they were 3.9862.12, 4.9661.45, 7.0861.68 and 7.9261.73, respectively. At 21 day, the DOX-PGMNPs-M treatment provided an effective inhibition of tumor growth that was comparable to those found for the other treatment strategies (p,0.05). It should be emphasized that at this time point (at 21 days), there was no significant difference between the effective inhibition of tumor growth in the DOX group and the DOX- PGMNPs group (p.0.05). However, at day 33 (after the start of the various treatment protocols), the inhibition of tumor growth was greatest in the DOX-PGMNPs-M group. The relative tumor volume in the DOX-PGMNPs-M group was 5.4661.48, in the DOX-PGMNPs group was 9.2261.51, in the DOX group was 14.861.67, and in the control group was 24.361.95 (for each p,0.01). It should be noted that a significant difference in the fractional percent of tumor volume was observed between the groups treated with DOX, with DOX-PGMNPs and with DOX- PGMNPs-M (p,0.05). Influence of DOX-PGMNPs on life span The survival rate of tumor-bearing mice treated with DOX- PGMNPs-M, DOX-PGMNPs, DOX and the control group decreased with the time. The survival rate of tumor-bearing mice treated with DOX-PGMNPs-M, DOX-PGMNPs, DOX and the control group were 75%, 66.7%, 33.3% and 16.7% following treatment for 33 days (Fig. 6A). The life span of tumor-bearing mice treated with DOX- PGMNPs-M, DOX-PGMNPs, DOX and the control group were 74.869.95, 66.1613.5, 31.363.31 and 25.8610.1 days, respec- tively (each p,0.05) (Fig. 6B). The life span of mice in the DOX- PGMNPs group and the DOX-PGMNPs-M group was signifi- cantly longer than of those in the DOX group or in the control group (each p,0.05). Furthermore, the life span of mice in the DOX-PGMNPs-M group was significantly longer than of those in the DOX-PGMNPs group (p,0.05). Discussion It is very critical for the drug adsorbed on the particles can release from carrier. Also the ideal drug delivery carrier is one from which the drug can release with a sustained and controlled Figure 5. Tumor growth curves of H22 tumor –bearing mice model that received different treatments indicated. Effective inhibition of the tumor growth can be seen resulting from the DOX- PGMNPs and DOX-PGMNPs-M treatment strategies. The same doxoru- bicin dose was injected to H22 tumor –bearing mice model for DOX, DOX-PGMNPs and DOX-PGMNPs-M. The same volume physiologic saline solution injected to H22 tumor –bearing mice model as control. *p,0.05; **p,0.01 compared with controls using Student’s unpaired t- test. doi:10.1371/journal.pone.0040388.g005 Drug Nanoparticle Carrier for Cancer Chemotherapy amount within given time. The loading and release patterns of DOX from PGMNPs showed that DOX could be efficiently loaded onto and released from PGMNPs (Fig. 1). The DOX loading ranged from 2.98% to 10.78% with the amount of DOX increase and the amount of maximum drug loading rate is about 107.8 mg/mg, which drug loading is 10.78% (Fig. 1A). The loading rate achieved saturation when the concentration exceeded 0.6 mg/ml. The results of drug release studies in vitro showed a period of rapid release in the first 10 hrs and drug release saturation after 20 hrs (Fig. 1B). Because of –OH group in DOX and PEG, the drug release behaviors are affected by the pH value and temperature. Based on the data, it was proved that the key interaction of DOX and PEG molecules modified on gold- magnetic nanoparticles is the hydrogen-bond [12]. In addition, the drug release behavior is in accord with the toxicity results of free DOX and DOX-PGMNPs in H22 hepatoma cells after 24 hrs exposure. Although the cell inhibition rate of DOX is modestly higher than that of DOX-PGMNPs, our studies show that DOX- PGMNPs display a similar toxicity profile as free DOX in H22 cells (Fig. 2A), thus indicating that DOX-PGMNPs have sufficient antitumor activity to inhibit tumor growth. In vitro cytotoxicity assays showed that PGMNPs were not significantly cytotoxic because 85.4% of H22 cells cultured in the presence of 2.0 mg/ml remained viable (Fig. 2B). The lack of any significant cytotoxicity being observed may be explained by the following: 1) the NPs have a gold shell and colloid gold is known to have low toxicity and good biocompatibility [25,26]; and 2) PEG is a biocompatible hydrophilic polymer that can improve the properties of NPs by decreasing their toxicity [27,28]. The cytotoxicity of DOX-PGMNPs was studied in vitro and the IC50 values of free DOX and DOX-PGMNPs were found to not be significantly different. We assume the cause that cell inhibition rate in DOX is lightly higher than the DOX-PGMNPs group is the DOX-PMGNPs exist first the release of DOX from the NPs; although it could not reach the inhibition effect as the same drug level as the free DOX group. Some DOX-PMGNPs could be uptaken by cells that led to potent cytotoxicty than free DOX as the same quality as which loaded in PMGNPs. Both factor are attributed to the cytotoxcity of DOX-PMGNPs are lower than DOX, although they had no significantly difference statistically. These results demonstrated that DOX-PGMNPs are potent cytotoxins towards H22 hepatoma cancer cells (Fig. 2A). Our results (from various treatments) indicated that the concentration of DOX was significantly lower in mouse livers compared with spleens (Fig. 3). Previous reports have shown that PEG-coated gold nanoparticles are accumulated in both the spleen and the liver [29,30]. Although the amount of nanoparticles should correlate with the amount of DOX in the system, this finding could be explained by the fact that DOX may be metabolized in the liver and, therefore, may be causing lower amounts of the compound to be detected in the liver in vivo. Magnetically targeted drug delivery system could improve therapy efficacy by increasing the drug concentration in the tumor while reducing systemic drug concentrations that produce systemic toxicities [31,32]. Antitumor activity of the DOX-PMGPNs-M treatment was observed by determining the relative tumor volume and the mouse life span (Figs. 5 and 6). The therapeutic potency and efficacy of the DOX-PGMNPs-M treatment was demonstrat- ed by tumor necrosis. The antitumor activities might be attributed to the various drug concentrations in the tumors due to the differences in the experiments (Figs. 3, 5 and 6). The DOX- PGMNPs-M therapy showed a significant increase of DOX efficiency when compared with DOX therapy alone, which is most likely because PGMNPs effectively carry DOX to the targeted site PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e40388