Fabrication of Silica-Coated Hollow Carbon Nanospheres

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Fabrication of Silica-Coated Hollow Carbon Nanospheres Encapsulating Fe3O4 Cluster for Magnetical and MR Imaging Guided NIR Light Triggering Hyperthermia and US Imaging Yun-Kai Huang, Chia-Hao Su, Jiu-Jeng Chen, Chun-Ting Chang, YuHsin Tsai, Sheng-Fu Syu, Tsu-Ting Tseng, and Chen-Sheng Yeh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04759 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016

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ACS Applied Materials & Interfaces

Fabrication of Silica-Coated Hollow Carbon Nanospheres Encapsulating Fe3O4 Cluster for Magnetical and MR Imaging Guided NIR Light Triggering Hyperthermia and US Imaging Yun-Kai Huang1, Chia-Hao Su2, Jiu-Jeng Chen1, Chun-Ting Chang1, Yu-Hsin Tsai1, Sheng-Fu Syu1, Tsu-Ting Tseng1, Chen-Sheng Yeh1,* 1

Department of Chemistry and Advanced Optoelectronic Technology Center, National Cheng

Kung University, Tainan 701, Taiwan 2

Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial

Hospital, Kaohsiung 833, Taiwan Keywords: yolk-shell, carbon, near-infrared, magnetism, ultrasound ABSTRACT

Iron oxide nanoparticles (IONPs)-carbon (C) hybrid zero dimensional nanostructures normally can be categorized into core-shell and yolk-shell architectures. Although IONP-C is a promising theranostic nanoagent, the in vivo study has surprisingly been less described. In addition, little effort has strived toward the fabrication of yolk-shell compared to the core-shell

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structures. In this context, we synthesized a yolk-shell type of the silica-coated hollow carbon nanospheres encapsulating IONPs cluster, which can be dispersed in aqueous solution for systemic studies

in

vivo,

via

the preparation

involving the

mixed

micellization,

polymerization/hollowing, sol-gel (hydration-condensation) and pyrolysis processes. Through a surface modification of the polyethylenimine followed by the sol-gel process, the silica shell coating was able to escape from condensing and sintering courses resulting in aggregation, due to the annealing. Not limited to the well-known functionalities in magnetical targeting and magnetic resonance (MR) imaging for IONP-C hybrid structures, we have expanded this yolkshell NPs as a near-infrared (NIR) light-responsive echogenic nanoagent giving an enhanced ultrasound imaging. Overall, we have fabricated the NIR sensitive yolk-shell IONP-C to activate ultrasound imaging and photothermal ablation under magnetically and MR imaging guided therapy.

Introduction The emerging one dimensional nanotubes and two dimensional graphene oxides have shed light on the applications in all aspect of fields including nanomedicine. These graphitic nanomaterials have given the capability for photo-activated therapy against malignant tumors as one of the important therapeutic treatments in biomedical applications because of their absorption in near-infrared (NIR) area.

1-8

Adjunct magnetic iron oxide nanoparticles (IONPs),

Fe3O4, provide adjuvant functionalities to implement magnetical targeting as well as the magnetic resonance (MR) imaging guided therapy. IONP-carbon (C) hybrid zero dimensional nanostructures are normally aware of core-shell and yolk-shell architectures. Among those structures, little effort has put into the fabrication of yolk-shell relative to the core-shell structures.

8-17

Although IONP-C is a prevalent hybrid nanostructure and can be developed to a


2

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promising theranostic nanoagent, the in vivo study has surprisingly been sketchily described. Until recently, the aggregated core-shell Fe3O4@C nanospheres with size of ~850 nm was intratumoral injection of adenocarcinoma A549 tumors to conduct photothermal ablation.8 Herein, we synthesized a silica-coated hollow carbon nanospheres encapsulating IONPs cluster (a yolk-shell structure denoted as SiO2/h-Fe3O4@C) to conduct magnetically and MR imaging guided NIR light activating photothermal hyperthermia and ultrasound (US) imaging in the systemic study. Not limited to the well-known functionalities in magnetic targeting and MR imaging, we have expanded this yolk-shell structure as a NIR-responsive echogenic nanoagent giving an enhanced ultrasound imaging. The presence of the void of the SiO2/h-Fe3O4@C has allowed us to load the low boiling point of perfluorohexane, affixing the visualization of the tumor US imaging. The heating effect derived from SiO2/h-Fe3O4@C when exposed to NIR light irradiation vaporized the perfluorohexane to afford echogenic source for US. In the course of this yolk-shell structure preparation, we have found that the pyrolysis process to form carbon shell was readily to create aggregated nanospheres. Through a surface modification of the polyethylenimine (PEI) followed by the sol-gel process, the silica shell coating on h-Fe3O4@C was able to escape from condensing and sintering courses resulting in aggregation, due to the annealing (Figure 1a). The dispersed yolk-shell nanospheres in aqueous solution were eligible for biomedical studies in vivo. Among the reported approaches, the occurrence of the aggregation has been commonly observed for the annealed C nanospheres and was highly depended on experimental conditions.

5-6,8-10,14,17-20

It might be the reason that the resulting

aggregation might have delayed the development of the IONP-C hybrid nanostructures applied for in vivo studies. Results and Discussion


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Synthesis and characterization of SiO2/h-Fe3O4@C Fe3O4 nanoparticles (NPs) (~20 nm) were synthesized following our previous method

21

with slight modification (Figure 1b). Sodium oleate (SO) and poly(ethylene glycol)-blockpoly(propylene glycol)-block-poly(ethylene glycol) (P123, Mn~5800) were used as double surfactants to form mixed micelles through the hydrophobic interaction 22. In consequence, Fe3O4 NPs were then blended into an SO/P123 aqueous solution to form micelles. Next, this solution was mixed with an acidic 2,4-dihydroxybenzoic acid (DA)/hexamethylenetetramine (HMT) solution to produce an emulsion. Through dynamic light scattering (DLS), the droplet size of the emulsion was determined to be approximately 150 nm. Under a hydrothermal condition (150 oC), HMT would decompose to formaldehyde and ammonium. Meanwhile, the formaldehyde product polymerized with DA on the surface of the emulsion droplets

