| Anti-breast cancer liposomes have obvious advantages on reducing the toxicity and increasing the intratumoral retention time,but there are still some limitations in prolonging the patient survival time.Based on the previous study for antitumor drugs delivery,it has been found that the abnormality of tumor microenvironment leading to a difficulty to deliver drugs to the deep tumor are the main reasons to the poor efficacy of liposomes.In this study,tanshinone ⅡA sodium sulfonate capable ofimproving blood circulation and removing blood stasis was introduced as the"microenvironment regulator";Besides,the folic acid modified microemulsions were used as the "main force" for targeting and consequently killing the tumor.We employed the sequential assembly technology to build a hierarchical "targeted-release" liposomal complex system,tanshinone ⅡA sodium sulfonate and celastrol microemulsion could be released at the peripheral tumor in the intimal stage:the former could normalize the tumor microenvironment and subsequently improve the drug delivery environment;the latter was able to penetrate into the deep tumor tissue and recognize the tumor cells by virtue of the small size and folic acid modification respectively,and thereby of releasing celastrol and coix seed oil to kill tumor cells synergistically and accurately.Under the guidance of of traditional Chinese medicine theory,we integrated the advantages of circulating blood and anti-tumor traditional Chinese medicine,and developed a liposomal delivery system with "deep penetration of tumor" and "accurate tumor targeting" for the treatment of breast cancer.It presented a new idea and method to deliver multicomponent of traditional Chinese medicine for the treatment of breast cancer.The specific results of this study are as follows:1.Preparation and physical and chemical characterization of folic acid modified celastrol microemulsion-tanshinone II A sodium sulfonate liposomes((FA)Cel-MEs/STS-LPs)In this chapter,we firstly prepared folic acid-modified celastrol microemulsions,characterized the physical and chemical properties and then evaluated the encapsulation efficiency of tanshinone II A liposomes,celastrol liposomes,tanshinone II A sodium sulfonate liposomes.Besides,we also co-embedded folic acid modified celastrol microemulsions and tanshinone II A sodium sulfonate into liposomes and characterized its physicochemical properties.1.1 Preparation and Characterization of Celastrol microemulsions(Cel-MEs)and folic acid modified celastrol microemulsions((FA)Cel-MEs)Celastrol microemulsions(Cel-MEs)and folic acid modified celastrol microemulsions((FA)Cel-MEs)were prepared by "one step creates microemulsions",the preparated microemulsions were clear and transparent,with particle sizes of 27.32±0.24 nm and 38.96±0.32 nm,respectively.PDI were 0.051±0.003 and 0.064±0.008;Zeta potentials were-11.0±0.9 mV and-12.2±1.1 mV,respectively.The microemulsion with uniform size and shape was observed by transmission electron microscopy(TEM).1.2 Optimization and characterization of tanshinone ⅡA liposomes,celastrol liposomes and tanshinone ⅡA sodium sulfonate liposomesThin film dispersion film and reverse evaporation method were used to evalualte the encapsulation efficiency of tanshinone ⅡA liposomes,celastrol liposomes and tanshinone II A sodium sulfonate liposomes.Celastrol liposomes and tanshinone ⅡA sodium sulfonate liposomes had a higher encapsulation efficiency under the reverse evaporation method than the thin film dispersion.Both two preparations had a similar result in the encapsulation efficiency of liposomes were celastrol liposomes>tanshinone II A sodium sulfonate liposomes>tanshinone ⅡA liposomes.Celastrol liposomes prepared by 80%phospholipid