| China has the highest incidence rate of liver cancer throughout the world. At present, the main treatment measures include surgery, radiotherapy and chemotherapy, of which chemotherapy is of the most importance. Liver cancer is hard to be cured partly due to the drug resistance of cancer cells, which enables the cells free from being eliminated and result in the relapse and metastasis of liver cancer. Another commonly accepted reason is defined as cancer stem cell theory, which assumes that anti-tumor drugs can only kill cancer cells effectively but not cancer stem cells due to drug resistance, resulting the rising percentage of cancer stem cells in carcinoma tissues and the possibility of relapse and metastasis of liver cancer.We proposed two ways to permanently cure liver cancer based on these two reasons. The first way is to use different kinds of anti-tumor drugs in combination to cure drugs by expanding the anti-cancer spectrum. The other way is to use anti-tumor-stem-cell drugs to prevent liver cancer from relapse and metastasis by killing cancer stem cells. Based on these two ways, this research adopted a strategy of using anti-tumor drugs and anti-tumor-stem-cell drugs in combination to cure liver cancer permanently.Salinomycin(SAL) is known as an antibacterial and coccidiostat ionophore therapeutic drug, which can selectively kill multiple kinds of cancer stem cells including liver cancer stem cells. The sodium salt form of Salinomycin(SAL-Na) has similar effect. Doxorubicin(DOX) is a commonly used clinical drug for liver cancer. Considering the physicochemical property of these two drugs, nano-liposomes will be used in this research to load thesertwo drugs and deliver them to the tumor location targetedly, which reduce the toxic and side effect of these two drugs at the same time.The methodology of quantitative analysis of SAL-Na and DOX by using HPLC-DAD was established. The linear equation of SAL-Na is "A=419.4C-226.7 (r2= 0.9994)" with the linear range between 7.8 to 1000 μg/mL. The linear equation of DOX is "A=13814C+192.8 (r2=0.9997)" with the linear range between 0.19 to 50 μg/mL. The linear correlation, specificity, inter-day precision, intra-day precision, recovery rate and stability the quantitation analyzing method has been assessed and validated as reliable. This has paved the way for the assay of encapsulation efficiency, drug loading rate and in vitro releasing of drug-loaded nano-liposomes.Four kinds of liver cell lines were selected in this research. Experimental performance including cell culture, passage, cryopreservation and thawing were mastered. HepG2 tumorsphere(HepG2-TS) was screened as liver cancer stem cells by culturing HepG2 cells in serum-free medium. HepG2-TS was identified by evaluating the yield, oncogenicity and expressing of CD-133 in different passages of HepG2-TS. Results show that the yield of HepG2-TS in the third passage doubles that of HepG2 cells, the oncogenicity of HepG2-TS is 100 times higher than that of HepG2 cells on the model of nude mice and the expressing of CD133 in HepG2-TS is 5 times higher than that of HepG2 cells. Based on these data, HepG2-TS can be used as liver cancer stem cells for following experiments. As the result of in vitro cytotoxicity-test shows, SAL-Na would sensitize DOX in liver cancer cells, simultaneous using SAL-Na and DOX to kill liver cancer cells perforTS better than using them in turn and SAL-Na can selectively kill HepG2-TS while DOX have little effect on HepG2-TS but can only kill HepG2 cells. In addition, the ideal proportions for synergic effect of DOX and SAL-Na were defined as 1:1 and 1:5.SAL-Na-loaded nano-liposomes(SLN), DOX-loaded nano-liposomes(DLN) and SAL-Na-DOX-loaded nano-liposomes(SDLN) were successfully prepared by applying film dispersion method. The preparing prescription was also optimized as HSPC:CHOL:DSPE-PEG-2K=85:10:5. The structure, particle size and Zeta potential of the prepared nano-liposomes were assayed by dynamic laser scatter instrument and transmission electron microscope(TEM). Results show that the nano-liposomes we prepared were equally distributed with legible lipid bilayer structure, particle size around 100 nm and Zeta potential around -35 mV. The in vitro releasing of drug-loaded nano-liposomes were evaluated considering the stability of SAL-Na and DOX in the dissolving medium. Results show that SDLN, DLN and SLN all have slow-release effect to some extent.The intakes of drug-loaded nano-liposomes and free drugs in HepG2-TS and HepG2 cells were investigated by using DOX as fluorescence probe. Results show that intakes of drug-loaded nano-liposomes were higher than intakes of free drugs in HepG2-TS and HepG2 cells, indicating that drug-loaded nano-liposomes would kill liver cancer cells and stem cells targetedly. Cytotoxicity-test of blank liposomes, drug-loaded liposomes and free drugs were also investigated. Results show that blank liposomes have little effect on HepG2-TS and HepG2 cells, drug-loaded liposomes can keep the activity of free drugs and SLN-DLN and SDLN can keep the activity of free SAL-Na and DOX, indicating that SDLN-DLN and SDLN both have powerful killing effect on liver cancer cells and stem cells.The pharmacokinetics of SDLN, SLN-DLN and F-SAL-Na-DOX in SD mice were investigated by HPLC-TS and HPLC-DAD. Results show that SLN-DLN plays better in controlling the release of SAL-Na and DOX while SDLN plays better in keeping the loading proportion of DOX and SAL-Na. Secondly HepG2 liver cancer nude mice model was established. The anti-tumor effect and drug toxicity of PBS, blank liposomes, F-SAL-Na, SLN, F-DOX, DLN, F-SAL-Na-DOX, SLN-DLN and SDLN were investigated. Results show that blank liposomes have no significant toxicity and SLN-DLN and SDLN perform the best among these groups with the anti-tumor rate of 76.46±5.49% and 71.55±5.26% respectively. SLN-DLN and SDLN have the highest anti-tumor activity without significant toxicity. At last, anti-cancer activity of all the prescriptions mentioned above has been tested by culturing HepG2-TS from the cancer tissues in vitro. Results show that SLN-DLN and SDLN can inhibit the number and volume growth of HepG2-TS, indicating that SLN-DLN and SDLN prescription possess strong inhibition activity upon liver cancer stem cells.In summary, SLN-DLN and SDLN can both simultaneously play killing effect on liver cancer cells and stem cells with the best activity, among which SDLN has the advantage of simple formulation over SLN-DLN. This provides a prospective clinical choice for the therapy of liver cancer. |
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