22

, yielding a hollow polymer

sphere containing Fe3O4 particles (h-Fe3O4@P) with average size of ~138 nm (Figure 1c). The hollow structure of the nanosphere was formed under the elevated temperature. The TEM image illustrates that the nanospheres feature a yolk-shell structure with a thickness of ~22 nm containing several Fe3O4 NPs aggregated in a form of cluster. If the h-Fe3O4@P was directly used to calcine at 500°C in argon, this carbonization produced a highly aggregated hollow carbon spheres (h-Fe3O4@C), as seen by a TEM image (Figure S1). The carbon shells were seriously fused. Thus, before pyrolysis, polyethylenimine (PEI) was adsorbed onto the surface of h-Fe3O4@P to positively charge its surface, followed by employing a sol-gel approach to form a silica shell coating on the surface of the sphere (Figure 1d). To evidence the SiO2 shell, the polymer layer of the SiO2 coated h-Fe3O4@P (SiO2/h-Fe3O4@P) was removed through aerobic pyrolysis to leave the SiO2 layer intact, which was verified to be approximately ~6 nm in thickness (Figure S2); the SiO2/h-Fe3O4@P NPs were ~134 nm in size, and the SiO2/h-Fe3O4@P


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shell was ~20.8 nm in thickness. A slight decrease in size from h-Fe3O4@P (138 nm) to SiO2/hFe3O4@P (134 nm) was observed. SiO2/h-Fe3O4@C was calcined under Ar at 650 oC to derive evenly dispersed NPs (Figure 1e), and as this TEM image shows, the nanoparticles (SiO2/hFe3O4@C) were ~113 nm in size and ~18 nm in thickness. After carbonization, the decrease in particle size was assumed to have resulted from the removal of certain functional groups from the polymer when the particles underwent pyrolysis

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. A high resolution transmission electron

microscopy (HRTEM) analysis (Figure 2a) of the Fe3O4 core of the NPs enabled observing the (220) and (533) planes of Fe3O4 (Figure 2b), indicating that Fe3O4 remained within the core. The HRTEM image of the carbon shell characterized the shell as an amorphous carbon structure (Figure 2c), as further evidenced by X-ray diffraction (XRD) (Figure 3b). The mapping images of the SiO2/h-Fe3O4@C showed the presence of the C, O, Si, and Fe elements (Figures 2d–h). A high-angle annular dark-field imaging (HAADF) was also performed to confirm a yolk-shell structure (Figure S3). We have performed pyrolysis at various temperatures, 500°C, 650°C, and 800°C, and found that SiO2/h-Fe3O4@C, which was obtained under pyrolysis at 650°C, revealed absorbance at near-infrared (NIR) wavelengths more effectively than did that obtained under pyrolysis at 500°C (Figure 3a). As Figure 3a displays, when the pyrolysis temperature increased from 500°C to 650°C, the yellowish-brown surface of SiO2/h-Fe3O4@C became black, indicating the recovery of the conjugated π network graphite 2. In addition, if the pyrolysis temperature increased to 800°C, then the carbon (002) plane could be observed through XRD, suggesting a high crystallinity of this carbon layer. However, Fe3O4 NPs were no longer to retain inside the hollow carbon spheres, as seen in the inset TEM image of Figure 3b. It seems that the Fe3O4 NPs have gone through a dissolution process and infiltrated out of carbon shell to recrystallize and


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attached outside the carbon layer. We therefore conducted pyrolysis at 650°C for the further experiments. We undertook a series of characterization on SiO2/h-Fe3O4@C. FT-IR spectra revealed weakened signals from several functional groups of the calcined SiO2/h-Fe3O4@C, indicating that some functional groups were removed from the material during the pyrolysis (Figure S4a). However, the Si-O-Si stretching signal appeared at 1110 cm-1, suggesting that the SiO2 shell layer remained during the pyrolysis from SiO2/h-Fe3O4@P to SiO2/h-Fe3O4@C. The Raman spectrum of SiO2/h-Fe3O4@C reveals the G-band and D-band at 1592 and 1320 cm−1, respectively (Figure S4b), confirming the presence of Csp2 and Csp3 in SiO2/h-Fe3O4@C. Their presence suggested the existence of graphitic carbon (G-band) and amorphous (partially hydrogenated) carbons (D-band) within the structure of the sphere, 24 as evidenced by a small CH stretching signal that occurred at 2900 cm−1 in the IR spectrum (Figure S4a). Magnetisms measured with a superconducting quantum interference device, SiO2/h-Fe3O4@C exhibited a higher saturated magnetic magnetization (142 emu/g[Fe]) than did the carbon shell-free Fe3O4 (114 emu/g[Fe]) (Figure S4c). This might be attributable to how Fe3O4 NPs aggregated in a form of cluster within the carbon spheres. Both structures exhibited the superparamagnetic property. A Brunauer–Emmett–Teller (BET) analysis yielded a type IV hysteresis loop, and the pore surface area and pore size of SiO2/h-Fe3O4@C were 117.8 m2 g-1 and 3.8 nm, respectively (Figure S4d). In addition, a broad band ranging from 40-80 nm was seen and reflected to the interior cavity of ~77 nm as determined by TEM for SiO2/h-Fe3O4@C. The BET confirmed the porous nature of the SiO2/h-Fe3O4@C prepared in this study, which can be loaded with various substances. To conduct in vitro and in vivo experiments, we treated the surface of SiO2/h-Fe3O4@C with polyethylene glycol (PEG) through the EDC/NHS method to increase the biocompatibility


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of the material. In order to modify PEG, APTES was firstly conjugated on surface, giving a 1622 cm-1 N-H signal (Figure S5a). The surface charge of the particles changed from -25.8 mV to +24.1 mV because of the presence of NH2 groups (Figure S5b). Subsequently, the –COOH end group of PEG was used to form amide bond with –NH2 of APTES and the other terminus –NH2 was exposed outward for SiO2/h-Fe3O4@C. PEG modified NPs displayed positively +22.3 mV. FT-IR spectrum revealed that the PEG coated SiO2/h-Fe3O4@C exhibited a C-O stretching signal overlapping with that of Si-O at 1110 cm-1, but an additional bump C-H signal was observed at 2870 cm-1 (Figure S5b), indicating the attachment of PEG to the surfaces of the particles. The success of this surface modification was further verified by the concurrently increased particle sizes, which were observed through DLS (Figure S5c). The stability illustrated that PEG coated SiO2/h-Fe3O4@C remained stable in various solutions such as water, PBS with a pH of 7.0, PBS with a pH of 5.0, cell culture Dulbecco’s Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS); these mixtures were observed for a week. PBS with a pH of 5.0 was used to emulate the endosomal and lysosomal environment in the cells, and PBS with a pH of 7.0 was used to emulate the environment outside the cells. As seen in Figure S6, the morphology of the PEG coated SiO2/h-Fe3O4@C NPs did not change for 7 days, indicating that they exhibit extremely high structural stability for at least 7 days. Photothermal efficacy in cells for SiO2/h-Fe3O4@C Because we would like to take the advantage of its NIR absorbance, particularly at 808 nm for NIR triggering hyperthermia and US imaging under an 808-nm diode laser irradiation, molar extinction coefficient (ε808nm) based on iron concentration at 808 nm was determined as 1616.6 M−1cm−1 for PEG coated SiO2/h-Fe3O4@C, which is equivalent to a mass extinction coefficient of 28.9 Lg−1cm−1 (Figure 4a); hence, its mass extinction coefficient is more favorable


