| Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the second most frequent causes of cancer death worldwide. Liver cancer burden, however, is not evenly distributed throughout the world. China alone accounts for more than 50％of the world’s cases. Surgical treatment is considered the most effective method to treat both the HCC and the underlying liver disease from which most cases of HCC develop. However, more than 80％of patients with HCC are diagnosed at advanced stages of the disease because of the long incubation period, high malignant degree and rapid development of tumor. Chemotherapy is still one of the most important therapy methods for HCC. While, the five-year survival for liver cancer in china is only around 10.1％. Molecular targeted therapy is now the new method and tendency in the treatment of HCC.Sorafenib is a small multi-kinase inhibitor, it significantly improved overall survival (OS) and time to progression compared with placebo in patients with advanced HCC in a randomized, placebocontrolled, phase Ⅲ trial as well as in a trial conducted in the Asia-Pacific region. Sorafenib was approved by FDA (U.S. Food and Drug Administration) for the treatment of HCC in 2007 based on the survival advantage of pivotal clinical trial studies, and it was recommended as the first-line drug for HCC therapy by multiple guides such as NCCN (National Comprehensive Cancer Network) and AASLD (American Association for the Study of Liver Diseases). Currently, sorafenib is administered orally and the recommended dosage is 400 mg, twice daily. Dose reduction or temporary discontinuation occurs frequently due to the unavoidable adverse events, such as gastrointestinal irritation (even major bleeding episodes), diarrhea, and hypertension. Therefore, effective delivery and reduction of systemic side effects of sorafenib become impotent for apply of sorafenib in HCC treantment. Rapid growth in nanotechnology towards the development of nanomedicine products holds great promise for cancer therapy.As one promising approach, lipid-based nanosuspensions (LNS) show unique advantages. First, the injectable phospholipids used as the stabilizer ensure the better biocompatibility. Second, nanosuspensions showed high drug loading capacities. Third, sorafenib has a high lipophilicity (log P= 3.8); thus, phospholipids is an appropriate stabilizer to ensures the great compatibility with sorafenib, Furthermore, LNS selectively permeate the tumor vasculature and remain in the tumor interstitium for an extended period of time led to the characterization of the tumor-selective EPR effect, thus exhibit higher accumulation in tumor tissues.In the present study, sorafenib-loaded lipid based namosuspensions (sorafenib-LNS) were prepared to be expected to enhance therapeutic efficiency and reduce gastrointestinal irritation of sorafenib, simultaneously. The main methods and results were as follows:1. Determination of drug content of sorafenib-LNSHigh Performance Liquid Chromatography (HPLC) was chosen for the determination of sorafenib content in vitro, and the methodology of HPLC determination was examined. The specificity, the linearity, RSDs of intra-day and between-day, the accuracy all satisfied the need of analysis.2. Preparation and characterization of sorafenib-LNS) and lyophilized sorafenib-LNS in vitroSorafenib-LNS were prepared by the nanoprecipitation method. The formulation and process were optimized by the single-factor research and orthogonal design, respectively. The drug loading of sorafenib-LNS was 10.55±0.16%. The mean particle size and poly-dispersity index (PDI) of the three batches of sorafenib-LNS were (164.5±4.5) nm and 0.202±0.015, respectively. The zeta potential was (-11.0 ± 0.2) mV. Mannitol (5％, W/V) was added to the fresh prepared sorafenib-LNS as lyoprotectant to get the lyophilized sorafenib-LNS.The in vitro release profile of sorafenib was evaluated by the dialysis bag diffusion technique. Phosphate buffered saline (PBS, pH=7.4) was chosen as the release medium, and of Tween-80 (1％,w/v) was added as solubilizer. The centrifuge tubes were placed in a shaking incubator with a stirring speed of 100 rpm at a temperature of 37 ℃. Cell growth inhibition efficiency was assessed on HepG2 and BEL-7402 cells by MTT assay. Hemolysis assay was also implemented. The cumulative release rate of sorafenib from sorafenib-LNS within 10 h was relatively quick which may was caused by the large surface area-to-volume ratio of LNS. Thereafter, the sorafenib was released relatively slow within the initial 48h. This sustained release could mainly result from the gradual dissolution of the lipid skeleton of the LNS. Sorafenib-LNS exhibited higher cytotoxicity than sorafenib solution against HepG2 and Bel-7402 cells at high concentrations(5μM) (p<0.01). Furthermore, the IC50 value of sorafenib-LNS was significantly lower than that of sorafenib solution in both cell lines (p<0.01). These results suggest that LNS is a promising nanocarrier for improving the in vitro cytotoxicity of sorafenib. In addition, no hemolysis or aggregation phenomenona was observed for the different concentrations of sorafenib-LNS, indicating the good hemocompatibility under preliminary experimental conditions.3. Biodistribution, in vivo anti-tumor study, and in vivo irritation evaluation of sorafenib-LNSBiodistribution experiments were carried out in Kunming mice, previously inoculated with H22 cells. To observe the biodistribution of LNS formulation over time, real-time near infra-red fluorophore (NIRF) imaging was used to monitor the particle fate. A NIRF dye, DiR, replaced sorafenib in the formulation. Furthermore, the in vivo anti-tumor efficacy evaluation, histological observation, and in vivo vascular irritation were also investigated.In H22-bearing Kunming mice, the results of biodistribution experiments demonstrated that The overall targeting efficiency (Te) of LNS in the tumor was increased from 5.96％(free sorafenib) to 7.28%(1.22 times), and the relative targeting efficiency (re) and the maximum concentrations (Ce) in the tumor were 2.26 and 1.67, respectively, indicating the tumor accumulation property of LNS. The tumor accumulation property was further verified in the in vivo imaging study. Sorafenib-LNS (9 mg/kg) exhibited higher anti-tumor efficacy compared with the sorafenib oral group (18 mg/kg) (p<0.05) and the sorafenib injection group (9 mg/kg) (p<0.05), respectively. The in vivo vascular irritation study, hemolysis assay and the normal cytoarchitecture of the tissue in the histopathological study indicated the good biocompatibility of sorafenib-LNS in vivo.In this study, sorafenib-LNS were successfully prepared, which hold several advantages including uniform size distribution, high drug loading, and good stability. Sorafenib-LNS could achieve significant accumulation in tumors, and exhibited increased therapeutic efficacy both in vitro and in vivo. Thus, these results suggested that sorafenib-LNS provided promising approach for intravenous administration of sorafenib. |
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