Silica-modified Fe-doped calcium sulfide nanoparticles for in vitro and in vivo cancer hyperthermia

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J Nanopart Res (2011) 13:1139–1149 DOI 10.1007/s11051-010-0106-0


Silica-modified Fe-doped calcium sulfide nanoparticles for in vitro and in vivo cancer hyperthermia Steven Yueh-Hsiu Wu • Kai-Chiang Yang Ching-Li Tseng • Jung-Chih Chen • Feng-Huei Lin

Received: 9 March 2010 / Accepted: 24 July 2010 / Published online: 9 October 2010 Ó Springer Science+Business Media B.V. 2010

Abstract In this study, sulfide-based magnetic Fe-doped CaS nanoparticles modified with a silica layer were investigated for cancer hyperthermia. A polyvinyl pyrrolidone polymer was used as the coupling agent. The developed nanoparticles contained 11.6 wt% iron concentration, and their X-ray diffraction pattern was similar to those of CaS and Fe–CaS nanoparticles. The average particle size was approximately 47.5 nm and homogeneously dispersed in aqueous solutions. The major absorption bands of silica were observed from the FTIR spectrum. The magnetic properties and heating efficiency were also examined. The specific absorption ratio of nanoparticles at a concentration of 10 mg/mL at 37 °C in an ethanol carrier fluid was 37.92 W/g, and the nanoparticles would raise the temperature to over 45 °C within

S. Y.-H. Wu  K.-C. Yang  J.-C. Chen  F.-H. Lin Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan K.-C. Yang Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan C.-L. Tseng  F.-H. Lin (&) Division of Medical Engineering Research, National Health Research Institutes, Zhunan, Taiwan e-mail: [email protected] J.-C. Chen National Science Council, Taipei, Taiwan, ROC

15 min. A cytotoxicity analysis revealed that the nanoparticles had good biocompatibility, which indicated that the nanoparticles did not affect cell viability. The therapeutic effects of the nanoparticles were investigated using in vitro and animal studies. Cells seeded with nanoparticles and treated under an AC magnetic field revealed a percentage of cytotoxicity (60%) that was significantly higher from that in other groups. In the animal study, during a hyperthermia period of 15 days, tumor-bearing Balb/c mice that were subcutaneously injected with nanoparticles and exposed to an AC magnetic field manifested a reduction in tumor volume. The newly developed silica-modified Fe–CaS nanoparticles can thus be considered a promising and attractive hyperthermia thermoseed. Keywords Calcium sulfide  Iron-doped magnetic nanoparticles  Hyperthermia  Silica  Surface modification  Targeted tumor  Nanomedicine

Introduction In the past few years, magnetic nanoparticles have become an attractive tool in many biomedical applications such as cell separation, magnetofection, drug delivery systems, magnetic resonance imaging (MRI), and magnetic fluid hyperthermia (Won et al. 2005; Harisinghani et al. 2003; Johannsen et al.



2005a, b; Jordan et al. 1999). Among these, tumor hyperthermia therapy is one of the most attractive applications, as it raises the temperature in a targeted tumor area to 41–45 °C in order to reduce the viability of malignant cells without damaging the normal cells, a result that is possible due to the poor thermo-resistance of cancer cells (Pankhurst et al. 2003). Many researchers have carried out in vitro and in vivo experiments for cancer hyperthermia therapy by using magnetic nanoparticles. Iron oxide nanoparticles coated with an aminosilane-type shell were injected intratumorally into rat prostates under an AC magnetic field operating at a frequency of 100 kHz and a variable field strength of 0–18 kA/m (Johannsen et al. 2005a, b). Dextran- or aminosilanecoated iron oxide nanoparticles that were used on glioblastoma multiforme in a rat tumor model have also been reported (Jordan et al. 2006). Resovist magnetic nanoparticles were used to study the survival rates of cells in different conditions and at different temperatures, and localized heating was applied to a mouse colon tumor model (Tseng et al. 2009). Moreover, the application of magnetic hydroxyapatite powders in a localized hyperthermia therapy for murine colon cancer has also been reported (Hou et al. 2009). Although many magnetic nanoparticles have been developed as thermoseeds for magnetic hyperthermia, many of these magnetic nanoparticles have been oxide-based materials (Drake et al. 2007; Wu et al. 2007). Magnetic nanoparticles based on sulfide- or sulfate-based phosphors, which yield good biodegradability or biocompatibility and optimum surface properties for later biomolecule immobilization, have rarely been studied. In our previous study (Wu et al. 2010), Fe-doped CaS (Fe–CaS) nanoparticles that have a high heating efficiency, homogeneous particle size, and good biocompatibility were successfully synthesized using a co-precipitation method. The developed nanoparticles could be stored in an absolute ethanol solution and could also be dispersed by ultrasonication. The problem of aggregation in aqueous solution, however, caused concern among researchers, which limited its further application in the physiological environment. In addition, problems usually arose during the use of magnetic nanoparticles for biomedical purposes. For example, the particles were easily recognized as foreign bodies and removed from blood circulation due to their low


