Codelivery Of Salinomycin And Chloroquine Using Nanoliposomes For Targeting Both Liver Cancer Cells And Cancer Stem Cells In Vitro

China is the country with high incidence of hepatocellular carcinoma which is lack of effective treatment at present and it is urgent to discover new therapeutic methods. Liver cancer stem cells(LCSCs) are considered to be the main cause of recurrence, metastasis and multi-drug resistance of hepatocellular carcinoma, so it is crucial for treatment of hepatocellular carcinoma to eradicate LCSCs. Recently, it has been reported that Salinomycin(SAL) has effects on various Cancer stem cells(CSCs) and it can inhibit proliferation and induce apoptosis of human hepatocellular carcinoma cells in vitro and in vivo. Therefore, we use SAL as the chemotherapy drug to eradicate LCSCs.However, evidence exists indicating that non-CSCs in the tumor can spontaneously and stochastically turn into CSCs de novo, undermining the efficacy of therapeutic strategies that only target CSCs. Hence, it is crucial to eliminate CSCs and non-CSCs simultaneously for effective cancer therapy. Recently, combination therapy has been developed for efficient therapy. At present, a lot of studies have demonstrated that autophagy inhibitor in combination with chemotherapy drugs could be effective in the treatment of tumors. Therefore, we combined SAL with chloroquine(CQ), which is an autophagy inhibitor, to eliminate LCSCs and non-LCSCs simultaneously through the inhibition of autophagy by CQ, as well as reduce the toxic side effects of SAL by decreasing its dosage.However, the two drugs in combination with three functions can be generated, i.e. synergism, additivity and antagonism. Therefore, we need to optimize their best synergistic ratios. But, owing to the differences in pharmacokinetic profiles of the drugs in a combination regimen, the synergistic drug ratios may not be attained in the tumor site. The use of nanoliposomes has been shown to maintain the desirable, synergistic drug ratios of the encapsulated drug combinations to achieve increased antitumor activity. Studies have demonstrated that simultaneous delivery systems show more significant therapeutic efficiency than administration of single drug loaded nanoparticles. More importantly, there is evidence demonstrating that co-delivery of two agents may have a synergistic effect on cancer therapy, which is not observed with a simple physical mixture of two individual drug loaded nanoparticles. Therefore, SAL and CQ were co-encapsulated at synergistic ratio in a nanoliposome formulation to enhance the antitumor effects and reduce the toxicity of SAL.In the first part, HepG2 tumor spheres(HepG2-TS) were isolated from HepG2 cells with characteristics of stem cells which are cultured in stem cell conditioned medium. Then the cytotoxicity of SAL and CQ were investigated in HepG2 cells and HepG2-TS. The IC50 values of SAL were 5.200±0.719 μM and 0.321±0.079 μM in HepG2 cells and HepG2-TS, respectively. The IC50 values of CQ were 10.531±1.322 μM and 18.687±3.018 μM in HepG2 cells and HepG2-TS, respectively. These results indicated that SAL had a selectively effect on HepG2-TS. Finally, we examined the activity of SAL and CQ combinations with different concentrations and ratios in HepG2 cells and HepG2-TS. As a result, we confirmed that the optimum synergistic ratio of combination was SAL:CQ=1:5(mol/mol).In the second part, we have established a method for determination of the contents of SAL and CQ in vitro, which was then evaluated for its methodology. The results showed that the concentrations of SAL and CQ had a good linear relationship with their peak areas in the concentration range of 10 ~ 1280 μg/ml and 5 ~ 320 μg/ml, respectively. The linear regression equation was A=1028C-661.1(R2=1) for SAL and A=38435C-46890(R2=0.9999) for CQ. Moreover, the specificity, precision and accuracy of the method can meet the requirements. Therefore, it was adopted as a method for the determination of drug contents in the following experiments.In the third part, nanoliposomes composed of the hydrogenated soybean phospholipid(HSPC), cholesterol, and DSPE-PEG2000(85:10:5 mole ratio) were prepared by ethanol injection method and ammonium sulfate gradient method. Then, blank nanoliposomes(NL), SAL loaded nanoliposomes(SNL), CQ loaded nanoliposomes(CNL) and SAL and CQ loaded nanoliposomes(SCNL) were prepared. The nanoliposome obtained was of uniform dispersion and round in shape whose size was about 120 nm and zeta potential was-13.7 ~-17.1 mV. The encapsulation efficiency(EE) of SAL in SNL was about 10% and the drug loading(DL) was about 3%. EE of CQ in CNL was about 70% and DL was about 10%. EE and DL of SAL and CQ in SCNL were similar to SNL and CNL. Furthermore, the molar ratio of SAL and CQ was about 1:5 in SCNL.In the fourth part, we evaluated the effects of drugs and nanoliposomes on HepG2 cells and HepG2-TS. CCK8 and Annexin V-FITC/PI double staining were used to detect the cytoxicity and apoptosis, respectively. Besides, we also conducted the clone formation experiment to evaluate the proliferation of HepG2 cells. The results showed that SAL combined with CQ in the synergistic ratio could significantly increase the cytoxicity effects on HepG2 cells, promote apoptosis and inhibit proliferation compared with SAL or CQ alone, but had no enhancement on HepG2-TS. Moreover, SNL combined with CNL or SCNL alone exhibited the strongest cytoxicity effects on HepG2, inducing apoptosis and proliferation inhibition, as well as HepG2-TS. There was no statistical difference between SNL combined with CNL and SCNL.In the fifth part, we firstly used Western-Blot to measure the basal autophagy flux in HepG2 cells and HepG2-TS. It was found that autophagosome synthesis was signifiantly greater in HepG2 cells than in HepG2-TS. Next, we examined the different groups(SAL, CQ, SAL+CQ, NL, SNL, CNL, SNL+CNL and SCNL) effects on the basal autophagy flux of HepG2 cells and HepG2-TS. It was found that each group had a strong reduction in the level of the basal autophagy flux in HepG2 cells, which was not observed in HepG2-TS. This explains the results of the fourth part, that is because of the high levels of the basal autophagy flux in HepG2 cells, while autophagy plays a protective role in tumor cells, therefore, it can enhance the antitumor activity through the autophagy inhibition when SAL combined with CQ in HepG2 cells. However, owing to the low levels of the basal autophagy flux in HepG2-TS, so the inhibition of autophagy can not enhance its antitumor activity.In summary, SCNL had strong effects on liver cancer cells and LCSCs in vitro. In addition, it was preliminary demonstrated that CQ could enhance the antitumor activity of SAL by inhibiting autophagy in HepG2 cells.