drug which is used to treat cancers including: sarcoma, small cell lung cancer, germ cell tumors, lymphoma, and ovarian cancer [9–14]. Though SAHA and DDP have been proven to be effective therapeutic efficacy, they are also can cause serious side effects. The conventional methods for delivering chemothera- peutic agents fail to achieve therapeutic concentrations of drugs, despite reaching toxic systemic levels. Moreover, the improve- ment of the curative is accompanied by the resistance to the drugs [15–18]. So, the novel controlled drug delivery systems are necessary designed to deliver drugs at predetermined rates for predefined periods at the target organ and overcome the shortcomings of conventional drug formulations, therefore could diminish the side effects and improve the life quality of the patients. Thus, a suitable controlled drug delivery system is extremely important for chemotherapy. In our previous study, we prepared a new kind of biodegrad- able, temperature sensitive and injectable poly (ethylene glycol)- poly(e-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) hydrogel based on PEG and PCL which are biocompatible and have been used in several FDA approved products [19]. The PECE hydrogel was a flowing sol at low temperature and formed a non-flowing gel at body temperature, the sol-gel-sol transition behavior of it has been reported in our previously study. And the results indicated that PECE hydrogen displayed a temperature- dependent sol-gel-sol transition in normal saline. When the concentration was above corresponding critical gelatin concentra- tion, the aqueous solutions of PECE copolymer undergo a sol-gel- sol transition as the temperature increases [20,21]. PECE also was proved to be biocompatible, bioabsorbable and thermosensitive. The thermosensitive hydrogel system had been successfully used for honokiol, 5-Fu, and bFGF controlled delivery in cancer treatment and the results presented its great sustained drug release properties [22–25]. Based on the above results, the PECE hydrogel might have great potential applications as an injectable controlled drug delivery system. In this report, we investigated the combined effects of HDAC inhibitors SAHA and DDP in an oral squamous cell carcinoma tumor model using the PECE hydrogels for controlled drug delivery. Our results suggested that the SAHA combine DDP delivered by PECE hydrogel have a significant therapeutic efficacy in murine model. This strategy had potential application in chemotherapy for the oral squamous cell carcinoma. Materials and Methods Animals and Cells Female athymic nude mice (4–6 weeks) were obtained from the Shanghai SLAC laboratory animal co. ltd (Shanghai, China). All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal experiments were approved by the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, China). Oral squamous cell carcinoma cells HSC-3 cells were main- tained in Dulbecco’s modied Eagle’s medium (DMEM; Gibco, USA) containing 10% fetal calf serum (Gibco, USA), penicillin (100 U/L) and streptomycin (10 mg/L). HOK16E6E7 cells, a human immortalized oral keratinocyte cell line, was cultured in keratinocyte growth medium (KGM) containing 0.15 mM of calcium, supplemented with EGF (Gibco, USA), both at 37uC in an atmosphere containing 5% CO2. Anti-Tumor Effect of Controlled Drug Delivery Reagents SAHA was purchased from (Alexis Corp., San Diego, CA, USA), and was dissolved in DMSO as stock solution. The maximum volume (%) of DMSO in the experiment was less than 0.1%. DDP was purchased from Jintai Pharmaceutical Co. Ltd. (Liaoling, PR China) and dissolved in PBS. Poly (ethylene glycol) methyl ether (MPEG, Mn = 550), e-caprolactone (e-CL), hexamethylene diisocyanate (HMDI), and stannous octoate (Sn(Oct)2) were purchased from Sigma-Aldrich Chemical Co. (USA). Synthesis of PECE Hydrogel PECE copolymer were synthesized and purified as reported previously [19]. Briefly, PEG-PCL diblock copolymers were prepared by ring opening polymerization of e-CL initiated by MPEG using stannous octoate as catalyst; PEG-PCL-PEG triblock copolymers were synthesized by coupling PEG-PCL diblock copolymers using HMDI as coupling agent. The obtained PECE copolymer was dissolved in AR grade dichloromethane and reprecipitated from filtrate using AR grade petroleum ether. Then the mixture was filtered and vacuum dried to constant weight at room temperature. The obtained PECE copolymers were characterized by FTIR (NICOLET 200SXV, Nicolet, USA), 1H-NMR (Varian 400 spectrometer, Varian, USA), and GPC (Agilent 110 HPLC, USA). Preparation of Injectable Thermosensitive Composite Hydrogel The drugs loaded injectable thermosensitive hydrogel was prepared similar to the protocol reported previously [26,27]. First, PECE copolymer was well dissolved in water at certain temperature and cooled to 4uC to form sol. Then, SAHA and DDP solutions were mixed into the PECE sol to form homogeneous solution, and the concentration of PECE was kept at 30 wt%. The prepared drugs loaded hydrogel was inhaled into injector and injected into or around the focus of infection in animal. Thus, composite sol turned into gel state and acted as depots for sustained release of drugs in situ when the cold sol is warmed to body temperature (37uC) in vivo. At last, for the degradation of the composite hydrogel, the introduced DDS was gradually emanated from body. Cytotoxicity Assay of PECE Copolymer Cytotoxicity of PECE triblock copolymer to HOK16E6E7 and HSC-3cells was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide (MTT) assay. Cells were seeded on 96-well plates at a density of 104 cells/well in their usual culture media. After 24 h, different amounts of PECE copolymer were added 0 mg, 10 mg, 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg each well, respectively. The normal saline was used as the control groups. Cytotoxicity studies were performed using MTT assay after 24 h, 48 h or 72 h. The absorbance of the samples was measured at 570 nm. The cell cytotoxicity of PECE copolymer is defined as the relative viability, which is the ratio of the number of live cells to that of the control cells (100%). Murine Tumor Models and Treatment The female nude mice were inoculated subcutaneously into the right flank regions with 26106 HSC-3 cells/mouse resuspended in 0.1 ml of DMEM medium without serum and antibiotics. When tumors were palpable one week later, the mice were randomly assigned to six independent treatment groups (6 mice per group): PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e33860