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biocompatibility (Gref et al. 1995). These magnetic nanoparticles were, therefore, stabilized with other materials that would act as a barrier to diminish their toxicity, prevent aggregation, create a surface that offers greater flexibility for specific objectives, and would also enable them to remain in the body for a longer time. To modify the surface, many materials were widely used as a polymeric coating material, such as dextran, polyethylene glycol, polystyrene, amphiphilic block copolymers, and silica (Berry et al. 2003; Gupta and Wells 2004; Xu et al. 2002; Kim et al. 2005; Lu et al. 2002). In this study, the developed Fe–CaS nanoparticles would be surface-modified to bear a hydrophilic polymer chain to prevent particle aggregation. Surface modifications not only improved the particles’ suspensibility but also enhanced their biocompatibility in a biological environment. Furthermore, the modified surface provides more functional groups that can conjugate with specific ligands or biomolecules. Several types of magnetic particles have been coated with a silica layer using the sol–gel method. The layer of coating not only improved their heating efficiency but also improved their suspensibility in aqueous solution (Kaman et al. 2009). A general method for nanoparticles coated with a silica layer has been developed previously (Graf et al. 2006). In this method, the silica layer was coated onto the surface of nanoparticles of various materials such as gold, maghemite, quantum dots, etc., facilitated by a polyvinyl pyrrolidone (PVP) polymer. PVP is an amphiphilic polymer that bonds to the particle surface through their hydrophobic interaction; the hydrophilic part of the polymer would act as nucleation sites for further silica shell growth. In this study, the developed Fe–CaS magnetic nanoparticles were coated with a silica layer by the sol–gel method, and PVP polymer was used as a coupling agent; the PVP polymer adsorbed onto the particle surfaces, and hence, the surfaces were further covered with a silica shell through hydrolysis and the condensation mechanism of tetraethyl orthosilicate (TEOS). The crystal structure of modified Fe–CaS nanoparticles was investigated using an X-ray diffractometer (XRD). The core–shell morphology of magnetic nanoparticles was observed using a transmission electron microscope (TEM). Fourier transform infrared spectroscopy (FTIR) was used to identify their surface properties. Their magnetic properties were

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measured by a superconducting quantum interference devices (SQUID) magnetometer. The calorimetric heating experiments were carried out with an AC magnetic field at a frequency of 750 kHz and a field strength of 10 Oe. The material’s mediated cytotoxicity was tested by 3T3 fibroblasts using lactose dehydrogenase (LDH) assays. To clarify their modification ability, the organic dye fluorescein isothiocyanate (FITC) was further immobilized onto the surface of SiO2-modified Fe–CaS nanoparticles (Fe–CaS–SiO2) with the help of 3-aminopropyltrimethoxysilane (APS) and was examined under a UV–Vis spectrometer. The in vitro cell hyperthermia experiment utilized CT-26 colon cancer cells which were treated with Fe–CaS–SiO2 nanoparticles and an AC magnetic field, and which were compared with other control groups to evaluate their heating efficiency. In the animal study, Balb/c mice bearing colon tumors were treated with Fe–CaS–SiO2 nanoparticles and a magnetic field. Their temperature profiles and variations in tumor size were recorded to estimate the therapeutic effect during a treatment period of 15 days.


