is changed across the LCST [29e33]. Importantly, the LCST of PNIPAM can be altered by incorporating co-monomer units such as acrylamide and methacrylic acid [34e38]. Shah et al. [34] coated poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-coAm) on MnFe2O4 magnetic nanoparticles. The LCST of this thermoresponsive copolymer was around 39 C when the molar ratio of acrylamide and NIPAM was 1:15 [34]. In poly(N-isopropylacrylamide-co-methacrylic acid) (P(NIPAM-co-MAA)) coated magnetic mesoporous nanoparticles, the LCST of P(NIPAM-coMAA) shifted from 33.9 C to 44.4 C when the MAA content was increased from 0 mol % to 3 mol % [35]. These results suggest the use of PNIPAM-based copolymer coated MMSNs constructs as

promising multifunctional platforms favoring temperature controlled drug release and hyperthermia upon exposure to an AMF. In the present work, we constructed a chemo- and magnetic hyperthermia therapeutic platform comprising of thermoresponsive P(NIPAM-co-MAA) as release “gate-keepers” and MMSNs as drug containers and thermo-seeds. P(NIPAM-co-MAA) copolymer was coated onto the surface of MMSNs via precipitation polymerization of NIPAM and MAA on methacrylate groups modified MMSNs (MMSN-MPS). The model anticancer drug, doxorubicin hydrochloride (DOX), was loaded into MMSN@P(NIPAM-co-MAA) nanoparticles. This was achieved by dispersion of nanoparticles at a greater LCST, causing structural collapse of P(NIPAM-co-MAA) copolymer. When DOX-loaded MMSN@P(NIPAM-co-MAA) (DOX-MMSN@P(NIPAM-co-MAA)) nanoparticles were treated under an AMF, MMSNs could generate heat to hyperthermia temperature, which was higher than the LCST, triggering simultaneous DOX release. Our results indicate the use of MMSN@P(NIPAM-co-MAA) nanoparticles as a promising platform with chemo- and magnetic hyperthermia therapy. 2. Experimental details 2.1. Materials Magnetic Fe3O4 nanoparticles of particle size 15e20 nm were synthesized via co-precipitation method [39]. Tetraethyl orthosilicate (TEOS), triethanolamine (TEA), ethanol, hydrochloric acid (HCl, 36e38%), potassium dihydrogen phosphate (KH2PO4), sodium hydroxide (NaOH), ferric chloride (FeCl3$6H2O), ferrous chloride (FeCl2$4H2O), methacrylic acid (MAA), and potassium persulfate (KPS) were obtained from Sinopharm Chemical Reagent Co., Ltd, (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from Sangon Biotech Co., Ltd, (Shanghai, China). Hexadecyltrimethylammonium p-toluenesulfonate (CTAT), Nisopropylacrylamide (NIPAM), N, N0 -methylenebisacrylamide (MBA), and 3-(trimethoxylsilyl) propyl methacrylate (MPS) were purchased from Sigma-Aldrich. Ultrapure water was obtained from Millipore pure water system (Shanghai, China). 2.2. Preparation of MMSN@P(NIPAM-co-MAA) nanoparticles MMSNs were prepared as previously described [11]. Briefly, 1.0 g of Fe3O4 nanoparticles was dispersed in 90 ml of water. To this, 1.71 g of CTAT and 1.0 g of TEA were added and stirred vigorously at 80 C until they dissolved completely. Subsequently, 14.0 ml of TEOS was rapidly added to the above solution and the mixture was allowed to react for 2 h. Brown colloidal nanoparticles were separated with a magnet, washed several times with ethanol, and dried in vacuum at 60 C for 24 h. Finally, MMSNs were obtained after calcination of dried brown colloidal nanoparticles at 540 C for 7 h. Before coating P(NIPAM-co-MAA) on MMSNs, MPS was used to modify MMSNs with methacrylate groups. Briefly, 500 mg of MMSNs were suspended in 100 ml of anhydrous ethanol by ultrasonication. Then, 2.0 ml of MPS was added to the mixture and stirred for 24 h at room temperature. The suspension was filtered, washed extensively with ethanol to remove the residual MPS, and was dried in vacuum at 60 C for 24 h to obtain MPS-modified MMSNs (MMSN-MPS). Coating of P(NIPAM-co-MAA) on MMSNs was performed by precipitation polymerization method as described previously [35]. Briefly, MMSN-MPS were dispersed in 60 ml of water in a threeneck flask equipped with a reflux condenser. To this, 100 mg of NIPAM, 2.4 mg of MAA, and 7 mg of MBA were added and the mixture was heated to 70 C under a nitrogen atmosphere. After


