Preparation Of PH-sensitive Antitumor Liposomes And Their Controllable And Sustained Drug Releases

As a potential drug delivery system in the early 1970s,the liposome has become one of the most important branches in the field of namomedicine and clinical application.Liposome has shown its prospective benefits in the medical and cosmetic as well as food industry for half a century.However,there still are several unsolved hadicaps to conquer.Nowadays,most of the researches focus on the embedding or suface modification on the lipidic membrane,the exploring of the liposomal aqueous inner core seems to be neglected.In the process of treatment of drug combinations,the side effects have not been attracted enough attention while pursueing higher therapeutic effect.Aiming at the current issues,the exploration on liposomal inner core and multicompartmental structure for drug combiation based on the antitumor liposomes were investigated in this thesis.A series of pH-sensitive liposome drug delivery systems were prepared from single compartment to multicompartments.And the loading content ranges from single drug component to drug combination.The in vitro drug release behavior from the drug delivery systems and their inhibitor y effect with corresponding drug component or drug combinations on tumor cells were also carried out.Firstly,pH-sensitive asymmetric liposome drug carriers were constructed.Exploiting the self-assembly of amphiphilic molecules(TPS)was used to replace the inner leaflet of lipid bilayer to make asymmetric liposomes.First,the TPS capsules were prepared in a microfluidic chip.The size of the asymmetric liposomes was determined by the flux ratio of oil phase to aqueous phase,the optmized flow velocities of oil phase and aqueous phase were 3?L/min and 1?L/min,respectively.Once the TPS capsules were formed,1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)molecules self-assembled on the surface of the TPS capusel,thus the asymmetric liposomes were formed.The stability of asymmetric liposomes was influenced by surfactant.The water in oil emution was unstable when the surfactant was insufficient,while the self assembly of lipid was hindered when the surfactant was excessive.The optmized amount of surfactant span 80 was 1wt%.The asymmetric liposomes were pH-sensitive.This property could stabilize the loaded drug in the circulation and dropped off the cargo rapidly at the target sites.The half maximal inhibition concentration of 5-fluouracil(5FU)loaded asymmetric liposomes on HeLa cells was 0.58?mol/L,while that of free drugs was 1.47?mol/L.This result validated the potential application of asymmetric liposomes as drug delivery system.Secondly,a pH-sensitive liposomes encapsulating aqueous two phase system were constructed.20 mol%Cholesterol(Chol)was doped in DPPC liposome to enhance the stability.3 wt%polyethelene glycol(PEG,20 kDa)ad 3.35%dextran(500 kDa)were chosen to make aqueous two phase system(ATPS),because of their relatively large difference in molecular weight,which drives marcomolecular repulsion.ATPS separated to two phases below the transition temperature 65?,but merged into one beyond 65?.Soluble drug such as DOX,has different partition coefficiency in each phase,DOX concentration was 8.3±0.4?g/mL in PEG phase,and 6.2±0.6?g/mL in dextran phase,while the average DOX concentration was 7.5?g/mL.Under acidic stimulus condition,DOX was released hierarchically from ATPS liposomes.The total releasing time from ATPS liposomes was 6 hour longer than that from the conventional liposomes.The ATPS liposomes improved the intake by th e cancer cells.The half maximal inhibitory efficiency of DOX loaded ATPS liposomes on HeLa cells was 0.018?mol/L,the therapeutic efficacy was 27.5 times higher than free drug.Meanwhile,ATPS liposomes had no cytotoxicity on Human umbilical vein endothelial cells.Thirdly,a pH-sensitive multicompartmental vesicles(MCVs)were constructed.MCVs were prepared to carry multiple drug components.DOX and STS were encapsulated in separated 200 nm DPPC/20 mol%Chol liposomes,and then encapsulated together in 1?m DOPC/20 mol%Chol liposomes.The encapsulating efficacy of MCVs was 55%evaluated by flow cytometry.DOX concentration and STS concentration were 0.49±0.043?mol/mL and 0.52±0.026?mol/mL respectively in MCVs.It will cause myocardial cell injury during the process of DOX treating cancer cell.The introduction of STS will not only exert synergistic effect with DOX,but also alleviate the injury by DOX.Bax,Bcl-2,and cleaved-caspase3 were chosen to assess the protective effects of STS on mice cardiac myocytes by western blotting,(DOX,STS)-MCVs achieved 90%cardiac myocyte survival rate while the cancer cell inhibitory remained 60~70%.Finally,a pH-sensitive MCVs were constructed.The MCVs were used to carry drug combination of DOX and 5FU using the same protocol mentioned above.At equivalent DOX concentration 1?mol/L,the inhibitory efficacy of DOX-5FU-LUVs was 65%,while that of DOX-5FU-MCVs was 71%.The animal model demonstrated that DOX-5FU-MCVs exhibited significant tumor inhibitory effect.On the 22 day of treatment,tumor volume of(DOX,5FU)-MCVs treated mice was 519±65 mm~3,while the tumor volume of control group was 858±373 mm~3.Slice detection were carried out on major organs by H&E staining method,no obvious abnormality or inflammation was observed.