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than those of reduced graphene oxide (~20 nm in size) (24.6 Lg−1cm−1) and gold nanorods (13.9 Lg−1cm−1).2 An 808-nm diode laser at different output powers was used to irradiate PEG coated SiO2/h-Fe3O4@C with a Fe concentration of 100 ppm and to monitor temperature increase in the solutions (Figure 4b). We found that the temperature increased with increasing output power of the laser and used 0.8 W cm-2, at which the end temperature rose to approximately 60 oC after 10 min exposure, as a condition for in vitro and in vivo experiments. Pure water without NPs receiving laser irradiation only reached 29 oC after 10 min exposure. MTT assay was used for examining cytotoxicity to identify the biological compatibility of the PEG coated SiO2/hFe3O4@C NPs. HeLa cell line (human cervical cancer cell line) was examined as a model. Incubation the cells for 24 h at 37 °C observed that the survival rate of the HeLa cells was nearly at 100%, even after the iron concentration reached 500 ppm (Figure 4c), showing high biocompatibility. Because the NPs contain iron oxide, an external magnet may be added to increase the material dosage accumulation within the cells through magnetic attraction. Therefore, the Cytoviva dark field and fluorescence images were performed for the cells treated PEG coated SiO2/h-Fe3O4@C NPs with or without magnet. For magnetic attraction, the magent was applied for 5 min and then NPs with HeLa cells were incubated with additonal 2 h. Without magnet, NPs purely incubated with cells for 2 h. Under magnetism, this characteristic enabled PEG coated SiO2/h-Fe3O4@C to accumulate rapidly on the surface of a cell to increase the cell’s uptake compared to the nil magnet group observed from dark field mode (Figure S7). In vitro photothermal efficacy in cell viability indicated that the significant poisoning effect was observed when HeLa cells treated with PEG coated SiO2/h-Fe3O4@C received 808-nm diode laser irradiation for 10 min under magnetism (5 min) resulting in survival rate down to 55% (Figure 4d) that is consistent with the observation of the enhanced red fluorescence of the dead


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HeLa cells (Figure 4e). Following the aforementioned process, cells received laser ablation after 2 h incubation with or without magnet. MR imaging behavior of SiO2/h-Fe3O4@C containing PFH The Fe3O4 NPs enveloped in PEG coated SiO2/h-Fe3O4@C are superparamagnetic and can be applied to conduct magnetic resonance (MR) imaging guided targeting. The hollow cavity enabled us to load hydrophobic perfluorohexane (PFH) to expand PEG coated SiO2/h-Fe3O4@C with additional functionality as echogenic source for US imaging. US provides the real-time monitoring treatments with noninvasive and painless procedure to patients and has been extensively employed for disease diagnosis. The loaded amount of PFH was measured to ~0.26 mmole/mg per particle with the encapsulation efficiency as ~2.6%. PEG coated SiO2/hFe3O4@C containing PFH NPs showed high compatibility when incubated with HeLa cells for 24 h (Figure S8). The relaxivity values (r2) were evaluated using 1.4T Minispec Contrast Agent Analyzer mq60 system for both PEG coated SiO2/h-Fe3O4@C and PEG coated SiO2/h-Fe3O4@C containing PFH in 1.5% agarose gel at various iron ion concentrations and determined as 424.7 and 202.7 mM−1sec−1, respectively (Figure 5a and b). The liquid PFH loaded into the cavity showed the effect for the water accessibility toward Fe3O4 NPs resulting in the decrease of the relaxivity. However, the resulting PEG coated SiO2/h-Fe3O4@C containing PFH still has better relaxivity compared to commercial MRI contrast agent (r2=98.3 mM-1sec-1 for Feridex and 151.0 mM-1sec-1 for Resovist. 25 Next, the PEG coated SiO2/h-Fe3O4@C containing PFH materials (10 mg kg-1) were injected intravenously through the tail vein into living mice to investigate its accumulation on tumor treated with and without magnetism. A T2 contrast images depict the changes in the tumor over time (Figure 5c). Specifically, 1 h after the injection, the signal reached the most darkened, suggesting that PEG coated SiO2/h-Fe3O4@C containing PFH had its


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greatest accumulation 60 min after administration. Therefore, we used the 1-h timescale as a temporal parameter for the in vivo treatments that followed. For magnetic attraction, the magnet was applied for 30 min after intravenous injection and then waited for additional 30 min representing post-injection 1 h, and so forth for post-injection 2 h, 3 h, and 24 h. In addition, as Figures 5d and e illustrate, when treated with magnetism, the tumor shown in the T2 image became more blackened than the liver did, whereas the liver that were treated without magnetism were more blackened. These results indicate that the material could be effectively drawn by magnetism to accumulate on its target tumor.

Photothermal efficacy in vivo for SiO2/h-Fe3O4@C containing PFH To perform NIR-responsive hyperthermia in vivo, phosphate-buffered saline (PBS) and PEG coated SiO2/h-Fe3O4@C containing PFH (20 mg kg-1) were injected intravenously through the tail vein into the mice. Consistent with the aforementioned MRI-guided treatment approach, the tumor in the mice were treated 1 h after administration for laser irradiation, and the changes in the tumors treated with and without magnetism were compared. As Figure 6a and b suggests, the tumors treated with PEG coated SiO2/h-Fe3O4@C containing PFH, magnetism, and an 808nm laser (0.8 W cm-2 in 10 min exposure) were eradicated completely after post-injection 20 days compared to those treated only with PBS and a laser or with PEG coated SiO2/h-Fe3O4@C containing PFH and a laser. These results supported the effectiveness of the MRI-guided photothermal therapy proposed in this study.