mechanically stirring for 30 min, 0.5 ml of KPS solution (0.1 mg/ml) was rapidly added and the reaction was allowed to proceed for 7 h. Finally, MMSN@P(NIPAM-co-MAA) nanoparticles were collected by centrifugation and washed several times with water and ethanol to remove unreacted monomers.

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100, and 200 mg/ml of MMSN@P(NIPAM-co-MAA) nanoparticles in a total volume of 100 ml of media per well. After 4 h and 48 h of incubation, 10 ml of CCK-8 solution was added to each well and the cells were further incubated for 3 h. The optical density (OD) was then measured as absorbance at 450 nm using a microplate reader (Bio-Rad 680, California, USA).

2.3. DOX loading and release To evaluate the drug delivery property of MMSN@P(NIPAM-coMAA) nanoparticles, DOX was used as a model drug. DOX was dissolved in PBS at a concentration of 0.5 mg/ml. Sixty milligrams of MMSN@P(NIPAM-co-MAA) nanoparticles were dispersed in 10 ml of DOX solution and stirred at 45 C for 24 h in dark. After the suspension cooled to room temperature, the DOXMMSN@P(NIPAM-co-MAA) nanoparticles were centrifuged and washed with PBS to remove the DOX adsorbed on the surface of the nanoparticles. To estimate the DOX loading capacity, all the supernatants were collected and analyzed by UVevisible spectrophotometer at a wavelength of 481 nm. To investigate the release of DOX from DOX-MMSN@P(NIPAMco-MAA) nanoparticles, release solutions with pH 7.4 or pH 5.0 were prepared. The prepared release solutions were maintained at a temperature of 37 C or 50 C. Briefly, 15 mg of DOXMMSN@P(NIPAM-co-MAA) nanoparticles were dispersed into 1 ml of release solution with continuous shaking at 150 rpm. At specified time intervals, the release system was centrifuged and 20 ml of the supernatant was analyzed by UVevisible spectrophotometer to determine the amount of DOX released. After each analysis, the used supernatant was replaced with the same amount of fresh release solution. Before experimental analysis, a calibration curve was recorded by measuring the absorbance values of DOX at 481 nm. 2.4. Magnetic heating capacity of MMSN@P(NIPAM-co-MAA) nanoparticles The magnetic heating capacity of MMSN@P(NIPAM-co-MAA) nanoparticles was evaluated using a DM100 System (NanoScale Biomagnetics, Spain). MMSN@P(NIPAM-co-MAA) nanoparticles were dispersed in water at a concentration of 10 mg/ml. Subsequently, 1.0 ml of the MMSN@P(NIPAM-co-MAA) suspension was added to the test vessel and fixed under an AMF. The magnetic field strength was set to range from 120 to 180 Gauss and the frequency was fixed at 409 kHz. The specific absorption rate (SAR) was calculated to evaluate the magnetic heating capacity of MMSN@P(NIPAM-co-MAA) nanoparticles. 2.5. Cell culture In the present study, HeLa cell lines were used to test the in vitro cytotoxicity and cellular uptake of MMSN@P(NIPAM-co-MAA) nanoparticles. HeLa cell lines were maintained in MEM medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were cultured in complete medium at 37 C with 5% CO2. 2.6. In vitro cytotoxicity of MMSN@P(NIPAM-co-MAA) nanoparticles In vitro cytotoxicity of MMSN@P(NIPAM-co-MAA) nanoparticles was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan). Before cell seeding, MMSN@P(NIPAM-co-MAA) nanoparticles were dispersed in MEM medium at a concentration of 1 mg/ml. HeLa cells were seeded in 96-well plates at a density of 5 103 cells per well. The cells were incubated with 0, 25, 50, 75,

