Figure 2. Cytotoxicity profiles of PGMNPs, DOX and DOX- PGMNPs upon H22 cell lines upon H22 cell lines determined by MTT assay. (A) H22 cells suspension were treated by DOX or DOX- PGMNPs conjugate. (B) H22 cells suspension were treated by PGMNPs or DOX-PGMNPs conjugate. doi:10.1371/journal.pone.0040388.g002 (Fig. 3). Therefore, our results show that DOX concentrations in tumors of mice from the DOX-PGMNPs-M group were significantly higher than those from the DOX-PGMNPs group or the DOX group (each p,0.05). The percent injected dose per gram tissue in blood of mice from the DOX-PGMNPs-M group, the DOX-PGMNPs group, and the DOX group were 0.765%, 0.876%, and 1.07%, respectively (for each group p.0.05). The percent injected dose per gram tissue in livers from the DOX- PGMNPs-M group, the DOX-PGMNPs group, and the DOX group were 1.23%, 1.33% and 0.402%, respectively. Therefore, no significant difference was observed between the DOX levels found in mouse livers of the DOX-PGMNPs and DOX- PGMNPs-M groups (p.0.05). However, the liver DOX concen- tration appeared to be much lower in mice of the DOX group compared with mice in the DOX-PGMNPs-M or DOX-PGMNPs groups (each p,0.05). Interestingly, the highest DOX concentra- tions were found in the spleen. The percent injected dose per gram tissue in from mouse spleens were 3.52%, 6.49% and 0.634% reported from the DOX-PGMNPs-M, the DOX-PGMNPs, and the DOX groups, respectively (each p,0.05). Very low concen- trations of DOX were observed in the other organs (e.g., the heart, lung and kidney) for all three groups (DOX, DOX-PGMNPs, and DOX-PGMNPs-M). Histological studies Tumor tissue was investigated using HE staining. Most necrotic cells and vacuoles were observed in tumors from the DOX- PGMNPs-M group. A few necrotic cells were observed in tumors from the DOX and DOX-PGMNPs groups, while only viable tumor cells were observed in tumors from the control group (Fig. 4). Drug Nanoparticle Carrier for Cancer Chemotherapy Figure 3. The percent injected dose per gram tissue in tumor, plasma and main organs of H22 tumor–bearing mice that received different treatments. The percent injected dose per gram tissue in the tumors of mice in the DOX-PGMNPs-M group was much higher than that in tumors of mice in the DOX-PGMNPs group or the DOX group. DOX(H22 tumor-bearing mice that treated with doxorubi- cin; DOX-PGMNPs (H22 tumor-bearing mice that treated with DOX- PGMNPs conjugate suspension); DOX-PGMNPs-M (H22 tumor-bearing mice injection DOX-PGMNPs conjugate suspension and subjected to a 0.5 tesla EMF focused onto the tumor for 0.5 hr).*p,0.05; **p,0.01 compared with controls using Student’s unpaired t-test. doi:10.1371/journal.pone.0040388.g003 Influence of DOX-PGMNPs on tumor growth Tumor growth curves resulting from different drug treatments of H22 hepatoma cell tumor-bearing mice are shown in Figure 5. The relative tumor volume of DOX-PGMNPs-M group, DOX- PGMNPs group, DOX group and the control group were determined 21 days after the start of various treatment protocols; Figure 4. Histological assay of tumors of H22 tumor-bearing mice that received different treatments. (hematoxylin and eosin stain, 4006 magnifications). A large number of necrotic cells as well as vacuoles were observed in tumors in mice of the DOX-PGMNPs-M group (D), whereas only a few necrotic tumor cells could be seen in tumors in mice of the DOX (B) or DOX-PGMNPs groups (C). Mostly viable tumor cells were observed in the tumors of the mouse control group (A). doi:10.1371/journal.pone.0040388.g004 PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e40388