Echogenic behavior of SiO2/h-Fe3O4@C containing PFH


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Another important demonstration is to transform NP into a NIR-sensitive echogenic nanoagent. PFH is a low boiling point liquid (~58 oC) and can be readily vaporized to provide the echogenic source when the NIR laser irradiates the PEG coated SiO2/h-Fe3O4@C containing PFH generating heat. The PFH-loaded particles were placed onto a microscope slide, and under the irradiation of an 808-nm diode laser beam at an output power of 0.8 W cm-2, bubbles were observed (Figure 7a–d). These bubbles reflected the vaporization of PFH, which resulted from the photothermal-induced-hyperthermia of the particles. Longer laser exposure period led to more bubbles yielded. Next, a micro-ultrasonic device (Visual Sonics, 40 MHz, B-mode) was employed to investigate the US imaging of PEG coated SiO2/h-Fe3O4@C containing PFH (0.5 mg mL-1), which was injected into agarose gel. When the material was irradiated using the 808nm laser (0.8 W cm-2 in 10 min exposure), the B-mode images captured during and after laser irradiation displayed the significantly enhanced white spots (Figure 7e and g) and the signal remained for 30 min. On the contrary, the group received no laser irradiation can still observe the US phantom because of the high impedance mismatch between solid and US transparent agarose gel, 26-29 but the corresponding grey-scale signal continued to drop and cannot last for 30 min. The bright spots were likely to be bubbles resulting from PFH vaporization, indicating the material’s potential for US contrast agent applications. A further observation of the ultrasonic images of the material containing PFH (20 mg kg-1) in living mice receiving an intratumoral injection showed the same trend upon laser irradiation. However, no US signal was detected for the groups from either PEG coated SiO2/h-Fe3O4@C containing PFH (no laser) or PEG coated SiO2/h-Fe3O4@C + laser (Figure 7f and h). For the mice administrated through a tail vein injection under magnetism, the image of the tumor was clearly enhanced after laser irradiation as well (Figure 8). Considering the NIR light triggering echogenicity in tumors, our design was able


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to last longer echogenic signal up to 30 min after receiving laser irradiation as compared to the reported results that only presented US signal for 12 min when using PLGA NPs to contain PFH.30

Toxicity of SiO2/h-Fe3O4@C containing PFH The toxicity of PEG coated SiO2/h-Fe3O4@C containing PFH to biological organisms was found that judging from the mice body weight, hematological analysis of liver and kidneys, and histological results revealed no apparent toxicity up to 20 days (Figures S9-S11). The biodistribution suggests that the Fe concentrations in the organs of the PEG coated SiO2/hFe3O4@C containing PFH-treated mice were comparable to those in the organs of the PBStreated mice 20 days after administration (Figure S12). This indicates that all the particles might have been vented or degraded and confirmed the nontoxicity of PEG coated SiO2/h-Fe3O4@C containing PFH.

Conclusion We have successfully synthesized a SiO2 coated hollow carbon nanosphere encapsulating Fe3O4 NPs. Coating with a SiO2 layer could endure high temperature pyrolysis to escape from aggregating that has endowed the promising potential for systemic studies. The resulting NPs possessed the appreciable absorption coefficient at 808 nm in the NIR region, giving the effective photothermal conversion for killing of cancer cells upon an 808-nm diode laser irradiation. Moreover, coupled with magnetism and MR contrast imaging, the NPs provided a


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targeted therapy for tumors in a living organism. Importantly, the cavities of the particles could encapsulate hydrophobic and highly volatile PFH for the expansion of the functionality with additional US imaging. Overall, we have firstly presented the NIR-responsive yolk-shell carbon nanospheres and applied for in vivo hyperthermia coupled with dual MR and US imaging.

Experimental Procedures Materials: All reagents were of analytical purity and used without further purification. Iron (III) acetylacetonate (Fe(C5H7O2)3, 99.9%), trioctylamine (TOA, [CH3(CH2)7]3N,98%), oleic

acid

(OA,

CH3(CH2)7CH=CH(CH2)7COOH,

90%),

sodium

oleate

(SO,

CH3(CH2)7CH=CH(CH2)7COONa, 99%), perfluorohexane (PFH, CF3(CF2)4CF3, 99%), Nhydroxysulfosuccinimide sodium salt (NHS, C4H4NNaO6S, 98%), and 3-(4,5-dime-thylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT, C18H16BrN5S, 97.5%) were used as purchased from Sigma-Aldrich. Hexamethylenetetramine (HMT, C6H12N4, 99%), polyethyleneimine branched (PEI, 30%, MW=70000), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, C8H17N3HCl, 98%) were obtained from Alfa Aesar. Tetraethyl orthosilicate (TEOS, 98%) and (3-aminopropyl)-triethoxysilane (APTES, 99%) were bought from Acros. Poly(ethylene

glycol)-block-poly(propylene

glycol)-block-poly(ethylene

glycol)

(P123,

Mn~5800), and 2,4-dihydroxybenzoic acid (DA, C7H6O4,97%) were purchased from Aldrich. H2N-PEG-COOH (PEG, MW=3400) was acquired from Nanocs. Ammonia solution (NH3(aq), 28~30%) was obtained from Showa. Toluene was purchased from Macron. Hydrochloric acid (HCl, 36%) and nitric acid (HNO3, 70%) was bought from BASF. Ethanol (EtOH, 99.9%) was purchased from J.T.Baker. Dulbecco's modified eagle medium (DMEM, high glucose, pyruvate),