2.7. Cellular uptake of DOX-MMSN@P(NIPAM-co-MAA) nanoparticles To investigate the cellular uptake of DOX-MMSN@P(NIPAM-coMAA) nanoparticles, 1.0 105 HeLa cells were seeded in a 35-mm Petri dish and cultured for 12 h to promote cell adhesion. After incubation, cells were washed with PBS and 1.0 ml of DOXMMSN@P(NIPAM-co-MAA) suspension in MEM was added to the cells at a concentration of 100 mg/ml. After 4 h of incubation, the cells were washed three times with PBS to remove the remaining nanoparticles and dead cells. The nuclei of HeLa cells were stained with 1.0 ml of methanol solution containing 40 ,6-diamidino-2phenylindole (DAPI, 1.5 mg/ml) for 15 min at 37 C. Finally, cells were washed several times with PBS and observed under a confocal laser-scanning microscope (Leica, SP5, Hamburg, Germany). 2.8. Therapeutic efficacy of DOX-MMSN@P(NIPAM-co-MAA) nanoparticles To investigate if the DOX-MMSN@P(NIPAM-co-MAA) nanoparticles had synergistic therapeutic efficacy of hyperthermia and chemotherapy, an AMF was applied to treat HeLa cells after incubation with the DOX-MMSN@P(NIPAM-co-MAA) nanoparticles. Typically, HeLa cells were seeded in a sample vial at a cell density of 5 104 cells per vial. After 8 h of incubation, the cells were washed with PBS and DOX-MMSN@P(NIPAM-co-MAA) suspension (100 mg/ ml in MEM) was added to the sample vial for cellular uptake. Corresponding concentrations of blank HeLa cells, free DOX solution, and MMSN@P(NIPAM-co-MAA) suspension were treated with the same amount of DOX-MMSN@P(NIPAM-co-MAA) suspension and used as controls. After 8 h of incubation, cells were moved to the DM100 System for treatment under an AMF with 180 Gauss and 409 kHz. After 20 min of treatment, 10 ml of CCK-8 solution was added to the sample vial, and the cells were further incubated for 2 h to measure cell viability. 2.9. Characterization Wide-angle X-ray diffraction (WAXRD) patterns were obtained on a D8 ADVANCE powder diffractometer (Bruker, Germany) using Cu Ka1 radiation (1.5405 Å). Scanning electron microscope (SEM) observations were carried out with a FEI Quanta 450 field emission setup. Transmission electron microscopy (TEM) images were obtained with a Tecnai G2 F30 electron microscope (FEI, Netherlands) operated at an acceleration voltage of 300 kV. The particle sizes were measured by dynamic light scattering (DLS) with a Malvern zeta-sizer Nano-ZS90. UVeVis analysis was performed on a NanoDrop2000C spectrophotometer (Thermo Fisher Scientific, USA). N2 adsorptionedesorption isotherms were obtained on a Micromeritics Tristar 3020 automated surface area and pore size analyzer (Micromeritics) at 77 K under continuous adsorption conditions. BrunauereEmmetteTeller (BET) and BarretteJoynereHalenda (BJH) methods were used to determine the surface area and pore size distribution. Magnetization curves were carried out using LakeShore 7407 vibrating sample magnetometer (VSM, Lake Shore, USA) at 298 K. Fourier transform infrared (FTIR) spectra were recorded on a LAM750(s) spectrometer in transmission mode. Thermogravimetric (TG) analysis was performed on a DMA-8000

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dynamic mechanical thermal analyzer in N2 atmosphere at a flow rate of 20 ml/min and a heating rate of 10 C/min.

3. Results and discussion 3.1. Preparation of MMSN@P(NIPAM-co-MAA) nanoparticles MMSNs were prepared using CTAT as a structure-directing agent as reported previously [11]. As shown in Fig. 1, MMSNs had good dispersity and the particle size was about 100e150 nm. DLS analysis revealed a relatively narrow particle size distribution for MMSNs. The average hydrodynamic particle size was estimated to be 190 ± 8 nm. In this method, a salvation layer formed around the nanoparticles contributed to the larger particle size as compared to that obtained by SEM observation. Additionally, TEM images clearly revealed the presence of mesoporous channels and Fe3O4 nanoparticles in MMSNs, which was in accordance with the previously published data [11]. To coat thermo-responsive P(NIPAM-co-MAA) copolymer on MMSNs, MPS was used to modify MMSNs, leading to the formation of carbon-carbon double bonds on the surface of

