(a) mice treated with 100 ml normal saline (NS), (b) mice treated with 100 ml blank hydrogel, (c) mice treated with 100 ml SAHA, (d) mice treated with 100 ml DDP, (e) mice treated with 100 ml combine SAHA with DDP (SAHA-DDP), (f) mice treated with 100 ml SAHA and DDP in thermosensitive hydrogel (SAHA- DDP/PECE). SAHA and DDP were peritumorally administered two times at the first dose and 14 days later. Tumor diameters were measured every 3 days, and tumor volume was calculated using the formula: Tumor size = (length) 6 (width)2 6 0.52. All the animals were observed after administration, including the general conditions (the activity, energy, hair, feces, behavior pattern, and other clinical signs), body weight, and mortality. Mice were sacrificed 14 days after the last treatment. Tumors were excised and weighted, half of which were snap frozen immediately for western blot assay, and anthers were fixed in 4% formalde- hyde. Toxicity Assessment Possible side effects were observed through weight, appetite, diarrhea, life span, and behavior until they were sacrificed. Organs such as heart, liver, spleen, lung, and kidney were collected, fixed in 4% paraformaldehyde solution and made into 4 mm sections which were stained with hematoxylin and eosin (H&E) and observed by two pathologists in a blinded manner under a microscope. Immunohistochemistry and TUNEL Assay Mouse anti-human PCNA antibody (1:500, BD, USA) was applied for detecting the tumor cell proliferation on the paraffin section. Microvessel of tumor tissues was detected by Anti-CD34 antibody (1:500, BD, USA) on lung frozen tissue section. TUNEL detection Analysis of apoptotic cells in tumor tissue was measured by the TUNEL Apoptosis Detection Kit (Upstate LabChem Inc, USA) following the manufacturer’s directions. Three equal-sized fields (at 6 400 magnification) were randomly chosen and the mean number of green fluorescence-positive cells was counted. Western Blot Analysis To prepare lysates from dissected in vivo tumors, samples were snap frozen in liquid nitrogen immediately after animal sacrifice and stored at –80uC before use. The lysates were incubated on ice in RIPA lyses buffer for 30 min before being homogenized by a mortar and pestle. Apoptosis related proteins were detected by anti-Caspase-3 antibody, anti-Caspase-8 antibody, anti-Caspase-9 antibody (Cell Signaling, USA). Iso- lation of histones was performed as described previously [28]. Total and acetyl-Histone H3 were detected by Anti-Histone H3 polyclonal antibody and Anti-acetyl-Histone H3 polyclonal antibody respectively. The primary antibody was detected with HRP-conjugated anti-mouse secondary antibody and developed with an enhanced chemiluminescence detection kit (Pierce, USA). Equal protein loading was confirmed by detection of GAPDH (R&D, USA). Statistical Analysis Data are expressed as the mean 6 SD. Statistical analysis was performed by Student’s t test for comparing two groups and by ANOVA for multiple group comparisons. p values less than 0.05 were considered to be statistically significant. All statistical tests were two-sided. SPSS 16.0 was used for all statistical analyses. Anti-Tumor Effect of Controlled Drug Delivery Results Synthesis and Characterization of PECE Copolymers The PECE triblock copolymer was synthesized according to Fig.1. FTIR, 1H-NMR and GPC were used to characterize the chemical structure of the PECE copolymer (data not shown) [19]. The Mn and PEG/PCL block ratio of PECE triblock copolymer calculated from 1H-NMR spectra was 3630 and 1100/2530 respectively. Cytotoxicity Assay of PECE Copolymer Firstly, We wanted to verify whether the prepared PECE copolymer has cytotoxicity to the human oral normal and cancer cell line, so HOK16E6E7 (an immortalized human oral keratinocyte cell line) and HSC-3 ( a human tongue squamous cell carcinoma cell line) were chose in the cytotoxicity assay of PECE copolymers. Fig. 2 exhibits the HOK16E6E7 and HSC-3 cells viability at the presence of PECE copolymer at different concentrations. The cell viability decreased with increase of PECE copolymer amount. But the HOK16E6E7 and HSC-3 cells viability was yet higher than 82% and 65%, respectively, even when the input PECE copolymers were 400 mg per well. According to Fig. 2, the HSC-3 cell viability of PECE copolymer was higher compared to HOK16E6E7 group. Thus, the PECE copolymers prepared in this paper could be regarded as a biocompatible with low cell cytotoxicity controlled drug delivery systems. Antitumor Activity of SAHA-DDP/PECE In Vivo In the mice model of oral squamous cell carcinoma xenografts, the female nude mice received 2 6 106 HSC-3 cells via subcutaneously into the right flank regions. Mice were assigned to six groups, receiving NS, PECE, SAHA, DDP, SAHA-DDP and SAHA-DDP/PECE, respectively. Two combination-therapy groups, especially the group treated with SAHA-DDP/PECE were significantly reduced the growth of tumors compared with other control groups. The growth delay of tumors in mice injected with SAHA-DDP/PECE continued until the day of sacrifice, reaching a mean volume of 62.43 mm3, while tumors of mice injected with NS, PECE, SAHA, DDP and SAHA- 3 DDP grew persistently, resulting in mean volumes of 148.60 mm , 187.60 mm3, 249.71 mm3, 279.41 mm3 and 284.32 mm3 (Fig. 3B and C, p , 0.05). Consistent with the data of tumor volume, the mean weight of tumors of mice treated with SAHA-DDP/PECE was 0.04 6 0.01 g, accounting for 22.18% of that of mice treated with NS (0.21 6 0.10 g), 22.42% of mice treated with PECE (0.20 6 0.04 g), 25.22% of mice treated with SAHA (0.18 6 0.04 g), 33.54% of mice treated with DDP (0.14 6 0.02 g) and 43.98% of mice treated with SAHA-DDP (0.10 6 0.01 g). To provide evidence that the drugs were detectable at the tumor site and the duration of drug presence following drug delivery, we Figure 1. Synthesis of PECE copolymers. doi:10.1371/journal.pone.0033860.g001 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e33860