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antibiotic-antimycotic (PS), non-essential amino acids solution (NEAA), and 0.25% trypsinEDTA were obtained from Gibco. Fetal bovine serum (FBS) was used as purchased from HyClone. Calcein, AM (C46H46N2O23) was acquired from Invitrogen. Propidium iodide (PI, C27H34I2N4) was bought from BD Biosciences.Water was obtained by using a Millipore direct-Q deionized water system throughout all studies. Characterization: Morphology of nanoparticles (NPs) was characterized by a transmission electron microscopy (TEM, Hitachi H-7500). High resolution TEM image and elemental analysis of the NPs were observed on a high-resolution transmission electron microscopy (HR-TEM, JEOL JEM-2100F at 200 KV). Concentrations of the NPs were quantified by an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Jobin Yvon JY138 Spectroanalzer). Zeta potentials and hydrodynamic diameters of NPs were measured by a dynamic light scattering spectrometer (DLS, Malvern, UK Zetasizer Nano ZS90). UV−vis spectra were recorded on the UV−vis spectroscopy system (Agilent 8453). X-ray diffraction patterns of NPs were obtained by a X-ray diffractometer (XRD, Shimasz Cu Kα radiation (λ = 1.5418 Å, 30 kV, 30 mA). Porous properties of NPs were characterized by Micromeritics ASAP 2020 surface area and pore size analyzer. Magnetic properties of the NPs were detected by a superconducting quantum interference device vibrating sample magnetometer (SQUID, Quantum Design MPMS). T2 relaxation rates were measured by the 1.4T Minispec Contrast Agent Analyzer mq60 system (Bruker Optik GmbH, Germany). The quantification of cell viability was done using an enzyme-linked immunosorbent assay reader (ELISA reader, Thermo Scientific Multiskan EX). Fluorescence images were taken by an inverted routine microscope (Nikon, ECLIPSE TS-100). FT-IR spectra of the polymersomes was measured by


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Fourier transform infrared spectrometer (FT-IR, JASCO 200E). Raman spectra was measured by Microscopes Raman Spectrometer (Renishaw) Preparation of Fe3O4 NPs: The Fe3O4 NPs were prepared following our previous study with slight modification. In brief, iron(III) acetylacetonate (1.42 g) was mixed with oleic acid (0.55 mL) and trioctylamine (20 mL) in a two neck flask. To remove water at 150 oC for 30 min and degas at 120 oC for 30 min, the solution was heated to 305 oC and kept at this temperature for 30 min. Collected the particles with magnet and centrifuged when the solution was cooled down, and washed with toluene for three times. Finally, the Fe3O4 particles were dried at room temperature and stored for next step. Preparation of h-Fe3O4@P NPs: As prepared Fe3O4 (160 mg) was dispersed in toluene, and then washed with ethanol for two times. Water (40 mL) with dissolved SO (0.146 g) and P123 (0.087 g) was mixed with Fe3O4. After Fe3O4 was dispersed in SO/P123 solution, aqueous solution (120 mL) containing DA (0.37 g) and HMT (0.28 g) was added. The mixed solution was sealed in Teflon-lined stainless steel autoclave to heat at 150 oC with rising rate of 1 oC min-1 and kept at this temperature for 2 h. The resulting hollow polymer sphere containing Fe3O4 particles (h-Fe3O4@P) NPs were collected and washed by water for 3 times. SiO2 coated h-Fe3O4@P (SiO2/h-Fe3O4@P) NPs: The resulting h-Fe3O4@P NPs were dispersed in water (120 mL) mixed with 0.3% PEI (12 mL). After shaking for 4 h, particles were centrifuged and washed with water, and then re-dispersed in H2O (300 mL). NH4OH (3.2 mL) and TEOS (2mL) were added to the solution under vigorous shaking for 24 h. Finally, particles were collected and washed by 1:1 EtOH:DDW solution for twice, followed by 99% EtOH rinsed for twice. SiO2/h-Fe3O4@P NPs were dried in vacuum for further use.


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Preparation of SiO2/h-Fe3O4@C NPs: Dried SiO2/h-Fe3O4@P NPs were heated under Ar atmosphere. The pyrolysis steps were set as following (step 1 → step 2); step 1: heated to 350 oC with heating rate 5 oC min-1 and kept for 2 h. step 2: heated to 650 oC with heating rate 2 oC min-1 and kept for 6 h. For the other parallel experiments, the pyrolysis was operated at either 500 or 800 oC in step 2. The SiO2/h-Fe3O4@C NPs were washed by 1:1 EtOH:DDW solution for twice and then 99% EtOH rinsed for twice. PEG Surface modification of SiO2/h-Fe3O4@C: SiO2/h-Fe3O4@C (2 mg) was dispersed in EtOH (10 mL) and added APTES (300 µL). After shaking for 18 h, the solution was washed by EtOH and then re-dispersed in PBS buffer (pH 7) (2 mL). 10 mM EDC/water (100 µL), 10 mM NHS/water (100 µL) and 10 mM PEG/water (100 µL) were added. After 4 h incubation, the PEG coated SiO2/h-Fe3O4@C NPs were washed with water and stored for further use. Encapsulating of PFH in PEG coated SiO2/h-Fe3O4@C: PEG coated SiO2/h-Fe3O4@C (1 mg) was mixed with PFH liquid (2 mL) and sonicated in ice bath for 30 min. Particles was separated from the residual PFH with magnet and then re-dispersed in water. After centrifugation and sonication in room temperature for three times, particles were re-dispersed in water and stored at 4 oC. In vitro temperature elevation experiments: PEG coated SiO2/h-Fe3O4@C (100 µL) in water was added to 96-well culture plate. An 808 nm diode laser with different power was used to irradiate solutions. The temperature was measured by the thermocouple (TES 1319A-K type). Cell culture and cytotoxicity experiments: HeLa cells (human cervical cancer cell lines) were cultured in medium of DMEM containing 0.1 mM NEAA, 1% penicillin/streptomycin (PS), and 10% FBS in the incubator at 37 °C and 5% CO2. 8×103 cells/well were cultured in 96-


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well culture plate for 24 h. PEG coated SiO2/h-Fe3O4@C containing PFH (100 µL) with different concentration was added to cells with medium and incubated for 24 h. After that, MTT assay was used to determine cell viability. In vitro cellular uptake experiments: 5×103 HeLa cells/well were cultured in 96-well culture plate for 24 h, followed by treatment of PEG coated SiO2/h-Fe3O4@C (100 or 200 ppm of Fe concentration) with or without magnetic attraction for 5 min. Cells were further incubate for 2 h and then washed with PBS for three times. Finally, aqua regia was added to dissolve NPs and Fe concentration was determined by ICP-AES. For the Cytoviva imaging, 5×103 Hela cells/well were cultured in 8-well chamber slides for 24 h, followed by treatment of PEG coated SiO2/h-Fe3O4@C with or without magnetic attraction for 5 min. Cells were further incubated for 2 h and then washed with PBS for three times. After staining with Hoechst and Alexa Fluor 488 phalloidin, the chamber slides were sealed and observed by Cytoviva spectrometer under dark field and fluorescence imaging. In vitro cell viability with laser irradiation: 8×103 HeLa cells/well were cultured in 96well culture plate for 24 h, followed by treatment of PEG coated SiO2/h-Fe3O4@C (200 ppm of Fe concentration) with or without magnetic attraction for 5 min and then incubated for 2 h. After washing by PBS and adding fresh medium, cells were irradiated with 808 nm diode laser (0.8 W cm-2) for 10 min. Cell viability were determined by MTT assay. For the cell fluorescence image, 5×104 HeLa cells/well were cultured in 8-well chamber slides for 24 h, and followed by the above-mentioned processes. Live and dead cells were stained with Calcein AM and propidium iodide, respectively, and observed with fluorescence microscopy.