Another precursor solution consisting of iron and calcium ions was prepared in another three-neck flask. Superfluous FeCl24H2O (0.087 g) and CaCl2 (0.2447 g) (with an Fe/Ca molar ratio of 0.2) were dissolved in 100 mL of absolute ethanol. The previous two solutions were individually deoxygenated by nitrogen gas at a flow rate of 200 mL/min to maintain a reductive atmosphere. The sulfide precursor solution was added drop-wise into the iron/calcium precursor solution and vigorously stirred for 3 h to allow for nucleation and nanocrystal growth. The excess ethanol was removed by evaporation at 70 °C until the mixture was a slurry. The slurry was then added to 100 mL of THF solution for Fe–CaS precipitation. The precipitate was collected by centrifugation at 3,000 rpm for 10 min, and was further washed with absolute ethanol several times to remove any residual THF solution. It was then freeze-dried overnight. The collected precipitate was soaked at 800 °C in a platinum crucible under a nitrogen atmosphere for 1 h and was then gradually cooled to 200 °C to prevent CaSO4 formation. Silica modification of Fe–CaS nanoparticles

Materials and methods

Synthesis of silica-modified Fe–CaS nanoparticles (Fe–CaS–SiO2)

Reagent and chemicals Tetraethoxysilane (TEOS, 98%, #131903), polyvinylpyrrolidone (PVP-10, average molecular weight 10 kg/mol), 3-aminopropyl-trimethoxysilane (APTS, 97%, #281778), fluorescein isothiocyanate (FITC, 90%, #F3651), ammonia hydroxide (28 wt% NH3 in H2O, #380539), ammonium iron sulfate (#F3754), and tetrahydrofuran solution (THF, #34865) were purchased from Sigma-Aldrich. Absolute ethanol (99.5%) was obtained from Nihon Shiyaku (Japan). All chemicals were used as received without further treatment. Synthesis of Fe–CaS nanoparticles Fe–CaS nanoparticles were synthesized by a wetchemical process. Briefly, two precursor solutions were prepared separately. First, a sulfide precursor solution with excess Na2S9H2O (0.3002 g) was dispersed in 100 mL of absolute ethanol in a threeneck flask followed by sonication for 30 min.

Fe–CaS–SiO2 nanoparticles were prepared according to the well-established Sto¨ber method (Sto¨ber et al. 1968) with the help of PVP-10 as a surface stabilizing agent. Fe–CaS nanoparticles at a concentration of 0.5 mg/mL were dispersed in 10 mL of absolute ethanol and then ultrasonicated for 10 min. 110 mg/mL of PVP-10 was dissolved in 2.5 mL of absolute ethanol and then sonicated for 30 min. The PVP-10 and Fe–CaS nanoparticles solutions were mixed and stirred for 24 h at room temperature at 600 rpm to permit the adsorption of PVP-10 onto the surface of the nanoparticles. The PVP-10-adsorbed Fe–CaS nanoparticle solutions were washed with water/acetone mixed solutions (w/w 1/10) several times and centrifuged to remove excess PVP-10. The PVP-10adsorbed Fe–CaS nanoparticles were re-dispersed in 10 mL of absolute ethanol followed by the addition of 1 mL of ammonium hydroxide. After the addition of ammonium hydroxide for 10 min, 100 lL of TEOS solution was added and stirred at 600 rpm for another 24 h. The silica-modified Fe–CaS nanoparticles were



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washed with absolute ethanol and collected by centrifugation at 3,000 rpm. The prepared silica-modified Fe–CaS nanoparticles were then stored in absolute ethanol solutions for later use.

nanoparticles were used in each scan. The applied magnetic field was in the interval from 10-4 to 104 Oe for measurement of the hysteresis loop. The M–H curves were in units of emu/g versus Oe.

