The nanoparticles can be coated with hydrophilic polymers, such as polyethylene glycol (PEG), which are used to disperse drug particles in order to increase their half-life in the blood and to minimize or prevent protein adsorption, thus avoiding RES clearance [21,22]. PEG is a flexible hydrophilic polymer that can be used as a shell-forming segment. The dense PEG shell allows for a high degree of biocompatibility and also endows the micelle with a stealth character in the blood compartment, consequently achieving a long circulation time [21–23]. PEG-modified GMNPs (PGMNPs) have been synthesized and characterized as drug carriers, which have a saturated magnetization of 34 emu/g with an average diameter of 50 nm and were homogeneous suspension without aggregation in PBS [12]. Doxorubicin (DOX)-based chemotherapy exhibited only a modest antitumor activity with tolerable adverse effects in patients with advanced hepatocellular carcinoma (HCC) [24]. The purposes of our study were 1) to investigate the kinetics of DOX-PGMNPs conjugation and the release of DOX from DOX- PGMNPs in vitro; 2) to assess the cytotoxicity of the DOX- PGMNPs conjugate using a mouse hepatoma cell line (H22) in vitro; and 3) to evaluate the therapeutic efficiency of the DOX- PGMNPs conjugate using an EMF-guided delivery and H22 hepatoma cell tumor-bearing mice in vivo. Materials and Methods Mouse hepatoma H22 cell line and tumor-bearing mouse model All animals were housed and handled according to the Northwest University Institutional Animal Care and Use Com- mittee guidelines and all animal work was approved by the appropriate committee (IACUC 0000125 and 0000125B-4). The protocol was approved by the local ethics committee (ethics committee, Northwest University 035/2009) and all animals received humane care in compliance with ‘‘The Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research and the ‘‘Guide for the Care and Use of Laboratory Animals’’ published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). The mouse hepatoma H22 cell line was grown in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37uC and 5% CO2. BALB/c mice (4- to 8-week-old males, body weight 2665 g) were housed (one mouse per cage) under specific pathogen-free conditions, maintained on a 12-hour light-dark cycle with controlled temperature (20–24uC), and were given sterile food. The tumor- bearing mouse model was generated by subcutaneous injection of 46105 H22 cells with 50 ml phosphate-buffered saline (PBS) into the shaved right flank. The treatment protocol required the mice to be anesthetized and these were anesthetized with ketamine (80 mg/kg) and xylazine (3 mg/kg), and acepromazine (2 mg/kg) via intraperitoneal injection (IP). If an additional dose was needed, ketamine alone was used. Synthesis of DOX-PGMNPs conjugates PGMNPs were synthesized and characterized as drug nano- carriers as previously described. PGMNPs with the diameter averaging ,50 nm were used in this study [12]. Approximately 0.2 ml of PGMNPs (10 mg/ml) suspensions was added to 5 ml centrifuge tubes, and magnetically separated with a magnetic separator (GoldMag Nanobiotec, Xi’an, China); the supernatant was discarded. A total of 2 ml of varying DOX concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mg/ml) was added to the PGMNPs precipitate. The mixtures were incubated in a shaker at room Drug Nanoparticle Carrier for Cancer Chemotherapy temperature for 4 hrs. The DOX-PGMNPs conjugate was magnetically separated and the drug content left in the superna- tant was measured with high-performance liquid chromatography (HPLC, Shimadzu 2010A instrument, Shimadzu, Japan). HPLC analysis was performed using a binary pump, column oven, and UV detector. LC solution software was used for the data analysis. The chromatographic separation was performed on an Inertsil H ODS-SP analytical column (150 mm64.6 mm, 5 mm, Shimadzu, Japan) with the column temperature set at 25uC. An isocratic elution was performed after 15 min using 0.1% acetic acid and acetonitrile in the ratio 72/28 (v/v). The flow rate was 0.8 ml/ min, the detector wavelength was set to 254 nm, and the injection volume was 20 ml. The drug loading (%) on the surface of the PGMNPs was calculated using the following equation: Drug loading (%) = (the mass of drug on PGMNPs/the mass of PGMNPs)6100%. DOX release study in vitro A total of 5 mg of the DOX-PGMNPs conjugate (DOX loading 8.2%) was suspended in 15 ml PBS (pH 7.4). The releasing medium was placed in a shaking incubator at 3760.5uC and 180 rpm for 15 min. PGMNPs were then separated, and an aliquot of the supernatant (0.5 ml) was retained. The concentra- tion of DOX in the supernatant was quantified by HPLC and the amount of DOX released from the PGMNPs was calculated. Drug release (%) from the DOX-PGMNPs conjugate was calculated at different time points using the following equation: Drug release (%) = (mass of drug in the supernatant/mass of drug originally conjugated onto the PGMNPs)6100%. Cytotoxicity of PGMNPs and the DOX-PGMNPs conjugate in H22 cell culture A volume of 180 ml of H22 cells were seeded in 96-well plates (4,000 cells/well). The following day, 20 ml DOX (0.4, 4, 8, 20 or 40 mg/ml) or 20 ml DOX-PGMNPs conjugate (4.88, 48.8, 97.6, 244 or 488 mg/ml) was added to H22 cell suspensions (DOX loading rate 8.2%). In both cases, the final concentration of DOX was the same (0.04, 0.4, 0.8, 2 and 4 mg/ml). The cells were treated with various DOX concentrations (from DOX solution or DOX-PGMNPs conjugate) in 96-well plates at 37uC and 5% CO2 atmosphere for 24 hrs. The cytotoxicity of equivalent amounts of PGMNPs (compared to DOX-PGMNPs conjugate) was evaluated as well. Control cells were cultured in medium only. Thiazolyl blue tetrazolium bromide (25 ml of 5 mg/ml) (MTT, AMRESCO Inc, Solon, OH) was added to each well and incubated for 4 hrs. The culture medium was removed from the wells and replaced with 150 ml dimethylsulfoxide (DMSO, BioTek, Winooski, VT). After shaking gently for 15 min at 25uC, the DMSO solution was transferred to centrifugation tubes and centrifuged at 2,000 rpm for 5 min. The absorbance was then measured at 570 nm with an ELx-800 Universal Microplate Reader (BioTek, Winooski, VT). The cell inhibition rate was calculated using the formula: cell inhibition rate = (12ATest/AControl)6100%; where ATest is the absorbance of the experimental wells and AControl is the absorbance of the control wells. The half-maximal inhibitory concentration (IC50; drug concentration corresponding to 50% growth inhibition) was calculated using SigmaPlot 9.0 software (Systat Software Inc., San Jose, CA) and the 4-parameter logistic function standard curve analysis for dose response. PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e40388