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In vivo evaluation of magnetic resonance image: All animal treatments and surgical procedures were performed in accordance with the guidelines of Chang Gung Memorial Hospital Laboratory Animal Center (Kaohsiung, Taiwan). All animals received humane care in compliance with the institution’s guidelines for maintenance and use of laboratory animals in research. All of the experimental protocols involving live animals were reviewed and approved by the Animal Experimentation Committee of Chang Gung Memorial Hospital Laboratory Animal Center. The diagnosis efficacy of PEG coated SiO2/h-Fe3O4@C containing PFH was evaluated using nu/nu nude mice, which was prepared by implanting subcutaneously the suspension of 8×106 HeLa cancer cells in medium (100 µL) into the dorsal flank of mice (6-8 weeks old; 17-19 g). After 8 days of tumor xenografts, the tumor volume was approximately 180-230 mm3, and the tumor-bearing mice were ready for studies. On the treatment, PEG coated SiO2/h-Fe3O4@C containing PFH were administrated by the intravenously injection at a dosage of 10 mg kg−1. The external magnetic field was applied for 30 min after injection (without magnet as sham control). The tumor-bearing mice were anesthetized using 2% isoflurane (Abbott Laboratories, Abbott Park, IL) mixed with 100% O2 delivered using a veterinary anesthesia delivery system (ADS 1000; Engler). Sequential MRI acquisitions were performed at a 9.4 T MR imager (Bruker BioSpec 94/20 USR) equipped with a high-performance transmitter-receiver RF volume coil. For T2-weighted imaging, T2-weighted coronal anatomic reference imaging were recorded using multislice turbo rapid acquisition with refocusing echoes (Turbo-RARE) sequence acquisition at pre-injection, 30 min, 1 h, 2 h, 3h, and 24h post-injection (with/without magnetic guiding) with the following parameters: field of view (FOV) for coronal imaging= 45.0×45.0 mm; matrix size = 256×256; spatial resolution = 0.176×0.176 µm; slice thickness = 1.0 mm; effective echo time


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(TE) = 28.0 ms; echo time = 9.333 ms; repetition time (TR) = 3500 ms; rare factor = 8; refocusing flip angle = 180 deg; number of averages = 5; number of repetitions (NR) = 1; total acquisition time = 9 min 20 s. In addition, the axial imaging with the following parameters: field of view (FOV) = 35.0×35.0 mm; matrix size = 256×256; spatial resolution = 0.137×0.137 µm; slice thickness = 1.0 mm; effective echo time (TE) = 28.0 ms; echo time = 9.333 ms; repetition time (TR) = 3500 ms; rare factor = 8; refocusing flip angle = 180 deg; number of averages = 5; number of repetitions (NR) = 1; total acquisition time = 9 min 20 s. The MR imaging signal intensities were measured using Image J 1.49 software. In vivo antitumor efficacy of PEG coated SiO2/h-Fe3O4@C containing PFH NPs: All animal treatments and surgical procedures were performed in accordance with the guidelines of National Cheng Kung University (NCKU) Laboratory Animal Center (Tainan, Taiwan). All animals received humane care in compliance with NCKU guidelines for the maintenance and use of laboratory animals in research. All of the experimental protocols involving live animals were reviewed and approved by the Animal Experimentation Committee of NCKU. The antitumor efficacy of PEG coated SiO2/h-Fe3O4@C containing PFH NPs was evaluated using nude mice (BALB/cAnN), which was established by implanting subcutaneously the suspension of 2×106 HeLa cancer cells in medium (100 µL) into the dorsal flank of mice (4 weeks old; 16-20 g). After 10 days of tumor xenografts, the tumor volume was approximately 20-60 mm3, and the tumorbearing mice were ready for studies. The tumor size was measured along the longest width and the corresponding perpendicular length. The tumor volume was calculated using the volume of an ellipsoid, where volume = 4π/3 (length/2×width/2×depth/2). This study assumed that depth = width and π= 3, resulting in volume = 1/2×length×(width)2. For the experimental details, all mice were divided into three


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groups. PBS or PEG coated-SiO2/h-C@Fe3O4 containing PFH NPs were administrated by intravenous injection at a dosage of 20 mg kg-1. The external magnetic field was applied for the group of PBS + laser and PEG coated-SiO2/h-Fe3O4@C containing PFH NPs + laser for 30 min after injection. The control experiment was done for PEG coated SiO2/h-Fe3O4@C containing PFH without magnetic attraction for laser exposure. 0.8 W cm-2 of 808 nm laser power was applied for all groups for 10 min irradiation with beam covering whole tumor region. The tumor size was observed every two days. In vitro and in vivo ultrasound imaging: For in vitro image, 3% agarose gel was prepared in 12-well cultural plate with an Eppendorf tube placed at the center of well in order to create a void where the solution was loaded. The solidified agarose gel was removed from the well and loaded with PEG coated SiO2/h-Fe3O4@C containing PFH colloids (0.5 mg mL-1). A transparent plastic wrap was covered on the top of the solution. Subsequently, a 0.8W cm-2 of 808 nm laser irradiated from the top of the void agarose gel with PEG coated SiO2/h-Fe3O4@C containing PFH colloids as a function of time. The contrast was monitored from the side of agarose gel exposed to Vevo 770 micro-US imaging system (Visual Sonics RMV-704, 40 MHz, B-mode). In vivo ultrasound imaging was evaluated using nude mice (BALB/cAnN). Following the aforementioned protocols the tumors were implanted on the mice. When the tumor-bearing mice were ready for studies, PEG coated SiO2/h-Fe3O4@C containing PFH NPs were administrated by intratumoral (10 mg kg-1) or intravenous injection (20 mg kg-1). For intratumoral injection, laser was applied right after injection for 10 min irradiation. Group without laser applied was set as control. In addition, PEG coated SiO2/h-Fe3O4@C in the absence of PFH received laser exposure was used as a control for comparison as well. Ultrasound image at tumor site was monitored at pre-injection, post injection 0 min (right before laser irradiation), and post-10 min, -20 min, and -


