Novel Lipid-based Nanosuspension Enhanced Synergistic Cancer Therapy Of Ceramide And Docetaxel

Cancer is the leading cause of deaths worldwide, seriously threatened human health. Due to the complexity of tumorigenesis and the difficulty of cancer therapy, combination therapy arose in response and was widely used in clinical cancer treatment. Rational combination therapy could generate synergistic anti-tumor effect, enhance therapeutic efficiency and reduce side effects. For combination therapy, it is critical to select the suitable agents to be combined. When evaluated in preclinical studies, some biologic agents plus cytotoxic drugs exhibit additive or synergistic activity without excessive toxicity, providing a promising direction for combination therapy.Ceramide (CE) is a naturally occurring membrane sphingolipid, which was considered as a novel biologic agent that mediate diverse cellular effects, such as cell growth, differentiation and death. Besides, CE could enhance the response of cytotoxic drug, demonstrating great potential in combination therapy. However, there are not systematic studies on the combination of CE with cytotoxic drugs, therefore, it is urgent to develop the CE-based combination therapy.Based on this situation, in our present study, we aim to develop the CE-based combination therapy, explore the synergy mechanism of CE and Docetaxel (DTX), and develop an innovative "two-in-one" lipid based nanosuspension (LNS) system to achieve the synergistic anti-caner efficiency. The main methods and results were as follows: 1. Screening of CE-based combination therapyThere is an urgent need to investigate and develop a comparatively comprehensive strategy on CE-based combination therapy. In present study, the in vitro anti-proliferation effects of CE combining with three traditionally and widely used anti-cancer drugs:DTX, Paclitaxel (PTX) and Doxorubicin (DOX) were correspondingly evaluated on four different cancer cell lines:murine malignant melanoma cell line (B16), human breast carcinoma cell line (MCF-7), human ovarian carcinoma cell line (SKOV3) and human hepatocellular carcinoma cell line (HepG2), respectively, by MTT assay. And then the combination index (CI) was further calculated to analyze whether there is a synergistic effect between CE and the chosen anti-cancer drug. To optimize the dosing schedule, the experiments of screening for optimal combination ratio and sequence of administration were subsequently carried out by MTT assay and CI assay. As the results showed, when comparing with the other anti-cancer drugs, DTX was most synergistic with CE on B16(CI=0.47) and MCF-7cells (CI=0.71). When CE and DTX combined with a molar ratio of0.5:1, the lowest CI values were obtained on B16(CI=0.31) and MCF-7cells (CI=0.48), respectively. There was no significant difference on the anti-proliferation effects between different sequential treatments (p>0.05). Taken together, simultaneous administration of CE and DTX with a molar ratio of0.5:1was finally determined as the optimal dose schedule for later studies.2. Study on the synergy mechanisms of CE+DTXTo confirm the synergy between CE and DTX, provide more scientific theoretical basis for later study, the cell apoptosis induction, Caspase-3activity, cell cycle arrest and cytoskeleton destruction were systematically studied to explore the mechanisms of synergy between CE and DTX. In order to verify the in vivo synergy effects, the in vivo antitumor efficacy of CE+DTX was also experimented. The results showed that CE could target the microfilament actin, leading to the polymerization and destruction of actin cytoskeleton, while DTX could target and disrupt the microtubules cytoskeleton. Significantly, CE plus DTX could cause a synergistic destruction of cytoskeleton, which resulted in a significant higher apoptosis and a significant higher arrest in G2/M arrest comparing with either agent alone (p<0.01). The in vivo antitumor study also verified the synergy between CE and DTX, as CE+DTX could achieve a significant higher antitumor efficacy (64.92±0.22%) than either agent alone (p<0.01). All these results established a proof-of-concept that a rational combination of CE and DTX could generate synergistic effects on cancer treatments.3. Determination of drug content of ceramide-docetaxel loaded lipid-based-nanosuspensionThe DTX concentration was determined by HPLC method and its methodology