Study On Anti-Cancer Stem-Like Cell Effects Of Liposomal Epirubicin-Metformin And ABC Phenomenon Upon Repeated Injectioh Of Liposomes

The cancer stem cell(CSC)theory suggests that the formation,recurrence and metastasis of tumor are caused by proliferation of CSC.Epirubicin hydrochloride(EPI),a synthetic anthracycline antitumor antibiotic,inhibits replication and transcription by embedding DNA strands.It also prevents topoisomerase II from flipping to achieve the purpose of inhibiting tumor cell growth and proliferation.The proliferation of CSC could be inhibited by metformin(MET)through multiple pathways.Therefore,epirubicin-metformin(EPI-MET)liposome was prepared by using transmembrane ammonium ethylenediaminetetraacetate-metformin(EDTA-MET-NH3)gradient.CD133+cells were sorted from S180 cell line by magnetic-activated cell sorting and the cancer stem-like cell characteristics was investigated.Furthermore,compared with EPI liposome prepared by the ammonium sulfate((NH4)2SO4)gradient,the anti-cancer stem-like cell effects of liposomal EPI-MET was investigated.Accelerated blood clearance(ABC)phenomenon of liposomes was studied in mice and rats upon repeated injection.In addition,we tried to modify liposomes with monosialoteterahexosyl ganglioside(GM1)in order to attenuate the ABC phenomenon produced by repeated injection of liposomes.The method of cation exchange fiber minicolumn centrifugation was employed to determine the encapsulation efficiency of liposomes.The preparation process was optimized through changing incubated temperature,incubated time,drug-to-lipid ratio,pH value,concentration of(NH4)2SO4,ammonium ethylenediaminetetraacetate and MET.The encapsulation efficiency of EPI-MET liposome was more than 97%under the optimal condition.The particle size of liposomes was measured based on the principle of dynamic light scattering.The results showed that the average particle size of EPI liposome and EPI-MET liposome prepared by(NH4)2SO4 gradient and EDTA-MET-NH3 gradient were 112.6±6.7 nm and 104.6± 5.9 nm,Zeta potentials were-23.75 ± 0.52 mV and-28.62 ± 0.74 mV,osmotic pressure were 272± 3.9 mmol·kg-1 and 290± 4.7 mmol·kg-1,pH value were 6.5± 0.1 and 6.9± 0.1.Long-term stability test showed that there were no significant changes in appearance,particle size or encapsulation efficiency when liposomes were stored at 2-8℃ away from light during six months.In vitro release test indicated that the rate of EPI release from liposomal EPI and liposomal EPI-MET was significantly reduced and slow rates of MET leakage from EPI-MET liposome was also observed.The CD133 antigen expression in S180 cell line was assayed by flow cytometry.The results indicated that approximately 1.0%of cells in the S180 cell line were CD133+cells.CD133+cells were sorted by magnetic-activated cell sorting and the majority of cells were in the G1 phase of mitosis,characteristic with higher proliferative potential in vitro and stronger tumorigenicity in vivo compared with CD 133-cells.In vitro cytotoxicity of drugs against CD133+ and CD133-cells were determined,and the results indicated that free EPI presents dose-dependent inhibition of tumor cell proliferation.Further results showed that the inhibitory effects of EPI on CD133-cells were 1.49-and 2.31-fold stronger than on CD133+cells when the doses varied from 5 to 20μmol·L-1.However,a greater level of cytotoxicity was observed in CD133+than CD 133-cells after incubation with free MET.Compared with the separate application of above two solutions,the mixed solution of EPI and MET can significantly inhibit the proliferation of CD133-cells and CD133+cells.Meanwhile,a statistically significant reduction in cell viability was observed after treatment with liposomal EPI-MET and it effectively inhibited the growth and recurrence of S180 murine sarcoma.The ABC phenomenon of liposomes upon multiple or sequential injection was investigated in Wistar rats and S180 tumor-bearing mice.The first injections of PEGylated liposomes induced the rapid clearance and leakage of subsequently injected PEGylated liposomal EPI when the doses were below 50 μmol phospholipids/kg and 1.2 mg EPI/kg.Of note,the clearance of EPI was two-to three-fold faster than the liposome itself.A similar phenomenon was observed upon sequential low-dose injection.This may contribute to the production of PEG-specific immunoglobuline M(IgM)and activation of complement.The ABC phenomenon and its accompanying rapid leakage and clearance of drug following repeated injection seriously affected the anti-tumor activity of liposomal EPI and EPI-MET.The incorporation of GM1 brings novel method to lighten the ABC phenomenon.