| Objective: Danshen,the dry root of Salvia Miltiorrhiza Bge,is a traditional Chinese medical herb that has been proved for thousands of years in China to activate blood circulation and remove blood stasis.Tanshinone IIA,one of the major effective components of Danshen.Sodium tanshinoneII-A sulfonate(DS-201),a water-soluble derivative of Tanshinone IIA which is a BKCa channel opener and a vasodilator.Clinically,DS-201 has been applied to treat ischemic cardiovascular and cerebrovascular diseases,such as coronary heart disease,angina pectoris,myocardial infarction,and so forth.DS-201 has effects on anti-atherosclerosis,reducing myocardial infarction area,increasing coronary blood flow,slowing heart rate,and strengthening the heart muscle.DS-201 for its hydrophilic property prohibits the membrane-penetrating process and thus limits the curative efficacies,although this property can guarantee their solubility in aqueous phase.So,it will affect DS-201 interacting with the target cells and decrease the curative effect.The research findings suggest that the vasodilatory effect of DS-201 is related to the activation of BKCa channelsat the intracellular side.The BKCa channel-activatingeffect of DS-201 used in intracellular is more than 100 times larger than used in extracellular.According to delivery purposes,liposomes as drug delivery vehicles can deliver water-soluble,liposoluble,and amphiphilic drugs to the tissue,cytomembrane,and intracellular sites.In this way,liposomes can improve therapeutic index,reduce drug-toxicity and side effects.The study intends to introduce a liposome-based drug delivery system which could efficiently deliver DS-201 into cells,thus significantly increase the BKCa channel-activating and vasorelaxing effects to improve the clinical application values of DS-201.The establishment of technologies and methods in this study are of great theoretical and practical value on the research of effective therapeutics,and especially popularizing the Chinese traditional medicine.Methods: ? The preparation and optimum screening of DS-201 liposomes:(1)The thin-film dispersion combined with probe sonication method was used to prepare DS-201 liposomes.(2)Refrigerated ultracentrifugation was used to separate lipids from free DS-201.(3)The amount of DS-201 was quantified by HPLC.(4)The uniform design was adopted to screen DS-201 liposomes examined the leakage rate as the evaluation indicator.? The evaluation of physical characteristics of DS-201 liposomes:(1)The morphology of the liposomes was observed by the transmission electron microscope(TEM).(2)The particle size distribution and zeta potential of the liposomes were measured via zeta potential and particle size analyzer.(3)The preliminary stability was measured by the variation of entrapment efficiency at one month interval,referred to as the leakage rate.Freeze-drying was used to prepare a lyophilized powder preparation of DS-201 liposomes,referred to as proliposomes.? The in vitro evaluationof biological activities of DS-201 liposomes:(1)The prepared GFP-loaded DS-201 liposomes were co-cultured with HEK-293 cells,and observed under a fluorescence microscope.(2)Patch clamp experiment was performed to determine whether liposome-entrapped DS-201 could activate BKCa channels on HEK-293 cells stably expressing hSlo.The experiments compared the BKCa single channel current orBKCa channels whole-cell currents before and after administating DS-201,blank-liposomes,and DS-201 liposomes,respectively.(3)Isolated rat mesenteric arteries were used for the vasomotor assay to determine whether DS-201 intracellularly delivered by liposome could exert a greater vasorelaxing effect on PE orPGF2? induced vascular constriction compared with DS-201 and blank-liposomes giving directly.Results: ? The concentration of soybean lecithin was 5 mg/ml,ratio of soybean lecithin to cholesterol was 4:1,probe sonication time was 25 min,and ratio of DS-201 to cholesterol was 7:1.They were the optimal formula screened by uniform design.?(1)The TEM images showed that most liposomes were spherical uniform particles,and unbroken structures.(2)The mean particle size value of the optimized liposomes was 206.93 ± 4.10 nm(n = 3);The zeta potential of the optimized liposomes was-36.79 ± 0.54 mV(n= 3).(3)The leakage rate was 4.64% owned by the lyophilized powder preparation of optimized liposomes at one month interval.?(1)After being co-cultured with GFP-loaded DS-201 liposomes for 24 h,HEK-293 cells were appeared the green fluorescence on cell membrane under the fluorescence microscope.(2)The DS-201 liposomes significantly increased the open probability of BKCa channel from baseline 0.013 ± 0.004 to 0.036 ± 0.011 at +40 mV membrane potential(n = 10,P< 0.05)in cell attached study,and also increased the current density from baseline 23.2 ± 4.4 pA/pF to 66.0 ± 15.2 pA/pF at +40mV membrane potential(n = 6,P< 0.05)in whole-cell recording study,compared with the direct extracellular administration of this drug.But for the other two groups,DS-201 and blank-liposomes,were both no significant difference in the variation of BKCa currents before and after administration.(3)About 60% inhibition of thePE orPGF2? induced vascular constriction,the DS-201 liposomes did possess significantly enhanced vasorelaxant effect on rat mesenteric artery,compared with around 20% inhibition of the directly administration of this drug(n = 6,P< 0.05).Conclusion:The DS-201 liposomes prepared in this study successfully delivered DS-201 into cells and thus significantly activated BKCa channels on Vascular Smooth Muscle Cells(VSMCs)and reversed the contraction induced by PE and PGF2? on rat mesenteric artery.The DS-201 liposome intracellular delivery enhanced the drug's bioavailability and vasorelaxing effects. |
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