MMSNs. This facilitated precipitation copolymerization of NIPAM and MAA. As shown in Fig. 1, although MPS modification did not influence the morphology, particle size, and mesoporous structure of MMSNs, some evident changes were seen on their surface after coating with P(NIPAM-co-MAA) copolymer. Coating of MMSN-MPS nanoparticles with P(NIPAM-co-MAA) led to the presence of a layer covering the surface of nanoparticles. This phenomenon was further confirmed by TEM images wherein, a thin layer could be observed on the outer surface of MMSNs. DLS analysis of MMSN@P(NIPAM-co-MAA) nanoparticles revealed a slightly larger particle size (255 ± 28 nm) compared to MMSNs (190 ± 8 nm) and MMSN-MPS (220 ± 15 nm) nanoparticles. These results are suggestive of the presence of P(NIPAM-co-MAA) copolymer coating on MMSNs. Furthermore, the mesoporous structure was retained in MMSN@P(NIPAM-co-MAA) nanoparticles, thus providing space for drug loading. To further confirm the procedure for preparing MMSN@P(NIPAM-co-MAA) nanoparticles, FTIR spectra, TG analysis, and N2 sorption were performed. Fig. 2 shows the FTIR spectra of MMSNs, MMSN-MPS and MMSN@P(NIPAM-co-MAA)

Fig. 1. Scanning electron microscope (SEM), transmission electron microscope (TEM) images and dynamic light scatting (DLS) particle size distribution of (A1-A3) magnetic mesoporous silica nanoparticles (MMSNs), (B1-B3) methacrylate groups modified MMSNs (MMSN-MPS) and (C1-C3) poly(N-isopropylacrylamide-co-methacrylic acid) coated MMSNs (MMSN@P(NIPAM-co-MAA)) nanoparticles.


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Fig. 4 shows N2 adsorption-desorption isotherms and pore size distribution of MMSNs, MMSN-MPS and MMSN@P(NIPAM-coMAA) nanoparticles. The surface area, pore volume, and pore size of MMSNs were 565 m2/g, 0.57 cm3/g, and 3.4 nm, respectively. After the modification of MMSNs with MPS, the amount of N2 adsorbed decreased significantly and accordingly, the surface area, pore volume, and pore size changed to 212 m2/g, 0.38 cm3/g, and 3.1 nm, respectively. This result confirms the modification of MMSNs by MPS. Furthermore, copolymerization of NIPAM and MAA on MMSN-MPS nanoparticles further decreased the surface area (198 m2/g), pore volume (0.32 cm3/g), and pore size (2.6 nm). This observation confirms the coating of P(NIPAM-co-MAA) copolymer on MMSNs. As expected, the thermo-responsive P(NIPAM-co-MAA) copolymer coating could cap mesopore outlets, thereby controlling the release of anticancer drugs for cancer therapy. 3.2. Magnetic heating capacity of MMSN@P(NIPAM-co-MAA) nanoparticles Fig. 2. Fourier transform infrared (FTIR) spectra of magnetic mesoporous silica nanoparticles (MMSNs), methacrylate groups modified MMSNs (MMSN-MPS), and poly(N-isopropylacrylamide-co-methacrylic acid) coated MMSNs (MMSN@P(NIPAMco-MAA)) nanoparticles.

Fig. 5A shows the magnetization curve of MMSN@P(NIPAM-coMAA) nanoparticles at room temperature. A very small hysteresis loop with a coercivity of 45 Oe and remanence of 0.3 emu/g was observed, suggesting the superparamagnetic behavior of

nanoparticles. Successful modification of MMSNs with MPS was confirmed by the presence of a stretching peak at 1718 cm1 indicating the presence of C¼O groups in MMSN-MPS nanoparticles. For MMSN@P(NIPAM-co-MAA) nanoparticles, characteristic peaks of P(NIPAM-co-MAA) were observed as several vibration peaks at 1542, 1460 cm1, and 1385 cm1, signifying the secondary amide C¼O stretching and deformation of methyl groups on eC(CH3)2. In addition, the shoulder peak of C¼O stretching at 1718 cm1 was attributed to the carboxylic acid groups of MAA in the MMSN@P(NIPAM-co-MAA) nanoparticles, which was consistent with the results reported by Chang et al. [35]. Thus, FTIR spectra indicated successful copolymerization of NIPAM and MAA on MMSNs. TG analysis revealed decrease in weight of MMSN-MPS and MMSN@P(NIPAM-co-MAA) nanoparticles, which was estimated to be about 9.3% and 18.4% from 200 C to 800 C, respectively (Fig. 3). These results suggest the presence of a polymer coating on MMSN@P(NIPAM-co-MAA) nanoparticles.

