A Novel Co-loading Vehicle For Delivering Both Nanoplatin Cores And Bmi-1 SiRNA To Treat Hepatocellular Carcinoma

Objectives: Hepatocellular carcinoma(HCC)has high malignance level and has difficulties in diagnosing at early phase.Besides,the therapeutic method is lacking.The five year survive rate of HCC is 10.1%.The problem of drug resistance attracts lots of attention and it is the reason for the poor outcoming in cancer treatment.The combination of different therapies was a common way to kill cancer cells with multiple targets.With the rapid development of nanotechnology,the combination therapy and the targeting nano pharmaceutics were introduced in the anti-cancer research which could enhance the effect and reduce the toxicity.In the current study,we established a drug delivery system for cisplatin and Bmi-1 siRNA with calcium phosphate nanoparticles.On the one hand,the nanoparticles can accumulate at tumor side via EPR effect and release drug in acidic environment,on the other hand Bmi-1 siRNA silences the drug-resistance related gene to inhibit the growth of cancer cells.Methods: 1.HepG2 cells were given cisplatin and gradually increased the concentration to 2 μM,and kept the concentration at 2 μM in 3 weeks.We determined Bmi-1 level in the cells exposed to cisplatin and we studied the cell sensitivity to drug with MTT assay.2.Preparation of(nanoplatin and siRNA co-loaded Ca P nanoparticles)NPSC: cisplatin,Bmi-1 siRNA and calcium phosphate were precipitated in w/o microemulsion and formed the cores with the diameter of 20 nm.Then DOTAP,cholesterol and DSPE-PEG2000 were coated on the surface to form the outer lipid.We determined morphology,size,encapsulation efficiency,silencing effect and stability of siRNA with the ways of FTEM,atomic absorption spectrophotometer and Western Blot.3.The study of effect in vitro and in vivo: we detected the biodistribution of NPSC by small animal imaging;we evaluated the delivery effect of NPSC by fluorescence labeling,MTT assay and Western Blot;we investigated the anti-tumor effect and safety of NPSC with HepG2 xenograft tumor bearing mice.Results: After incubating with cisplatin for several weeks,Bmi-1 level increased and the sensitivity of HepG2 to cisplatin declined.The inhibition rate of cisplatin increased when we silenced Bmi-1.NPSC had the diameter of 50 nm,and it released drug in the acidic environment.The encapsulation efficiency of cisplatin and Bmi-1 siRNA were 24.3 ± 5.4 % and 88.1 ± 3.5 %,respectively.Compared with free drug,NPSC achieved higher cell uptake level.The results of Western Blot assay suggested that Bmi-1 siRNA in NPSC effectively silenced Bmi-1 gene.NPSC elevated the concentration of drug in tumor tissue and showed significant inhibition of tumor growth in vivo.Besides,the cisplatin concentration in kidney of NPSC was lower than free drug which avoided the injury of kidney.Conclusions: These results suggested that NPSC enhanced the drug sensitivity of cancer cells by the combination therapy of cisplatin and Bmi-1 siRNA,and improved the targeting ability of free drug by lipid coated calcium phosphate nanocarriers.The introducing of nucleotide drugs in the nano delivery system provided strategy to overcome chemoresistance and side effects.