The Construction And Evaluation Of Drug Delivery System Based On Docetaxel Prodrug Self-assembled Nanoparticles And Liposomes

DTX is one of the promising new generations of antitumor drugs to exert its remarkable anticancer effects towards a broad variety of tumor types.However,the water-solubility of DTX is poor.Taxotere?,the commercial formulation of DTX with Tween 80 and 13%(w/w,ethanol/water)solution of ethanol as solvents caused serious adverse reactions such as hemolysis and hypersensitivity.The tumor targeting efficiency of DTX-Sol is not enough.In order to explore a safe and efficient drug delievery system,this research designed and synthesized the reduction-sensitive DTX prodrug with disulfide bond as linker that could self-assemble in water to form DSNPs.And we encapsulated DTX-S-S-VE into DSPE-PEG2000 modified liposomes to form DPLs.Because of EPR and reduction condition in tumor micoenvironment,nano-preparations demonstrated more effective on tumor targeting,and triggered the rapid release of DTX within tumor cells to achieve antitumor efficacy.The main contents of this thesis included the synthesis and identification of DTX prodrug,the preparation and properties,in vitro antitumor antivity experiments,in vivo pharmacokinetics,in vivo biodistribution experiments and in vivo pharmacodynamic behavior of nano-preparations.DTX and VE had been selected as the anticancer model drug and carrier,respectively.A reduction-sensitive DTX prodrug(DTX-S-S-VE)with disulfide bond as linker was synthesized.At the same time,a reduction-insensitive DTX prodrug(DTX-VE)was synthesized as the control.Their chemical structures were verified by FT-ICR MS,1H-NMR and FT-IR methods.Self-assembled nanoparticles of DTX prodrug was prepared by the molecular assembly technology.DPLs were prepared using the thin-film dispersed hydration method.The physicochemical properties of the nanopreparations were investigated.The EE and DL of DTX prodrug of DSNPs and DNPs were all over 97%and 75%.The NPs were spherical with uniform size.The EE and DL of DTX prodrug and diameter of DPLs were 97.6%±0.0300%,7.09%±0.227%and 93.1 ± 0.716 nm,respectively.DPLs were spherical with uniform size and clearly visible phospholipid bilayer.The in vitro release study of nanopreparations in pH 7.4 PBS was investigated by the dialysis method.And the DTX was released from the nanopreparations in a GSH concentration-dependent manner,released rapidly at high GSH concentration but slowly at physical condition.The release rate of DTX from DSNPs was much faster than DNPs,indicating that the DTX was easily released from DTX-S-S-VE with disulfide bond as linker.The in vitro release behavior of nanopreparations in rat plasma was investigated.These nanopreparations were stable under physiological and extracellular conditions,which provided an effective drug delivery system to improve the stability of DTX prodrug.The cell viability assay,cellular uptake,endocytosis pathway,cellular distribution,cell cycle and apoptosis on A549 cells and PC-3 cells of DTX-Sol,DSNPs,DNPs and DPLs were investigated.The blank vehicle showed no apparent toxicity.All the nano-preparations appeared a time-dependent and concentration-dependent inhibition on A549 cells and PC-3 cells.The UPLC-MS/MS methord was used to determine the uptake of preparations in A549 cells and PC-3 cells.All the nano-preparations were taken into the cells in a time-dependent and concentration-dependent manner.The uptake of DSNPs,DNPs and DPLs in both cells was much lower than that of DTX-Sol.The DSNPs and DPLs accumulated more DTX in both cells in comparison with DNPs.The endocytosis mechanisms of DSNPs and DPLs were energy-dependent,and the DSNPs and DPLs were endocytosised in both cells through multiple ways.In comparison with DTX-Sol,the cells treated with DSNPs and DPLs appeared the similar inhibition of cell cycle and apoptosis rate.DSNPs and DPLs appeared a much higher antitumor efficacy than DNPs.UPLC-MS/MS method was used to determine DTX-Sol,DSNPs,DNPs and DPLs in rat pharmacokinetics after intravenous injection.DNPs showed no improvement on the pharmacokinetic parameters of AUC(0-t)and t1/2 compared with DTX-Sol.The AUC(0-t)and t1/2 of DSNPs and DPLs were 6.14,14.5,9.91 and 1.36-fold higher than that of DTX-Sol,indicating that DSNPs and DPLs could significantly improve the blood concentration of DTX and prolong the blood circulation time of DTX.The tissue biodistribution behaviors of DSNPs in the A549 tumor-bearing nude mice were investigated by the in vivo fluorescence imaging and UPLC-MS/MS method.These results of the in vivo fluorescence imaging demonstrated that the DiR-DSNPs significantly changed the distribution performances of NPs in nude mice.The quantitative results of tissue biodistribution showed that DSNPs were more distributed into liver and spleen,while decreased the distribution of DTX in heart,lung,and kidney than DTX-Sol.The accumulation of DTX in tumor had no significant difference between DSNPs and DTX-Sol.The tumor uptake of DPLs was much higher than that of DTX-Sol and DSNPs,and this high tumor accumulation of DPLs would improve their therapeutic efficacy in vivo.The in vivo antitumor activity of DTX-Sol,DNPs,DSNPs and DPLs was assessed in A549 tumor-bearing nude mice model.The tumor inhibition rates of DSNPs and DPLs were 1.45 and 2.03-fold higher than that of DNPs;DPLs showed the best antitumor efficacy and decreased the toxicity compared with DTX-Sol.In comparison with DTX-Sol,DSNPs had lower tumor inhibition rate(p<0.01).Due to the reduced acute toxicity of the DTX and the improved quality of life of nude mice,the DSNPs showed better advantage for long-term cancer therapy.