Fig. 3. Thermogravimetric (TG) analysis of magnetic mesoporous silica nanoparticles (MMSNs), methacrylate groups modified MMSNs (MMSN-MPS), and poly(N-isopropylacrylamide-co-methacrylic acid) coated MMSNs (MMSN@P(NIPAM-co-MAA)) nanoparticles.

Fig. 4. (A) N2 adsorption-desorption isotherms and (B) corresponding pore size distributions of magnetic mesoporous silica nanoparticles (MMSNs), methacrylate groups modified MMSNs (MMSN-MPS), and poly(N-isopropylacrylamide-co-methacrylic acid) coated MMSNs (MMSN@P(NIPAM-co-MAA)) nanoparticles.

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3.3. Drug release from MMSN@P(NIPAM-co-MAA) nanoparticles

Fig. 5. (A) Magnetization curve of poly(N-isopropylacrylamide-co-methacrylic acid) coated magnetic mesoporous silica nanoparticles (MMSN@P(NIPAM-co-MAA)) nanoparticles measured at room temperature. (B) Magnetic heating capacities of MMSN@P(NIPAM-co-MAA) nanoparticles at a concentration of 10 mg/ml under an alternating magnetic field (AMF) with a frequency of 409 kHz and magnetic field strength of 120e180 Gauss.

MMSN@P(NIPAM-co-MAA) nanoparticles. The saturation magnetization value was estimated to be 6.2 emu/g. Encapsulation of magnetic Fe3O4 nanoparticles in MMSNs ensures the generation of potential heat from MMSN@P(NIPAM-co-MAA) nanoparticles under an AMF. Fig. 5B represents the magnetic heating capacities of MMSN@P(NIPAM-co-MAA) nanoparticles at a concentration of 10 mg/ml under an AMF at a frequency of 409 kHz and magnetic field strength of 120e180 Gauss. The temperature of the MMSN@P(NIPAM-co-MAA) solution increases under an AMF and the magnetic heating capacity is enhanced with the increasing magnetic field strength. At a magnetic field strength of 120 Gauss, the temperature increases from 30 C to 44.6 C within 15 min. However, when the magnetic field strength increases to 180 Gauss, the temperature reaches 64.2 C within 15 min. Normally, in magnetic hyperthermia therapy, tumors are heated to a temperature of 43 Ce48 C [40]. Cancer cells are destroyed whereas healthy cells do not undergo non-reversible damage within the range of hyperthermia temperature. Furthermore, magnetic heating zone applied in cancer therapy localizes within the tumor site and experiences rather high temperature hyperthermia, thereby enhancing the therapeutic efficacy [41]. Thus, our results indicate the potential use of MMSN@P(NIPAM-co-MAA) nanoparticles for magnetic hyperthermia.