Preparation of FITC-conjugated Fe–CaS–SiO2 (Fe–CaS–SiO2-FITC)

Measurement of iron concentration

FITC was first conjugated to APTS (to become so-called FITC-APTS) as follows: FITC (0.025 mmol) and APS (0.25 mmol) were added to 10 mL of ethanol and stirred at 600 rpm in darkness for 24 h. 0.5 mg/mL of Fe–CaS–SiO2 nanoparticles were dispersed in 5 mL of absolute ethanol and then thoroughly mixed with 100 lL FITC-APTS conjugates, 100 lL ammonia hydroxide, and 100 lL TEOS for 24 h in dark. The synthesized Fe–CaS– SiO2-FITC nanoparticles were washed with absolute ethanol to remove any residual FITC. Characterization of Fe–CaS–SiO2 nanoparticles X-ray diffraction X-ray diffraction patterns were recorded with an X-ray diffractometer (Rigaku, Rint-2200, Japan) in the 2h range from 20° to 70° at a rate of 4°/min under ˚ ) as 20 Am and 30 kV by using Cu Ka (k = 1.5406 A the source of radiation. The nanoparticles were packed into a sample holder, and data were collected using the continuous scan mode for crystal identification and the measurement of average grain size. FTIR analysis FTIR was used to characterize the functional groups on the synthesized Fe–CaS and Fe–CaS–SiO2 nanoparticles. Absorption spectra were recorded (FTIR, JASCO 410, Japan) from 4,000 to 400 cm-1 with 4 cm-1 resolution and 32 scans of each scan. Dried nanoparticles were mixed with potassium bromide (KBr) and compressed into pellets. Superconducting Quantum Interference Devices (SQUIDs) The magnetization data were determined using a SQUID magnetometer (MPMS XL-7, Quantum Design, USA) at 300 K in DC mode. 10 mg of


The Fe–CaS–SiO2 nanoparticles were placed in a preweighed glass vial and lyophilized overnight. The iron content of the nanoparticles was determined by an 1,10-phenanthroline colorimetric assay (Jain et al. 2008; Mykhaylyk et al. 2007). The absorption spectrum was measured at 510 nm using a UV–Vis spectrophotometer (V-550, Jasco, Japan). Cytotoxicity Cytotoxicity was evaluated using a lactate dehydrogenase assay (CytoTox 96Ò Assay, Promega, USA). The NIH-3T3 cells were seeded in 96-well plates at a density of 5 9 103 cells/well. After 24 h incubation, the cells were incubated with different concentrations of Fe–CaS–SiO2 (0.1–2 mg/mL). Cells incubated in a medium without Fe–CaS–SiO2 were used as the control group, and Triton-X 100 (total lysis) was adopted as the positive control. After 1 and 3 days of cultivation, the culture medium was assayed by the LDH assay kit according to the manufacturer’s protocol. In brief, 50 lL of the medium was transferred to a new enzymatic assay plate in which 50 lL of the LDH substrate solution was added and kept in the dark. After 30 min, 50 lL of stop solution was added to each well in the plate and read on microplate reader spectrophotometer (SpectraMAX M2; Molecular Devices, USA) at 490 nm optical density (OD). All experiments were repeated 6 times for statistical analysis. In vitro heating using Fe–CaS–SiO2 nanoparticles CT-26 colon carcinoma cells (1 9 105 cells/mL) were cultured in a glass vial with 500 lL of fresh medium (RPMI-1640) for 24 h. The medium was then replaced by a fresh supplement with a concentration of 2.5 mg/mL Fe–CaS–SiO2 nanoparticles and then incubated at 37 °C for 1 h. The cells were then inserted into a water-cooled copper coil (25 mm in diameter) to provide an external AC magnetic field at a frequency of 750 kHz and a field strength of 10 Oe.

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Cells were then kept in a temperature range 43–47 °C for 15 min. The supernatant medium was then collected, and its hyperthermia-mediated cytotoxicity was evaluated by an LDH assay. The optical density (O.D.) value from CT-26 cells in spontaneous release served as the control group, CT-26 cells incubated with a medium containing 15% lysis buffer was the positive control group, and the experimental groups were cells incubated with Fe–CaS–SiO2 nanoparticles that were treated with and without a magnetic field. The percentage of cytotoxicity was calculated using the following equation: Cytotoxicity ð%Þ ¼