had a highest entrapment efficiency among all the test groups.Tanshinone ⅡA sulfonate liposomes developed using 92%phospholipid had the highest entrapment efficiency and tanshinone ⅡA liposomes with 50%phospholipid had the highest entrapment efficiency among all the phospholipids.1.3 Preparation and Physicochemical Characterization of folic acid modified celastrol microemulsion-tanshinone II A sodium sulfonate liposomes((FA)Cel-MEs/STS-LPs)(FA)Cel-MEs/STS-LPs were prepared by thin film dispersion method.The mass ratio of microemulsion to liposome was 1:2,2:1,the particle size was 98.1±2.4 nm,99.3±1.0 nm and PDI were 0.221±0.012,0.224±0.005,respectively.The drug release rate of(FA)Cel-MEs/STS-LPs and Cel/STS-LPs was investigated by constant temperature constant velocity dialysis.The release of tanshinone II A sulfonate in(FA)Cel-MEs/STS-LPs and Cel/STS-LPs in vitro was consistent with the first-order release equation.Both in the early release of drug release rate faster,slow release in the late release until the platform period.The cumulative release rate of STS in both(FA)Cel-MEs/STS-LPs and Cel/STS-LPs were over 98%.The cumulative release of celastrol in Cel/STS-LPs was significantly higher than that in(FA)Cel-MEs/STS-LPs whichi was 91.5±2.8%and 68.3±3.7%,respectively.2.Cytotoxicity of folic acid modified celastrol microemulsion-tanshinone II A sodium sulfonate liposomes on MCF-7Blank LPs,blank MEs and blank MEs-LPs were nearly no toxic on MCF-7 and L-02 cells under predetermined dosage.The IC50 of blank LPs on MCF-7 cells and L-02 cells were 2377.0±425.0μg/mL and 2751.0±566.0 μg/mL,respectively.The IC50 of blank MEs-LPs on MCF-7 and L-02 cells was 1060.0±105.3 μg/mL and 718.0±47.4 μg/mL,respectively.The IC50 of blank MEs on MCF-7 and L-02 cells were 129.0±14.7 μg/mL and 120.4±12.0 μg/mL,respectively.Celastrol,Cel-MEs,Cel-LPs(SPC),Cel-LPs(DPPC+SPC),Mixture,(FA)Cel-MEs,Cel-MEs/STS-LPs and(FA)Cel-MEs/STS-LPs inhibited the proliferation of MCF-7 cells significantly and were positively correlated with the concentration of celastrol.Compared with Celastrol group,Cel-MEs had higher toxicity on MCF-7 cells,which were 1.046±0.132 pM and 0.765±0.127 μM at 24 h and 48 h,respectively.There was no significant difference among(FA)Cel-MEs,Cel-MEs/STS-LPs,(FA)Cel-MEs/STS-LPs and Mixture groups.Celastrol,Cel-MEs,Cel-LPs(SPC),Cel-LPs(DPPC+SPC),Mixture,(FA)Cel-MEs,Cel-MEs/STS-LPs and(FA)Cel-MEs/STS-LPs inhibited the proliferation of human lung cancer A549 cells significantly and had a positive correlation with the concentration of celastrol.3.Uptake of folic acid modified celastrol microemulsion-tanshinone II sodium sulfonate liposomes on MCF-7 cellsFITC mixture had stronger fluorescence intensity than FITC group,the fluorescence intensity of FITC was 33.12±0.5,FITC mixture were 119.5±11.6,indicating that a certain amount of excipients can significantly promote the uptake the cells;when FITC were prepared into microemulsion or liposomes,the cell uptake was significantly increased,FITC-MEs and FITC-LPs uptaken by cell uptake were 39.1 and 39.4 times than the FITC group,respectively.After the modification of folic acid,the cell uptake of microemulsion was further increased by 2.6 times,suggesting that the modification of folic acid indeeded enhance the uptake of MCF-7 cells,which may be associated with the folate receptor on MCF-7 cell surface.In addition,(FA)FITC-MEs-LPs were uptaken by MCF-7 cells was 1.6 times higer than FITC-MEs-LPs.4.Study on the induced MCF-7 cells apoptotic rate of folic acid modified celastrol microemulsion-tanshinone II sodium sulfonate liposomes by flow cytometryThe apoptosis of MCF-7 cells was induced by Celastrol,Cel-mixture,Cel-LPs,Cel/STS-LPs,Cel-MEs,(FA)Cel-ME,Cel-MEs/STS-LPs