| Lately,the mixed micelles have already obtained immense attraction owing to the advantages of better stability and higher drug loading capacity compared with the single micelle.PEG1000 vitamin E succinate(TPGS)is approved by USA FDA and extensively applicated in the drug release field.TPGS can improve the intracellular uptake and therapeutic effect in vitro and in vivo.The folate-modified Bletilla polysaccharide(FA-BSP-SA)amphiphilic polymer with the property of the spontaneous formation of nanomicelles in aqueous medium was synthesized by our group.FA-BSP-SA can enhance antitumor drug targeted delivery to the tumor sites and accelerbrate anti-tumor effect.In this study,the effects of TPGS on the physicochemical properties,drug loading,antitumor activity in vitro and pharmacokinetics of FA-BSP-SA micelles were investigated the potential of the the combination of TPGS and FA-BSP-SA as antitumor drug delivery carriersIn this study,based on the synthesis of an amphiphilic polymer of folic acid modified Bletilla striata polysaccharides,a pH-sensitive and active targeted mixed micellar drug delivery system composed of TPGS and folate-mediated stearic acid-modified Bletilla Striata polysaccharide was developed,which is expected to achieve superior the anti-tumor effect of doxorubicinThe effect of TPGS on the spontaneous aggregation of FA-BSP-SA in aqueous media was investigated.The doxorubicin-cholate complex was prepared by charge electrostatic interaction as a hydrophobic model drug and then directly loaded into the micelle.The effects of added TPGS amounts on the particle size,particle size distribution,entrapment efficiency and drug loading content of FA-BSP-SA nano-micelles were investigated.The effects of micelle concentration,TPGS amounts and different mass ratios(TPGS:FA-BSP-SA)on drug loading and entrapment efficiency were evaluated.The particle size,morphology and in vitro drug release behavior of nano-micelles under different pH conditions were investigated.The plasma concentrations of free doxorubicin(Dox),Dox-loaded FA-BSP-SA micelles and Dox-loaded FA-BSP-SA/TPGS mixed micelles were determined and the main pharmacokinetic parameters were compared.MTT assay and hemolysis determination were carried out to estimate the safety of FA-BSP-SA/TPGS mixed micelles.The inhibition for the 4T1 proliferation was conducted and intracellular uptake of Dox@FA-BSP-SA was determined using Fluorescent enzyme labeling instrumentThe results of aggregated assay showed the addition of TPGS clearly influenced the aggregation performance of FA-BSP-SA.The mass ratio of TPGS is 0%,12.5%,25%,50%,75%and 100%expressed the CAC values are 5.40,25.64,15.0,6.38,2.62 and 36.2?g/mL,respectively.When the mass percentage of TPGS is 50%,the CAC value of FA-BSP-SA/TPGS is similar to that of FA-BSP-SA.Under the same micelle concentration,the particle size of the mixed drug-loaded micelle at the mass ratio of 1:1 and 3:1(FA-BSP-SA:TPGS)was smaller than that of blank micelle while the particle size of the mixed micelle at 1:3(FA-BSP-SA:TPGS)was larger than that before drug was loaded in micelle.The reason may be related to the higher water solubility of TPGS and the lower CAC.There was no obvious change in Zeta potential.When the mass ratio of the micelles was 1:3,1:1 and 3:1(FA-BSP-SA:TPGS),the micelle concentrations of the smaller blank micelles were 1.0,1.0 and 0.5 mg/ml,respectively,but the micelle concentrations of the smaller micelles after drug loading were 1.0,0.5 and 1.0 mg/mL,respectively.The addition of TPGS affected the drug loading content and entrapment eff-iciency of FA-BSP-SA The FA-BSP-SA/TPGS100 mixed micelle along with the mass ratio of 3:1 at the concentration of 0.5 mg/mL exhibited the average particle siz of less than 160 nm,Zeta potential of about-15 mV,the drug loading content of 14.4%and the entrapment eff-iciency of 91.9%.Considering the CAC value,particle size,drug loading and entrapment efficiency of micelles together,FA-BSP-SA/TPGS with the mass ratio of 3:1 was selected for the further studyThe release results indicated the release rate of free doxorubicin is not affected by pH value,but the release rate of micelle obviously increased with the decrease of pH value.The release behavior of micelle pesented pH-sensitivity,which is beneficial to the drug release from micelle in acid tumor sites.The release curve in vitro was fitted by Ritger-Peppas model,and the release index m is less than 0.45,which accorded with the mixed mechanism of drug diffusion and skeleton dissolutionThe pharmacokinetics results demonstrated AUCo-? values of free doxorubicin,Dox@FA-BSP-SA micelle and Dox@FA-BSP-SA/TPGS mixed micelle were 12.53±1.88 mg·h/L,63.56±8.19 mg·h/L and 65.20±8.32 mg·h/L,respectively.The AUC0-? values of Dox@FA-BSP-SA micelle and Dox@FA-BSP-SA/TPGS mixed micelle were 5.07-and 5.21-fold higher than that of free doxorubicin.The MRT0-? was 4.12-and 3.83-time-longer than that of free doxorubicin,respectively.The elimination half-life(t1/2)of doxorubicin was 4.69-and 3.77-time-higher than that of free doxorubicin,respectively.Meanwhile,the clearance rate(Cl)was significantly decreased.The results showed that Dox encapsulated in FA-BSP-SA micelle and FA-BSP-SA/TPGS mixed micelles could prolong the circulation time of the drug in rats,maintain a long retention time and promote the in vivo absorption of doxorubicin,which may facilitate antitumor effectsMTT results proved that FA-BSP-SA and FA-BSP-SA/TPGS showed good biocompatibility.Within the concentration range of 0.25-1.0 mg/mL,the hemolysis rate of FA-BSP-SA and FA-BSP-SA/TPGS mixed micelles is less than 5%,which exhibited good safety.When the concentration of doxorubicin was 0.2-1.0 ?g/mL,the cell survival rate decreased with the increase of doxorubicin concentration.The cell survival rates of Dox@FA-BSP-SA micelle and Dox@FA-BSP-SA/TPGS mixed micelles with the doxorubicin concentration of 0.5-1.0 ?g/mL were significantly lower than that of free doxorubicin group(*p<0.001).The survival rates of 4T1 cells incubated with free doxorubicin,Dox/BSP-SA micelles and Dox@FA-BSP-SA/TPGS micelles were 55.62%,37.82%and 25.88%,respectively The results showed that the addition of TPGS into FA-BSP-SA micelles significantly fabricated the inhibitory effect of doxorubicin on tumor cells,and the anti-tumor effect was more obviousThe results of intracellular uptake indicated the addition of TPGS could increase the intracellular uptake of doxorubicin in micelles.The intracellular uptake the drug of in free doxorubicin,Dox@FA-BSP-SA and Dox@FA-BSP-SA/TPGS increased in a dose-dependent manner with the increase of doxorubicin concentration and the prolongate of incubation time.The rank of intracellular uptake was Dox@FA-BSP-SA/TPGS>Dox@FA-BSP-SA>free doxorubicin(*p<0.05,**P<0.01 or*p<0.001).In conclusion,the addition of TPGS can enhance Dox@FA-BSP-SA/TPGS the inhibitory effect on 4T1 cells. |
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