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30 min after laser irradiation using Vevo 770 micro-US imaging system (Visual Sonics RMV704, 40 MHz, B-mode) The focal zones of the US images have located at the center of the tumor site throughout experiments. For intravenous injection, external magnetic field was applied for 30 min after injection and then waited for additional 30 min, followed by 10 min laser irradiation. Ultrasound image at tumor site was monitored at pre-injection, before and post laser irradiation. In vivo blood analysis, H&E stain, and biodistribution: Tumor bearing mice were sacrificed on 1st and 20th days after injection of PEG coated SiO2/h-Fe3O4@C containing PFH NPs (20 mg kg-1). Blood was collected, added to heparin rinsed eppendorf, and centrifuged to obtain serum. Blood biochemistry analysis (ALP, AST, ALT, T-Bil, BUN, UA, and CRE) was determined by biochemical analyzer (FUJI DRI-CHEM 4000i). The tissues (heart, liver, spleen, lung, and kidney) were collected, washed twice with normal saline solution, and stored in 4% para-formaldehyde solution. Small piece of tissue was separated for H&E stain, which is made by tissue bank, National Cheng Kung University Hospital (Tainan, Taiwan). The other part of tissues were cut into small pieces and digested in aqua regia for 1 week. The iron content of the tissues was measured by ICP-AES. The group injected of PBS was also measured as a control.

ASSOCIATED CONTENT Supporting Information. Additional TEM image, FTIR spectra, raman spectra, SQUID, BET, DLS, zeta potential, particle stability, cytoviva image, MTT assay, body weight, blood biochemical analysis, H&E stain, and biodistribution. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *Correspondence author E-mail: [email protected] Funding Sources We appreciate the financial support from the Ministry of Science and Technology (MOST 1032113-M-006-008-MY2), and in port by the Headquarters of University Advancement at the National Cheng Kung University, which is sponsored by the Ministry of Education, Taiwan. ACKNOWLEDGMENT We appreciate the financial support from the Ministry of Science and Technology (MOST 1032113-M-006-008-MY2), and in port by the Headquarters of University Advancement at the National Cheng Kung University, which is sponsored by the Ministry of Education, Taiwan.

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(3) Mao, H. Y.; Laurent, S.; Chen, W.; Akhavan, O.; Imani, M.; Ashkarran, A. A.; Mahmoudi, M., Graphene: Promises, Facts, Opportunities, and Challenges in Nanomedicine. Chem. Rev. 2013, 113, 3407-3424. (4) Tu, X.; Ma, Y.; Cao, Y.; Huang, J.; Zhang, M.; Zhang, Z., PEGylated Carbon Nanoparticles for Efficient In Vitro Photothermal Cancer Therapy. J. Mater. Chem. B 2014, 2, 2184-2192. (5) Wang, Y.; Wang, K.; Zhang, R.; Liu, X.; Yan, X.; Wang, J.; Wagner, E.; Huang, R., Synthesis of Core-Shell Graphitic Carbon@Silica Nanospheres with Dual-Ordered Mesopores for Cancer-Targeted Photothermochemotherapy. ACS Nano 2014, 8, 7870-7879. (6) Wang, L.; Sun, Q.; Wang, X.; Wen, T.; Yin, J.-J.; Wang, P.; Bai, R.; Zhang, X.-Q.; Zhang, L.-H.; Lu, A.-H.; Chen, C., Using Hollow Carbon Nanospheres as a Light-Induced Free Radical Generator to Overcome Chemotherapy Resistance. J. Am. Chem. Soc. 2015, 137, 1947-1955. (7) Chen, Y.-C.; Chiu, W.-T.; Chen, J.-C.; Chang, C.-S.; Wang, L. H.-C.; Lin, H.-P.; Chang, H.C., The Photothermal Effect of Silica-Carbon Hollow Sphere-Concanavalin A on Liver Cancer Cells. J. Mater. Chem. B 2015, 3, 2447-2454. (8) Lee, H.-J.; Sanetuntikul, J.; Choi, E.-S.; Lee, B. R.; Kim, J.-H.; Kim, E.; Shanmugam, S., Photothermal Cancer Therapy Using Graphitic Carbon-Coated Magnetic Particles Prepared by One-Pot Synthesis. Int. J. Nanomed. 2015, 10, 271-282. (9) Wang, H.; Sun, L.; Li, Y.; Fei, X.; Sun, M.; Zhang, C.; Li, Y.; Yang, Q., Layer-by-Layer Assembled Fe3O4@C@CdTe Core/Shell Microspheres as Separable Luminescent Probe for Sensitive Sensing of Cu2+ Ions. Langmuir 2011, 27, 11609-11615.


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(10) Chen, J.; Guo, Z.; Wang, H.-B.; Gong, M.; Kong, X.-K.; Xia, P.; Chen, Q.-W., Multifunctional Fe3O4@C@Ag Hybrid Nanoparticles as Dual Modal Imaging Probes and Near-Infrared Light-Responsive Drug Delivery Platform. Biomaterials 2013, 34, 571-581. (11) Cheng, K.; Sun, Z.; Zhou, Y.; Zhong, H.; Kong, X.; Xia, P.; Guo, Z.; Chen, Q., Preparation and Biological Characterization of Hollow Magnetic Fe3O4@C Nanoparticles as Drug Carriers with High Drug Loading Capability, pH-Control Drug Release and MRI Properties. Biomater. Sci. 2013, 1, 965-974. (12) Wang, H.; Shen, J.; Li, Y.; Wei, Z.; Cao, G.; Gai, Z.; Hong, K.; Banerjee, P.; Zhou, S., Magnetic Iron Oxide-Fluorescent Carbon Dots Integrated Nanoparticles for Dual-Modal Imaging, Near-Infrared Light-Responsive Drug Carrier and Photothermal Therapy. Biomater. Sci. 2014, 2, 915-923. (13) Liang, D.; Wu, S.; Wang, P.; Cai, Y.; Tian, Z.; Liu, J.; Liang, C., Recyclable Chestnut-Like Fe3O4@C@ZnSnO3 Core-Shell Particles for the Photocatalytic Degradation of 2,5Dichlorophenol. RSC Adv. 2014, 4, 26201-26206. (14) Lei, C.; Han, F.; Sun, Q.; Li, W.-C.; Lu, A.-H., Confined Nanospace Pyrolysis for the Fabrication of Coaxial Fe3O4@C Hollow Particles with A Penetrated Mesochannel as A Superior Anode for Li-Ion Batteries. Chem.-Eur. J. 2014, 20, 139-145. (15) Sun, Q.; Guo, C.-Z.; Wang, G.-H.; Li, W.-C.; Bongard, H.-J.; Lu, A.-H., Fabrication of Magnetic Yolk-Shell Nanocatalysts with Spatially Resolved Functionalities and High Activity for Nitrobenzene Hydrogenation. Chem.-Eur. J. 2013, 19, 6217-6220.


