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

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.