and(FA)Cel-MEs/STS-LPs with the concentration of celastrol at 0.5,1.0,2.0 and 2.5 μg/mL.Apoptosis rate was positive with the concentration of celastrol.The apoptotic rates of Cel-MEs/STS-LPs,Cel/FA-MEs-STS-LPs at 0.5 μg/mL were 16.8±2.6%and 15.5±1.2%,respectively.The apoptotic rates of Cel-MEs/STS-LPs,Cel/FA-MEs-STS-LPs at 2.5 μg/mL were 51.6±0.2%and 50.3±4.1%,respectively.The apoptotic rates of Cel-MEs/STS-LPs,(FA)Cel-MEs/STS-LPs at the concentrations of celastrol of 5.0 μg/mL were 59.6 ±2.3%and 81.1±0.2%,respectively.It indicated that(FA)Cel-MEs/STS-LPs had obvious advantages at high concentrations compared to Cel-MEs/STS-LPs.5.MCF-7 cell 3D tumor model culture and penetration study in vitroMCF-7 cells 3D tumor was loose in the first three days,but became condense and stable in the following days.The structure of 3D tumor sphere was intact with a diameter of 600～700μm.The in vitro tumoral penetration of DiO-MEs,DiO-MEs-STS-LPs,DiO-MEs-LPs and DiO-MEs+STS-LPs were investigated with the DiO concentration of 2.5μM at 6 h and 12 h,respectively.The results showed the penetration capacity was obviously correlated with the incubation time.6、Breast cancer-targeting and antitumor efficacy of Cel/STS-LPs in vivo6.1 Evaluation of tumor targeting using DiD-LPs and DiD/STS-LPs in vivo in MCF-7 tumor-bearing nude mice.The biodistribution of DiD-LPs and DiD/STS-LPs was investigated using MCF-7 tumor-bearing nude mice model.The results showed that DiD-MEs,DiD-LPs and DiD/STS-LPs can specifically target the tumor site within 48 h.Among all groups,DiD/STS-LPs group showed the strongest fluorescence intensity in the tumor site,followed by DiD-MEs/STS-LPs group and DiD-MEs+STS group.DiD+STS group and DiD group showed the weakest fluorescence intensity.6.2 Antitumor efficacy of Cel-LPs and Cel/STS-LPs in vivoThe anti-tumor effect of Cel-LPs group and Cel/STS-LPs group was investigated using MCF-7 tumor-bearing nude mice.After treatment with various celastrol-related preparations,the,the tumor inhibitions of mice were improved significantly compared with the saline group,especially Cel/STS-LPs group exhibited the strongest tumor growth inhibition.Furthermore,serum immune indices were determined by using ELISA kits for MCP-1,IFNy,IL-2 and TGF-β.The results showed that the MCP-1 index of Cel-LPs group and Cel/STS-LPs group were significantly decreased compared with saline group(P<0.05).The content of IFNy,IL-2 and TGF-β among the groups had no obvious change,which the reasons still to be further discussed.7、Safety evaluation of Cel/STS-LPsSeveral indices representing the function of liver and kidney such as aspartate aminotransferase(AST),alanine aminotransferase(ALT),uric acid(UA),blood urea nitrogen(BUN)and CREA(creatinine)were detected by blood biochemical analyzer.The results showed that ALT,AST and renal function indexes UA,BUN and CREA had no pathological changes compared with normal saline group in nude mice.Only the celastrol group,the renal function index UA was significantly higher than that in the normal group and there was significant difference in the renal function index CREA between the Cel-LPs group and the normal group,but both indexes were in the normal range.Compared with the normal saline group,there were no significant difference in blood routine analysis among WBC,RBC,HGB and PLT.In addition,the results of HE staining showed that Celastrol group and Cel+STS group had certain inflammation in the liver,while the Cel-LPs group and the Cel/STS-LPs group had no inflammation in the liver.Lung and other organizations had no obvious abnormal suggested that the designed preparation had excellent therapeutic effect and good security. |
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