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(16) Liu, W.-j.; Liu, Y.-x.; Yan, X.-y.; Yong, G.-p.; Xu, Y.-p.; Liu, S.-m., One-Pot Synthesis of Yolk Shell Mesoporous Carbon Spheres with High Magnetisation. J. Mater. Chem. A 2014, 2, 9600-9606. (17) Zhang, H.; Zhou, L.; Noonan, O.; Martin, D. J.; Whittaker, A. K.; Yu, C., Tailoring the Void Size of Iron Oxide@Carbon Yolk-Shell Structure for Optimized Lithium Storage. Adv. Funct. Mater. 2014, 24, 4337-4342.

(18) Lu, A.-H.; Li, W.-C.; Hao, G.-P.; Spliethoff, B.; Bongard, H.-J.; Schaack, B. B.; Schueth, F., Easy Synthesis of Hollow Polymer, Carbon, and Graphitized Microspheres. Angew. Chem., Int. Ed. 2010, 49, 1615-1618. (19) Fu, J.; Xu, Q.; Chen, J.; Chen, Z.; Huang, X.; Tang, X., Controlled Fabrication of Uniform Hollow Core Porous Shell Carbon Spheres by the Pyrolysis of Core/Shell Polystyrene/Cross-Linked Polyphosphazene Composites. Chem. Commun. 2010, 46, 65636565. (20) Lu, A.-H.; Sun, T.; Li, W.-C.; Sun, Q.; Han, F.; Liu, D.-H.; Guo, Y., Synthesis of Discrete and Dispersible Hollow Carbon Nanospheres with High Uniformity by Using Confined Nanospace Pyrolysis. Angew. Chem., Int. Ed. 2011, 50, 11765-11768. (21) Li, W. P.; Liao, P. Y.; Su, C. H.; Yeh, C. S., Formation of Oligonucleotide-Gated Silica Shell-Coated Fe3O4-Au Core-Shell Nanotrisoctahedra for Magnetically Targeted and NearInfrared Light-Responsive Theranostic Platform. J. Am. Chem. Soc. 2014, 136, 1006210075. (22) Wang, G.-H.; Hilgert, J.; Richter, F. H.; Wang, F.; Bongard, H.-J.; Spliethoff, B.; Weidenthaler, C.; Schueth, F., Platinum-Cobalt Bimetallic Nanoparticles in Hollow Carbon


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Figure 1. a) Illustration of the synthesis of SiO2/h-Fe3O4@C following mixed micellization, polymerization/hollowing, sol-gel (hydration-condensation) and pyrolysis processes. TEM images of b) Fe3O4 NPs, c) h-Fe3O4@P, d) SiO2/h-Fe3O4@P, and e) SiO2/h-Fe3O4@C was shown. The clustering Fe3O4 NPs can be seen from TEM images.

Figure 2. a) HR-TEM image of SiO2/h-Fe3O4@C. The magnified images in red square boxes reveal b) core and c) shell locations showing the crystallization Fe3O4 with (220) and (533) planes and the amorphous carbon shell, respectively. e)- h) are the C, O, Si, and Fe elemental mapping images of SiO2/h-Fe3O4@C corresponding to d).


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Figure 3. a)UV-vis spectra of SiO2/h-Fe3O4@C NPs pyrolysis at 500 oC and 650 oC. The inset shows the colloidal colors from SiO2/h-Fe3O4@C NPs from 500 oC and 650 oC. b)XRD patterns of SiO2/h-Fe3O4@C obtained at 500 oC, 650 oC, and 800 oC pyrolysis using a Cu Kα radiation (λ = 1.5418 Å) source. The inset shows the NPs morphology obtained at 800 oC.


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Figure 4. a) The absorbance versus iron concentration of PEG coated SiO2/h-Fe3O4@C NPs to obtain the molar extinction coefficient at 808 nm. The inset shows the corresponding UV-vis spectra. b) Photothermal heating curve of PEG coated SiO2/h-Fe3O4@C NPs (100 ppm [Fe concentration]) as a function of laser power. c) Biocompatibility for HeLa cells incubated with different concentrations ([Fe concentration]) of PEG coated SiO2/h-Fe3O4@C for 24 h, and d) In vitro photothermal efficacy in cell viability for HeLa cells alone, HeLa cells with only laser exposure for 10 min, HeLa cells with materials under magnetic attraction for 5 min, HeLa cells with materials under laser exposure for 10 min, and HeLa cells with materials under magnetic


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attraction for 5 min with 10 min laser exposure. An 808 nm diode lasers was employed. All the experiments were triplicate. e) The fluorescence images of HeLa cells correspond to d). Live cells were stained with Calcein AM with green fluorescence and dead cells stained with PI with red fluorescence. Cells received laser irradiation after 2 h incubation with or without magnet. The “materials” shown on Figures represents as PEG coated SiO2/h-Fe3O4@C NPs and M as magnet.


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Figure 5. The r2 values of a) PEG coated SiO2/h-Fe3O4@C and b) PEG coated SiO2/h-Fe3O4@C containing PFH NPs were calculated by T2 relaxation (T2-1) rate versus iron concentration. c) In vivo T2-weighted images of PEG coated SiO2/h-Fe3O4@C containing PFH NPs monitored by 9.4 T animal micro-MRI system. The images were captured on the pre-injection, post-injection 30 min, 1 h, 2 h, 3 h, and 24 h after particles being injected intravenously. The MR imaging intensity was analyzed with and without magnet for d) and e), respectively. For magnetic attraction, the magnet was applied for 30 min after intravenous injection and then waited for additional 30 min representing post-injection 1 h, and so forth for post-injection 2 h, 3 h, and 24 h. White arrows indicate tumor sites.


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Figure 6. a) Tumor growth curves with different treatments (n=3) (*P

Fabrication of Silica-Coated Hollow Carbon Nanospheres

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