DOX biodistribution in tumor-bearing mice and chemotherapy experiments The H22 tumor-bearing mice were randomly divided into 4 groups; Group I (DOX group) was injected with 0.15 ml DOX dissolved in physiologic saline solution (0.82 mg/ml); Group II (DOX-PGMNPs group) was injected with 0.15 ml DOX- PGMNPs conjugate suspension (10 mg PGMNPs/ml, DOX loading 8.2%); Group III (DOX-PGMNPs-M group) was injected with 0.15 ml DOX-PGMNPs conjugate suspension (10 mg/ml, DOX loading 8.2%) using a 0.5 tesla EMF (0.5 T permanent magnet, Northwestern Polytechnical University, Xi’an, China) applied on the tumor for 2 hrs; and Group IV (Control group) was injected with 0.15 ml physiologic saline solution alone to serve as a control. The doses were normalized to 5 mg DOX per kg body weight when the tumor volume reached 50 to 100 mm3 (,10 days after tumor cell inoculation). The volume for 150 ml was injected via the tail vein and the treatment was carried out three times without interval in three weeks. DOX biodistribution measurement: mice (Group I, Group II, and Group III; n = 6 for each group) were sacrificed after 0.5 hr of treatment for measurement of DOX biodistribution. The blood, main organs (heart, lung, liver, spleen, kidney), and the tumor of each mouse were harvested for quantitative analysis of the DOX biodistribution. The blood was centrifuged at 5,000 rpm for 1 min at 4uC and the supernatant was obtained by adding 180 ml methanol and 20 ml of inner standard solution (0.2 mg/ml daunorubicin solution dissolved in methanol) to a 2-ml tube containing 200 ml plasma. For the organ (heart, lung, liver, spleen, kidney) and tumor samples, the frozen tissues were weighed and homogenized at 4uC in 0.9% NaCl to a total volume of 10 ml, and centrifuged at 5,000 rpm for 1 min at 4uC to retrieve the supernatant. A total volume of 200 ml of the plasma, or the supernatant of homogenized tissue, or the tumor samples was then mixed with 180 ml methanols and 20 ml inner standard solutions, followed by 0.4 g sodium sulfate. The mixture was homogenized by vortexing for 5 min and then incubated at 4uC for 1 hr. After the 1-hr incubation, the mixture was centrifuged at 12,000 rpm for 15 min. The supernatant (50 ml) was analyzed by HPLC and the quantity of DOX in the tissue and tumor samples was measured. Finally, the percent-injected dose per gram tissue of the tissue and tumor samples was calculated. Chemotherapy experiments: H22 tumor-bearing mice (4 groups; for each group, n = 18) were used and the treatment was performed as described above. The antitumor effect was assessed by the following steps: 1) six mice were sacrificed for each group on the 24th day following treatment. The tumors were harvested and fixed in 10% formalin, embedded in paraffin and sectioned (5 mm coupes) for hematoxylin–eosin (HE) staining for microscopic examination purposes; 2) tumor sizes (4 groups; for each group, n = 12) were measured by a caliper before therapy and every three days after treatment using the following equation: tumor volume (V, mm3) = (tumor length6tumor width)2/2. The life span of each animal was also recorded. The relative tumor volume was calculated from V = V/V0, where V0 is the tumor volume at the beginning of treatment. The endpoints of the antitumor effect were evaluated by the degree of tumor growth inhibition and life span after treatment. Statistical analysis Quantitative data were expressed as the mean 6 standard deviation (SD). The means were compared using Student’s t test where p values,0.05 were considered statistically significant. Drug Nanoparticle Carrier for Cancer Chemotherapy Results Efficiency of DOX loading on the surface of PGMNPs The amount of DOX conjugated to 2 mg PGMNPs was directly proportional to the amount of DOX added. The DOX loading ranged from 2.98% to 10.78% depending on the amount of DOX used (0.2 to 1.2 mg). The loading rate achieved saturation when DOX exceeded 0.6 mg (Fig. 1A). DOX release from DOX-PGMNPs conjugates in vitro The cumulative DOX release from DOX-PGMNPs conjugate is shown in Figure 1B. In the first 0.5 h, 20.4% of DOX was released while 60.1% of DOX was released in 8 hrs. Subsequently, the DOX release amounts were 79.3%, 90.2%, and 91.3% at 20 hrs, 48 hrs, and 72 hrs, respectively. The maximum DOX release (92.2%) from the surface of the PGMNPs was achieved at 100 hrs. Cytotoxicity of PGMNPs and DOX-PGMNPs The cell inhibition rate was determined using the MTT assay and the results are shown in Figure 2. The cytotoxicity of DOX and DOX-PGMNPs presented in Figure 2A, which shows that the cell inhibition rate increased with increasing drug concentration. The cell inhibition rate of DOX was modestly higher than that of the DOX-PGMNPs group (IC50 values were 0.53060.010 mg/ml and 0.65260.056 mg/ml (p.0.05), respectively). Moreover, when compared with DOX-PGMNPs group, PGMNPs alone did not affect H22 hepatoma cell proliferation/viability (Fig. 2B). DOX biodistribution in tissues The percent injected dose per gram tissue in tumors of mice from the DOX-PGMNPs-M group, DOX-PGMNPs group, and DOX group were 4.51%, 0.627% and 0.741%, respectively Figure 1. Efficiency of DOX loading onto the surface of PGMNPs and the behavior of DOX release from DOX-PGMNPs conjugates. (A) Efficiency of DOX loading onto the surface of PGMNPs was analyzed by HPLC. The amount of DOX conjugated to PGMNPs was positively correlated with the DOX mass added. (B) DOX release from DOX-PGMNPs conjugates was analyzed by HPLC. The slow, steady, and controlled release of DOX was observed. doi:10.1371/journal.pone.0040388.g001 PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e40388