Construction And Characteristic Of Sodium Tanshinone IIA Sulfonate Loaded Nano-erythrocytes Delivery System

In this study,a new drug delivery with high safety and good biocompatibility was prepared from erythrocyte,which was prepared to load sodium tanshinone IIA sulfonate(STS).Besides,the biological characteristic of this drug delivery system was investigated.The experiment is as follows.1 Establish an in vitro analysis method to detect STS-Nano-RBC.2 Firstly,under low osmotic conditions,the cytoplasm of red blood cell(RBC)was lost and became to erythrocyte ghosts,and then the ghosts were cleaved into vesicles by ultrasound method.At this time,STS were added into the vesicles,after a while,added into high osmotic solution,so that the system could return to isotonic condition.Then,the selection of drug loading temperature,low osmotic solution,STS concentration and the volume ration between STS and RBC were investigated with the index of STS encapsulation efficiency.The morphology,particle size,zeta potential,entrapment efficiency and protein content of STS-Nano-RBC were characterized by transmission electron microscopy,dynamic light scattering instrument,high performance liquid chromatography and gel electrophoresis methods 3 The in vitro release of STS-Nano-RBC was investigated by dialysis method.The pharmacokinetics and tissue distribution of STS-Nano-RBC were investigated by injecting STS-Nano-RBC through tail vein into rats.4 The uptake of STS-Nano-RBC by EA.hy926 cells was investigated by fluorescence microscopy and high performance liquid chromatography(HPLC)method.The pathways of STS-Nano-RBC uptake into EA.hy926 cell was investigated by incubating cells with different channel inhibitors or incubating cells at different conditions.5 By incubating EA.hy926 cells with different concentration of STS-Nano-RBC to investigate its cell viability.To investigate the protective effect of STS-Nano-RBC on oxidative stress-impaired cells,we first used 750 ?M H2O2 to establish the model of oxidative stress-impaired cells,then added 10-150 ?g/ml STS-Nano-RBC to intervention.The experimental results showed: 1 The in vitro analysis method for STS-Nano-RBC was established by high performance liquid chromatography(HPLC)method,which showed high accurate and high recovery rate.2 The result indicated that when the temperature was 4?,the low permeability solution was 10%×PBS,the concentration of STS was 1.0 mg/ml and the volume ratio of STS and erythrocyte ghosts was 2:1,STS-Nano-RBC had the best entrapment effiency.STS-Nano-RBC was spherical particles with particle size of 156 ± 5.8 nm and potential of-2.34 ± 0.67 mv.The encapsulation efficiency of STS-Nano-RBC was 33.15 ± 2.61%,and the protein content showed no significant difference with erythrocyte membrane.STS-Nano-RBC has good stability and safety.3 The result of in vitro release showed that STS injection group released 97% STS at 6h,while STS-Nano-RBC group released only 44%,besides,STS-Nano-RBC can be slowly released to 36 h.The result of in vivo release indicated the half-life(t1/2)of STS-Nano-RBC was 6.92 h,while the t1/2 of STS was only 2.23 h.The mean retention time(MRT)of STS-Nano-RBC was 8.14 h,while the MRT of STS was 2.23 h.The STS could not be detected in rat blood at 6 h after injection,while STS-Nano-RBC could be detected after 36 h injection.4 The cell uptake result of STS-Nano-RBC by fluorescence microscope showed that STS-Nano-RBC could be effectively penetrated into EA.hy926 cell.Quantitative detection of STS-Nano-RBC by HPLC method showed the same result,besides,the result also indicated 20.7% STS penetrated into cells after 2 h incubating,when the incubating time increase to 24 h,the STS penetrated into cell increased to 26.7%;while the STS-Nano-RBC group was only 5.4% at 2h,but with the incubating time increased to 24 h,STS-Nano-RBC penetrated into cell increased to 33.2%.The result indicated that STS-Nano-RBC could more effectively penetrated into cell.Incubating EA.hy926 cell with different channel inhibitors or incubating cells under different conditions to investigate the way STS-Nano-RBC involved into cell.The result showed that the process was not only affected by temperature,ATP,environmental pH and osmotic pressure,but also regulated by the protein of caveolin and clathrin.5 The result of cell viability showed that STS-Nano-RBC had little toxic to EA.hy926 cell.When the concentration of STS was or less than 100 ?g/ml,cell survival rate was or more than 75%.According to this result,we chose the concentration of 10-150 ?g/ml to study the protective effect of STS-Nano-RBC on oxidative stress impaired cells.The model of oxidative stress impaired cell was established by incubating EA.hy926 cell with 750 ?M H2O2 for 12 h.The experiment of investigating the protective effect of STS-Nano-RBC on oxidative stress impaired cell showed that at the concentration of 80-150 ?g/ml,STS-Nano-RBC group revealed superior oxidative stress protective effect than STS group.Therefore,we could make a conclusion that the preparation process of STS-Nano-RBC is simple,stable and safe.STS-Nano-RBC can prolong the cycle time of STS in rats and enhance the protect effect of STS on oxidative stress impaired cell.So,the Nano-RBC has promise to be a novel nano-drug delivery system in clinical practice.