还原敏感型透明质酸-甘草次酸偶联物胶束的制备及评价

Preparation and Evaluation of Redox-sensitive Hyaluronic Acid-glycyrrhetinic Acid Conjugate Micelles

目的 合成还原敏感型透明质酸-甘草次酸(hyaluronic acid-glycyrrhetinic acid,HSG)偶联物,对其自组装行为及安全性进行评价,以它为胶束载体增溶化疗药紫杉醇(paclitaxel,PTX),并考察其体外释药行为、细胞摄取行为与细胞毒性。方法 以透明质酸(hyaluronic acid,HA)为亲水性骨架材料,通过还原敏感型胱胺连接臂引入疏水甘草次酸(glycyrrhetinic acid,GA)制备HSG偶联物,应用^(1)H-NMR与FT-IR表征验证HSG的结构;从粒径及分布、临界胶束浓度、形态3个方面对偶联物的自组装行为进行表征,考察疏水段GA取代度对HSG胶束粒径、分布及其增溶PTX性能的影响;通过溶血性初步评估HSG偶联物的安全性;采用透析法考察载药胶束PTX/HSG在不同浓度谷胱甘肽(glutathione,GSH)介质中的释放行为;流式细胞仪检测HA和GA预孵育对细胞摄取HSG胶束的影响;MTT法考察PTX/HSG对HepG2的细胞毒性。结果 成功合成了一系列两亲性还原敏感型HSG偶联物,随GA取代度提高,HSG胶束粒径降低,确定HA∶GA=1∶2为最佳投料比,此时偶联物GA摩尔取代度为(7.83±1.24)%,粒径为(209.7±10.4)nm,临界胶束浓度为50.1μg·mL^(-1),透射电镜显示HSG胶束皆近似球形结构;HSG溶血性低于Tween 80,与聚氧乙烯蓖麻油相当。不同GA取代度的HSG偶联物对PTX均具有增溶效果,并随取代度增加,HSG胶束载体载药能力增强;HSG包载PTX后在高浓度GSH介质中有还原响应性快速释药能力;HSG与HA、GA预孵育后肿瘤细胞对其摄取量降低;PTX/HSG对HepG2的细胞毒性强于Taxol制剂。结论 所制HSG两亲性偶联物能形成胶束,安全系数高,具有还原触发快速释药与主动靶向的能力,可作为载体材料包载难溶性化疗药并有助于其疗效的发挥。

OBJECTIVE To synthesize redox-sensitive hyaluronic acid-glycyrrhetinic acid(HSG) conjugate,evaluate its self-assembly behavior and safety,and use it as a micellar carrier to solubilize chemotherapeutic drug substance paclitaxel(PTX),and investigate its in vitro drug release behavior,cellular uptake behavior and cytotoxicity.METHODS Hyaluronic acid(HA)was used as a hydrophilic skeleton material,and glycyrrhetinic acid(GA) was introduced through a redox-sensitive cystamine linker to prepare HSG conjugates,and the structure of HSG was confirmed by means of ~1H-NMR and FT-IR.The self-assembly behavior of the conjugate was characterized by particle size and its distribution,critical micelle concentration and morphology.In addition,the impact of hydrophobic segment GA substitution of the conjugates on the particle size,size distribution and the solubilization performance on PTX of HSG was evaluated.The safety of HSG conjugates was preliminarily assessed using hemolysis test.Dialysis method was used to investigate drug release behavior of PTX/HSG in different concentrations of glutathione(GSH) media.The effect of HA and GA pre-incubation on the uptake of HSG micelles was detected by flow cytometry.The cytotoxicity of PTX/HSG micelles on HepG2 cells was investigated by MTT assay.RESULTS A series of amphiphilic redox-sensitive HSG conjugates were successfully synthesized.As the degree of substitution of GA increased,the HSG micelle size decreased.It was determined that the best feeding ratio of HA∶GA was 1∶2.At this feeding ratio,the degree of GA moles substitution of the conjugates was (7.83±1.24)%,with a particle size of (209.7±10.4)nm and the critical micelle concentration value of 50.1μg·mL^(-1).Transmission electron microscope image showed that HSG micelles were all similar to spherical structures.The studies concluded that the hemolysis of HSG conjugates was equivalent to cremophor but lower than Tween 80;HSG conjugates with different degrees of GA substitution had a solubilizing effect on PTX,and as the degree of substitution of GA increased,the drug loading capacity of PTX into HSG micellar carrier increased.The PTX-loaded HSG micelles showed specific reduction-sensitive rapidly drug release ability to high concentration GSH.The uptake of HSG by tumor cells decreased after pre-incubation with HA and GA.PTX/HSG micelles exhibited stronger cytotoxicity to HepG2 cells than Taxol preparations.CONCLUSION The prepared HSG amphiphilic conjugates can form micelles with high safety factor,rapid drug release in response to reducing agent and active targeting ability.It can be used as a carrier material to encapsulate poorly soluble chemotherapeutics and facilitate their efficacy.