In the present study, DOX, a model anticancer drug was used to investigate drug release from MMSN@P(NIPAM-co-MAA) nanoparticles. The DOX loading capacity of MMSN@P(NIPAM-co-MAA) nanoparticles was 38.6 mg/mg as analyzed by UVeVis analysis. This was close to that of MMSNs [11e14], suggesting that the space for loading DOX molecules was provided by mesoporous channels. Fig. 6 shows the DOX release profile of DOX-MMSN@P(NIPAM-coMAA) nanoparticles in the release media at pH 7.4 or 5.0 and at a temperature of 37 C or 50 C. For the present analysis, a temperature of 50 C was used to evaluate the possibility of drug release within the hyperthermia temperature range. DOXMMSN@P(NIPAM-co-MAA) nanoparticles exhibited both temperature and pH dependent DOX release behavior. The release of DOX from the DOX-MMSN@P(NIPAM-co-MAA) nanoparticles was very slow in the medium at pH 7.4 (only ca. 9e12% of DOX release in 8 h at 37 C or 50 C) and faster in the medium at pH 5.0. This difference was because of the increase in temperature of the medium at pH 5.0 that accelerated DOX release from the DOXMMSN@P(NIPAM-co-MAA) nanoparticles. The release of DOX from DOX-MMSN@P(NIPAM-co-MAA) nanoparticles was 19% at 37 C and 44% at 50 C within 8 h. Evidently, P(NIPAM-co-MAA) coating plays an important role in the DOX release process. At 37 C (LCST), P(NIPAM-co-MAA) copolymer is in a collapsed, hydrophobic state, resulting in the opening of mesopores and enabling DOX present in the channels to diffuse out. A previous study had demonstrated weakening of electrostatic interaction and hydrogen bonding between DOX and the pore surface with decreasing pH, owing to the protonation of surface groups. Hence, DOX molecules were easily released into the medium at pH 5.0 from DOX-MMSN@P(NIPAMco-MAA) nanoparticles at 50 C. The present study proves that MMSN@P(NIPAM-co-MAA) nanoparticles could generate heat under an AMF to raise the surrounding temperature. When DOXMMSN@P(NIPAM-co-MAA) nanoparticles are internalized by cancer cells along with magnetic heating under an AMF, DOX release is accelerated by the open state of the mesopore outlets and the acidic

Fig. 6. Cumulative doxorubicin hydrochloride (DOX) release from DOX-loaded poly(Nisopropylacrylamide-co-methacrylic acid) coated magnetic mesoporous silica nanoparticles (DOX-MMSN@P(NIPAM-co-MAA)) nanoparticles at different pH and temperature conditions.


environment of the endosome/lysosome and cytosol (pH 5.0e5.5). This enhances the therapeutic efficiency of DOX-MMSN@P(NIPAMco-MAA) nanoparticles. Furthermore, DOX-MMSN@P(NIPAM-coMAA) nanoparticles release very small amounts of DOX into the medium at pH 7.4, thus, lowering the side effect of toxic drug delivery during circulation in the blood stream. 3.4. In vitro cytotoxicity and cellular uptake of MMSN@P(NIPAMco-MAA) nanoparticles Biocompatibility of drug carriers is essential for drug delivery. In this study, in vitro cytotoxicity of MMSN@P(NIPAM-co-MAA) nanoparticles in HeLa cells was evaluated using cell counting kit8 (CCK-8) assay. As shown in Fig. 7, HeLa cells showed low cytotoxicity when incubated with MMSN@P(NIPAM-co-MAA) nanoparticles for 4 h and 48 h, and no significant decrease in cell viability was detected even up to a concentration of 200 mg/ml. He et al. [42] reported low cytotoxicity of MSNs even at a concentration of 500 mg/ml. Previous studies reported biocompatibility between poly(N-isopropylacrylamide) and poly(N-isopropylacrylamide-co-

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acrylic acid) [43e45]. P(NIPAM-co-MAA) coating did not influence the biocompatibility of nanoparticles. These results suggest the use of MMSN@P(NIPAM-co-MAA) nanoparticles as carriers for drug delivery. Cellular uptake of MMSN@P(NIPAM-co-MAA) nanoparticles makes them suitable candidates for local hyperthermia treatment owing to the magnetic heating in cells. In addition, cellular uptake of MMSN@P(NIPAM-co-MAA) nanoparticles enhances the therapeutic efficacy because of enhanced intracellular drug delivery. In this study, DOX-MMSN@P(NIPAM-co-MAA) nanoparticles were incubated with HeLa cells for 4 h and analyzed by confocal microscopy. As shown in Fig. 8, red fluorescence was detected in HeLa cells indicating the presence of DOX-MMSN@P(NIPAM-co-MAA) nanoparticles, suggesting their cellular uptake by endocytosis. However, DOX-MMSN@P(NIPAM-co-MAA) nanoparticles were not internalized into the nucleus. Hence, loading of anticancer drugs in MMSN@P(NIPAM-co-MAA) nanoparticles for cancer therapy would enhance the therapeutic efficiency because of the combined effect of magnetic hyperthermia and controlled drug release. 3.5. Synergistic therapeutic effect of DOX-MMSN@P(NIPAM-coMAA) nanoparticles Synergistic effect of chemo-magnetic hyperthermia therapy was further investigated by quantitatively evaluating the cell viability of HeLa cells subjected to varied treatments, using the CCK-8 assay. Fig. 9 represents cell viability of HeLa cells in the presence or absence of AMF treatment. Magnetic field treatment did not influence cell viability of free HeLa cells. Though free DOX decreased cell viability to 58%, magnetic field treatment did not influence cell viability. As expected, HeLa cells did not show cytotoxicity in the absence of magnetic field treatment when incubated with MMSN@P(NIPAM-co-MAA) nanoparticles. Cell viability decreased to 42% after treatment under an AMF for 20 min proving the magnetic hyperthermia capacity of MMSN@P(NIPAM-co-MAA) nanoparticles to kill cancer cells. Interestingly, when HeLa cells were incubated with DOX-MMSN@P(NIPAM-co-MAA) nanoparticles for 8 h in the absence of magnetic field treatment, cell viability decreased to 76%. This is attributed to the release of DOX triggered by the acidic environment in cells. Cell viability was higher after treatment with DOX-MMSN@P(NIPAM-co-MAA) nanoparticles than when treated with free DOX, suggesting the release of only small quantities of DOX from DOXMMSN@P(NIPAM-co-MAA) nanoparticles in cells. However, when HeLa cells were treated with magnetic field after incubation with DOX-MMSN@P(NIPAM-co-MAA) nanoparticles, there was a significant decrease in cell viability to 23%. Conclusively, these results are suggestive that DOX-MMSN@P(NIPAM-co-MAA) nanoparticles had synergistic effect of chemo- and magnetic hyperthermia therapy. 4. Conclusions