was examined. The results showed that it was an easy, convenient and correct method to determine the DTX concentration in vitro. The CE concentration was determined by fluorescence spectrofluorimetry method and its methodology was also examined. The results showed that, with high sensitivity, good selectivity and convenience, fluorescence spectrometry can accurately determine the content CE.4. Novel lipid-based nanosuspension enhanced synergistic cancer therapy of ceramide and DocetaxelAlthough CE+DTX for cancer treatment has received increasing attention, both of them have difficulties in formulation due to their poor water solubility. For example, Duopafei(?) and Taxotere(?), the clinical formulation of DTX, is formulated in Tween-80and ethanol, which has been found to cause serious side effects such as hypersensitivity reactions, nephrotoxicity and cardiotoxicity. In our present study, a non-toxic "two-in-one" LNS system (LNS loaded with both CE and DTX, CE+DTX-LNS) was prepared with high pressure homogenization method and the lyophilized CE+DTX-LNS was subsequently prepared by freeze drying method. Particle size, size distribution, zeta potential and morphology were evaluated, respectively. In vitro drug release was assessed by the dynamic dialysis method. The in vitro synergistic anti-proliferation effect of CE+DTX-LNS was investigated by MTT and Caspase-3study. NBD-CE (10%, w/w) and Rhodamie-DTX (10%, w/w) labeled CE+DTX-LNS was prepared and the biodistribution of CE+DTX-LNS was evaluated by in vivo fluorescence imaging technology. Co-delivery of CE and DTX to same tumor cell with CE+DTX-LNS was evaluated by fluorescence microscope and FACS Calibur flow cytometer. The in vivo antitumor efficacy was also evaluated on B16tumor bearing mice to investigate the synergistic anti-tumor effect of CE+DTX-LNS and determine the relationship between the co-delivery efficiency and synergistic anti-tumor effect.The morphology of CE+DTX-LNS was approximately spherical and the particle size, zeta potential and drug loading were (108.1±3.42) nn,(-23.25±0.49) mV, and11.32±0.11%respectively. After lyophilized, the stability of CE+DTX-LNS was obviously improved. Both the release behavior of CE and DTX from CE+DTX-LNS were sustainedly and independently. Comparing with CE (or DTX) solution, the cytoxicity caused by CE-LNS (or DTX-LNS) was significantly increased (p<0.01), indicating the high delivery efficiency of LNS. The Caspase-3level activated by CE+DTX-LNS was significantly higher than that of CE-LNS and DTX-LNS (p<0.01), demonstrating the synergistic induction of apoptosis. CE+DTX-LNS could effectively deliver CE and DTX to same tumor cell, while the cells treated with the physical mixture of CE-LNS and DTX-LNS (CE-LNS+DTX-LNS) were separated into two cell populations, indicating that CE and DTX were separately delivered to different cancer cells. Both the cellular uptake of CE+DTX-LNS and CE-LNS+DTX-LNS was in a dose dependent manner. The highest cellular uptake of CE+DTX-LNS was72.6±4.2%, significantly higher than that of CE-LNS+DTX-LNS (64.5±2.7%, p<0.05), suggesting that the co-delivery of CE and DTX to same tumor cell was the basics for CE promoting the cellular uptake of CE+DTX-LNS by interacting with cell membrane. The in vivo fluorescence imaging results showed that, CE+DTX-LNS could effectively prolonged the in vivo circulation time and passively targeted to the tumor sites by EPR effect. The in vivo anti-tumor effiency of CE+DTX-LNS was93.94±2.77%, significantly higher than that of CE-LNS (33.22±5.43%, p<0.01), DTX-LNS (69.32±3.65%, p<0.01) and Duopafei(?)(59.91±4.54%, p<0.01), demonstrating the synergistic therapeutic effect in vivo. Meanwhile, the anti-tumor effiency of CE+DTX-LNS was also significantly higher than that of CE-LNS+DTX-LNS (84.94±3.76%, p<0.05), confirming that the co-delivery of CE and DTX to same tumor cell was the basis for synergistic therapeutical effect.In conclusion, this study made a comparatively comprehensive exploration in the field of CE-based combination therapy and the synergy mechanisms of CE+DTX were firstly elucidated, providing a theoretical basis for further study. Moreover, the CE+DTX-LNS prepared in our study could effectively co-deliver CE and DTX into same tumor cell and synergistically inhibit tumor growth, reduce toxicity, holding the great potential to be an appropriate choice for clinical application.