experiment  control  100 positive control  control

Fig. 1 XRD patterns of (a) Fe–CaS and (b) Fe–CaS–SiO2 nanoparticles

In vivo heating using Fe–CaS–SiO2 nanoparticles Twelve female Balb/c mice were purchased from an animal culture center (National Taiwan University Hospital, Taipei, Taiwan) and were maintained in accordance with the institute’s guidelines. 7 9 106 colon cancer cells (CT-26) were injected subcutaneously into the dorsum of the mice, and tumors were allowed to grow to a size of 8 9 10 mm2, which took about 10 days in which there was no other treatment. After this period, 300–500 lL of Fe–CaS–SiO2 nanoparticles dispersed in normal saline (150 mg/mL) were injected into the tumor tissue area of the experiment group (n = 6) through a syringe needle (24-gauge). The control group (n = 6) was not treated with the developed nanoparticles. All the mice were treated with an oscillation magnetic field (with a frequency of 750 kHz and a field strength of 10 Oe) immediately after receiving injections. Thermotherapy was applied for the first 2 days, after which it was given every other day until day 15. The temperatures of the mice’s tumors and rectums were recorded during this period.

Results The XRD pattern of Fe–CaS is shown in Fig. 1a. The major peaks of Fe–CaS indicated by 27.3, 31.7, 45.4, 56.4, and 66.1 were assigned to the (111), (200), (220), (222), and (400) families, respectively. The XRD pattern of Fe–CaS–SiO2 was similar to that of Fe–CaS (Fig. 1b). The XRD result showed that the

Fig. 2 FTIR spectrum of Fe–CaS–SiO2 nanoparticles

silica layer on the surface of Fe–CaS did not affect the crystal structure of the Fe–CaS cores. The FTIR spectrum of Fe–CaS–SiO2 is shown in Fig. 2. The absorption bands attributed to TEOS were traced from the pattern, such as the –O–Si–O– vibrational modes of the deformation at 461 cm-1, the SiO4 tetrahedron ring structure at 800 cm-1, the SiOH stretching at 956 cm-1, and the –Si–O–Si– stretching vibration centered at 1,085 cm-1. The bands at 570 and 1,649 cm-1 can be attributed to C–C and C=O bonding, respectively. The peak around 1,450–1,490 cm-1 can be attributed to the C–H bond. The broad peak in the range 2,800–3,800 cm-1 can be attributed to the hydroxyl group. These results indicate that the silica was formed onto the surface of Fe–CaS nanoparticles.



Figure 3a is a TEM (H-7100 Hitachi Instruments, Tokyo, Japan) picture of developed Fe–CaS–SiO2. The core–shell structures are clearly visible where a thin layer with greater translucency covers the outside of the developed nanoparticles. The nanoparticles displayed a quite homogeneous and uniform particle size with a narrow size distribution. The average size of the particles was about 47.5 nm, which is larger than the particle size of Fe–CaS nanoparticles (35.5 nm) (Wu et al. 2010). This larger particle size could be attributed to the thickness of the absorbed PVP-10 and to the polymerized SiO2 shell. From an EDS analysis (Fig. 3b), Fe, Ca, S, O, and Si could be traced on the surface of Fe–CaS–SiO2 nanoparticles. The high intensity of Si and O was due to the SiO2 layer. To examine the iron concentration in the Fe–CaS and Fe–CaS–SiO2 nanoparticles,

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ammonium iron sulfate in a concentrated range 0.5–6 g Fe/mL was used as the standard material. The iron content in Fe–CaS and Fe–CaS–SiO2 was 31.4 and 11.6 wt%, respectively. The magnetic properties of Fe–CaS and Fe–CaS– SiO2 were evaluated by their M–H curves, which are presented in Fig. 4. Figure 4a shows the M–H curve of Fe–CaS. Its saturated magnetization was 6.47 (emu/g) and the hysteresis loop area was 3,553 (emu g-1 Oe). The saturated magnetization and hysteresis loop area of Fe–CaS–SiO2, on the contrary, decreased to 2.13 (emu/g) and 72 (emu g-1 Oe), respectively, as shown in Fig. 4b. This reduction in magnetization and hysteresis loop area could be attributed to the diamagnetic PVP-10 and silica layer surrounding the magnetic cores. The decreased weight ratio of Fe–CaS in the developed Fe–CaS–SiO2 nanoparticles was another key factor in these two areas of reduction. The heating profiles of Fe–CaS–SiO2 nanoparticles with concentrations of 5 and 10 mg/mL in ethanol solution are shown in Fig. 5. The particles treated with an oscillation magnetic field could generate enough energy to heat the temperature to over 45 °C within 15 min. The curve fitting could be obtained from the exponential decay equations that were used to calculate the specific absorption ratio (SAR) values (Fortin et al. 2008). SAR ¼