Fig. 7. Effect of different concentrations of poly(N-isopropylacrylamide-co-methacrylic acid) coated magnetic mesoporous silica nanoparticles (MMSN@P(NIPAM-co-MAA)) nanoparticles on cellular cytotoxicity of HeLa cells after (A) 4 h and (B) 48 h of incubation, respectively, as measured by the Cell Counting Kit-8 assay.

In this study, a potential chemo- and magnetic hyperthermia therapeutic platform has been developed by coating thermoresponsive P(NIPAM-co-MAA) copolymer on MMSNs to obtain MMSN@P(NIPAM-co-MAA) nanoparticles. These nanoparticles could generate heat to increase the temperature within the hyperthermia temperature range upon exposure to AMF. DOX was used as a model drug and DOX-MMSN@P(NIPAM-co-MAA) nanoparticles exhibited temperature- and pH-responsive drug release behavior. Both, hyperthermia temperature and low pH environment accelerated DOX release. Cell culture results indicated

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Fig. 8. Confocal laser scanning microscope (CLSM) images of HeLa cells after 4 h of incubation with doxorubicin hydrochloride-loaded poly(N-isopropylacrylamide-co-methacrylic acid) coated magnetic mesoporous silica nanoparticles (DOX-MMSN@P(NIPAM-co-MAA)) nanoparticles: (A) bright field; (B) DAPI channel; (C) DOX channel and (D) merged from bright, DAPI and DOX channels.

hyperthermia, resulting in higher efficacy to kill cancer cells. Therefore, MMSN@P(NIPAM-co-MAA) nanoparticles are a promising platform for cancer therapy. Acknowledgements The authors gratefully acknowledge the support by grants from the National Natural Science Foundation of China (No. 51572172), the Shanghai Science and Technology Commission (NO. 17060502400) and the University of Shanghai for Science and Technology (No. 16KJFZ011, 2017KJFZ010). References

Fig. 9. Cell viability of HeLa cells after 8 h of incubation with free doxorubicin hydrochloride (DOX), poly(N-isopropylacrylamide-co-methacrylic acid) coated magnetic mesoporous silica nanoparticles (MMSN@P(NIPAM-co-MAA)) and DOXMMSN@P(NIPAM-co-MAA) suspensions. (DOX: 3.8 mg/ml, MMSN@P(NIPAM-co-MAA): 100 mg/ml) in the absence and presence of magnetic field treatment (n ¼ 3, *p < 0.05).

negligible cytotoxicity and efficient cellular uptake of MMSN@P(NIPAM-co-MAA) nanoparticles by HeLa cells. Interestingly, compared to free DOX and MMSN@P(NIPAM-co-MAA) nanoparticles, DOX-MMSN@P(NIPAM-co-MAA) nanoparticles exhibited synergistic effect with chemo- and magnetic

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