C dT yFe dt

where the specific heat of ethanol solution C is 2.44 J g-1 K-1, and yFe is the mass ratio of magnetic

Fig. 3 a TEM image of Fe–CaS–SiO2 nanoparticles and b the corresponded EDS pattern


Fig. 4 M–H curves of (a) Fe–CaS and (b) Fe–CaS–SiO2 nanoparticles

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Fig. 5 Heat profiles of Fe–CaS–SiO2 nanoparticles at concentrations of 5 and 10 mg/mL

Fig. 6 Cytotoxicity analysis by LDH assay at day 3 (one-way ANOVA, mean ± SD, n = 6)

core iron to ethanol solution, which is 1.47 9 10-3 for 10 mg/mL. The SAR value of 10 mg/mL is expressed as 131:46et=t0 , where t0 is the initial time (230 s). At body temperature (37 °C and t = 185), the SAR value is about 37.92 W/g. The cytotoxicity of Fe–CaS–SiO2 nanoparticles was evaluated by LDH assays. Figure 6 shows the results of LDH analysis for different concentrations of Fe–CaS–SiO2, which was conducted to check the cell survival rate on day 1 and day 3 (day 1 not shown). There were no statistical differences among all the test groups and the control group. We also checked the cell viability by the results of WST-1 test for different concentrations of Fe–CaS–SiO2, and there appeared no significant differences among all the experimental groups and the control group (data not shown). From the results of the LDH and WST-1


Fig. 7 Absorption spectra of (a) Fe–CaS–SiO2-FITC and (b) Fe–CaS–SiO2 nanoparticles

Fig. 8 LDH assay of CT-26 cells treated with Fe–CaS–SiO2 nanoparticles at concentration of 2.5 mg/mL (one-way ANOVA, mean ± SD, n = 6, * p \ 0.05: significantly different)

analysis tests, the developed Fe–CaS–SiO2 caused no harm to the 3T3 cells. After the developed nanoparticles conjugated to FITC, the absorption spectrum of the nanoparticles was analyzed with a UV–Vis spectrometer. The samples were thoroughly washed to remove any excess and free molecules before the analysis. The peak located at 504 nm was contributed by the FITC conjugation (Fig. 7a); such a peak was absent in the absorption spectrum of the Fe–CaS–SiO2 nanoparticles (Fig. 7b). The study of in vitro cell hyperthermia was analyzed by an LDH assay, as shown in Fig. 8. The CT-26 colon cancer cells treated with Fe–CaS–SiO2 nanoparticles and an external AC magnetic field



displayed a high O.D. value. The O.D. value in the control group and other experiment groups was much lower than in the group of CT-26 treated with Fe–CaS–SiO2 to which an external magnetic field was applied. In the animal study, the Balb/c mice were fixed inside a 50-mL centrifuge tube that was put into a coil. The tumor area and rectum were connected with a fiber thermometer for temperature detection. None of the experimental mice were anesthetized to avoid any possible interference from an anesthetic. The temperature around the tumor area (Fig. 9a) was raised to 44 °C within 7 min and then kept constant in the range 45–47 °C. During this time, the mice’s body temperature (Fig. 9b) increased just slightly but did not exceed 39 °C. Figure 10 shows the variations in tumor size that occurred during the period of hyperthermia for both the experiment group and the control group. The tumor size for the experiment group was slightly reduced after 1 day of treatment, then gradually shrank and formed black scar tissue on the tumor site beginning on day 3. This scar tissue enlarged from day 3–5 and became hard and flat, and in some cases became detached from the back from day 13–15. For the control group, the tumors displayed continual growth and necrosis developed in the center of the tumor interior. In the control group that was not treated with Fe–CaS–SiO2 nanoparticles, the tumor size became as large as 1.4 ± 0.14 cm3 in 15 days, as shown in Fig. 11.

Fig. 9 Temperature recordings of (a) tumor area and (b) mice rectum during the hyperthermia treatment


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Fig. 10 Tumor size variations of the control and experiment groups after thermotherapy

Fig. 11 Photographs of the tumor size of the a Fe–CaS–SiO2 nanoparticles induced group and the b control group

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Discussion The Fe–CaS nanoparticles with a high heating efficiency, homogeneous particle size, and good biocompatibility were synthesized based upon a previous study. However, the particles easily formed aggregates in aqueous solution and displayed a lack of useful functional groups on the surface, which made them difficult to use in biological applications. Although ultrasonication could be used to temporarily disperse the nanoparticles in an aqueous solution, the sedimentation rate would still be too high to prevent aggregation. The modification of their surface to not only overcome the above-mentioned problems, but to also create functional groups such as amine, to further conjugate with biomolecules, would greatly improve their applicability to the life sciences. Particularly in cancer hyperthermia, nanoparticles need to specifically target the tumor site after long-term transport in the blood circulatory system which delivers them into the cell body. The many functions involved in this process all rely upon the interactions between cell surface receptors and the immobilized functional domains on the nanoparticles. The crystal structure of Fe–CaS completely matched that of JCPDS card No. 080-464, which was identified as CaS. The crystal structure of Fe–CaS–SiO2 nanoparticles was similar to that of Fe–CaS, which was not changed by the silica layer. Although the intensity of all its peaks decreased slightly, as shown in Fig. 1, the location of the major peaks still matched that of JCPDS card No. 08-0464. It has been reported that amphiphilic, nonionic polymer PVP can be easily adsorbed to the surface of metals, metal oxides, silica, cellulose, and polystyrene (Graf et al. 2003). It is widely used as a coupling agent for surface modification. The surface of Fe–CaS nanoparticles, however, provided no useful functional groups for surface modification. The adsorbed PVP polymer was used for TEOS adsorption for silica layer development in the surface modification and was further linked with APS to provide an amino group for biomolecule conjugation. The silica layer precursor, TEOS, was hydrolyzed with the help of catalytic agent NH3OH to form a hydrophilic hydroxyl group. After this, the TEOS formed a –Si–O–Si– silane network by means of the Sto¨ber sol–gel process. The adsorbed site of the PVP polymer in solution could be due to the negatively


charged carbonyl group in the resonance structure of the pyrene ring, and it is suggested that the interaction between silica and PVP might occur through the weak hydrogen bond due to the carbonyl group of PVP and the silanol group of hydrolyzed TEOS that followed the acid–base route (Pattanaik and Bhaumik 2000). As seen in Fig. 2, the absorption band C=O and C–C belongs to the PVP polymer. The four bands at 1085, 956, 800, and 461 cm-1 were assigned to the –Si–O–Si–, Si–OH, SiO4, and –O–Si–O–, respectively, which resulted from the silane structure, and which were also shown in the FTIR spectrum. We believe that Fe–CaS was successfully silanized through the Sto¨ber sol–gel process. Many parameters affect the magnetic property and size of the hysteresis loop of nanoparticles, including size, shape, and defects in the crystal structure (Raming et al. 2002). Apart from these, the weight ratio of iron in nanoparticles might exert the greatest influence on the size of the hysteresis loop. As shown in Fig. 4, the size of the hysteresis loop of Fe–CaS– SiO2 nanoparticles was smaller than that of Fe–CaS, due to the iron content in Fe–CaS (0.314 g Fe/g) being much higher than it is in Fe–CaS–SiO2 (0.116 g Fe/g), as shown in the results of the iron concentration analysis. The low magnetization of Fe–CaS–SiO2 could also take into account the lower density of the ferromagnetic component (Fe–CaS) and the presence of an extensive part of the nonmagnetic silica layer (He et al. 2007). The magnetization of Fe–CaS–SiO2 was similar to that of Fe–CaS when it was expressed in terms of emu per gram of Fe–CaS content. The ferromagnetic/nonmagnetic weight ratio exerted a direct influence upon the heating efficiency and SAR of the nanoparticles. The weight percent of silica could be adjusted by the amount of TEOS precursor in order to manipulate the thickness of the silica layer and control the particle size (Lu et al. 2002; Yoon et al. 2005). Based on the iron ratio in the nanoparticles, the heating efficiency and SAR of Fe–CaS–SiO2 should be three times lower than that of Fe–CaS. However, the SAR values of the Fe–CaS nanoparticles and Fe–CaS–SiO2 nanoparticles were 45.47 and 37.92 W/g, respectively, which does not indicate such a great difference. We believe that the Brownian rotation due to the hydrophilic surface of the silica layer might be attributed to an increased SAR in Fe–CaS–SiO2 nanoparticles. The Brownian rotation in a specific



fluid could compensate for a decreased SAR caused by the lower iron ratio of the nanoparticles. It has been reported that only 3% of CT-26 cells survived at 45 and 46 °C after hyperthermia, 26% survived at 42 °C, and only 2% survived when the temperature was maintained at 47 °C (Tseng et al. 2009). As shown in the experimental data in Fig. 5, the nanoparticles could raise the temperature to over 45 °C within 15 min at a concentration of 10 mg/mL and to over 43 °C within 15 min at 5 mg/mL, which indicates that Fe–CaS–SiO2 nanoparticles can effectively reduce the survival rate of CT-26 cancer cells. The Fe–CaS–SiO2 nanoparticles could be further modified with other functional groups that increase the possibility of biomolecule conjugation. Organic dye FITC, for example, could be immobilized by the developed nanoparticles. The surface of Fe–CaS–SiO2 nanoparticles was conjugated with FITC through the interaction of an amino group on APS and of a carboxylic group on FITC. From analysis of the UV–Vis spectrum (Fig. 7), the peak located around 504 nm was identified as the emission band of FITC, which is indicative of successful modification. Many biomolecules in addition to FITC have also been immobilized onto the surface of silica-coated magnetic nanoparticles, such as poly(ethylene glycol), rhodamine B, iridium complexes, etc. (Yoon et al. 2005; Ren et al. 2007; Lai et al. 2008), which greatly extended their application in many areas of biomedicine. As shown in Fig. 8, the percentage of cytotoxicity of the group (cell-treated particles and field) was found to be 60%, which was significantly higher than in the other experiment groups (whose percentages were less than 5%). The Fe–CaS–SiO2 nanoparticles showed effective heating results. Generally, for the in vitro experiment, cells were usually incubated with magnetic nanoparticles at a concentration of several milligrams. The uptake of nanoparticles mostly involved several pictograms per cell, which could provide part of the heat energy for intracellular heating (Prasad et al. 2007). Moreover, the extracellular hyperthermia that was caused mainly by Brownian rotation and hysteresis loss mechanisms could also generate effective heat and play a vital role that contributed to cell necrosis or apoptosis. In the animal study, the mice’s core body temperature reached only to about 39 °C and was not affected by the magnetic heating, as is shown in Fig. 9. Localized tumor hyperthermia could be


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achieved during the treatment. The volume of the tumors in the control group grew almost 3.5 times greater than their initial size. Tumors that were not injected with magnetic nanoparticles did not generate heat. The central necrosis, as shown in Fig. 11b, was commonly due to poor nutrition supply to the fast growing tumor. In the hyperthermia treatment group, the volume of the tumors shrank significantly during the period of treatment and black, hard scar tissue formed over an area that was larger than the size of the tumor. We expected that the Fe–CaS–SiO2 nanoparticles in the injection site would gradually extend to the entire area of the tumor and spread to the other part of the dorsum. The migration of the nanoparticles away from the tumor site might have resulted in their spreading with a concentration gradient, which contributed to the generation of heat that charred the surrounding tissue.

Conclusions In conclusion, Fe–CaS nanoparticles were successfully synthesized and modified with a silica layer with the help of PVP polymer using the Sto¨ber sol–gel method. The developed nanoparticles could disperse in an aqueous solution and had a homogeneous particle size. SQUID and SAR analysis confirmed the effectiveness of their magnetic properties. Their heating efficiency in in vitro and in vivo experiments was satisfactory. The biocompatibility of the nanoparticles was good, as they exhibited low cytotoxicity in LDH assay. The conjugation of Fe–CaS nanoparticles with FITC was achieved, which indicated their capacity to immobilize numerous biomolecules. In addition, the results of an animal study revealed that these nanoparticles induced a significant reduction in tumor volume in mice. These combined results demonstrate that the developed nanoparticles can be regarded as a suitable material for a hyperthermia thermoseed.

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