TAT和PEG双修饰田蓟苷复合磷脂脂质体的工艺优化及体外评价

Optimization and in vitro evaluation of TAT and PEG co-modified tilianin-loaded composite phospholipid liposomes

目的筛选细胞穿膜肽(transcription activator,TAT)和聚乙二醇(polyethylene glycols,PEG)双修饰田蓟苷复合磷脂脂质体(TAT&PEG tilianin CPL,T&PTCPL)的最佳制备工艺,并考察其对心肌细胞H9C2的保护作用。方法采用薄膜分散-超声法制备T&PTCPL,利用3因素3水平的Box-Behnken设计,分别以总磷脂与田蓟苷质量比(X1)、DSPEPEG2000-TAT的浓度(X2)和水化体积(X3)为考察对象,包封率(Y1)、粒径(Y2)和多分散系数(PDI,Y3)为主要评价指标筛选T&PTCPL最佳配方,并以Na2S2O4建立H9C2细胞缺氧/复氧损伤模型,以超氧化物歧化酶(SOD)、肌酸激酶同工酶(CK-MB)、丙二醛(MDA)和乳酸脱氢酶(LDH)为指标,考察T&PTCPL对细胞缺氧/复氧损伤的影响,同时,考察其体外释放率(动态透析法)及人结肠癌Caco-2细胞对田蓟苷原料药和T&PTCPL的吸收情况。结果 T&PTCPL的最佳组合为X1=20,X2=1.7%,X3=3.2m L;包封率为(86.62±2.51)%,粒径为(149.7±8.2)nm,PDI为0.15±0.05。在体外细胞实验中,与模型组比较,原料药组及T&PTCPL组能显著提高SOD活性,减少MDA的含量及抑制LDH和CK-MB的释放(P<0.05),并且T&PTCPL的效果优于原料药。同时,T&PTCPL在48 h基本释放完全,累积释放率88.65%,Caco-2细胞对T&PTCPL的吸收效果较佳。结论 Box-Behnken实验设计法用于T&PTCPL的优化筛选是可行的,数学模型的预测值与实验观察值相符,并且T&PTCPL在体外释放中表现出较好的缓释效果,能促进田蓟苷在Caco-2细胞中的吸收,同时,提示T&PTCPL具有保护心肌缺血再灌注损伤作用。

Objective To optimize the preparation technology of transcription activator (TAT)and polyethylene glycols (PEG) co-modified tilianin-loaded composite phospholipid liposome (TAT &PEG tilianin CPL,T&PTCPL)and investigate its protective effect on cardiomyocytes.Methods The composite phospholipid liposome was prepared by thin film-ultrasonic method.A three- factor,three-level Box-Behnken experimental design was employed.The weight ratio of total phospholipid to tilianin (X1),the concentration of DSPE-PEG2000-TAT (X2),and hydration volume (X3)were observed.The encapsulation efficiency (Y1);particle size (Y2),and polydispersion coefficient (Y3)were evaluated to optimize optimal formula.In addition,hypoxia/reoxygenation model was established with Na2S2O4in H9C2cells.Superoxide dismutase (SOD)activity,malonaldehyde (MDA)level and release of lactate dehydrogenase (LDH)and creatine kinase-MB (CK-MB)were assessed to evaluate the effect of T&PTCPL,meanwhile,the in vitro release rate (dynamic dialysis method)and absorption rate of tilianin and T&PTCPL in Caco-2cell were examined.Results The optimal formula was as following:X1 =20,X2=1.7%,andX3=3.2mL;The encapsulation efficiency was (86.62±2.51)%,particle size was (149.7±8.2)nm and PDI was 0.15±0.05.Compared with model group,T&PTCPL and tilianin groups increased SOD activity, inhibited level of MDA,LDH and CK-MB leakage (P <0.05),and the effect of T&PTCPL group was better than tilianin group, meanwhile,T&PTCPL was completely released at 48h,with a cumulative release of 88.65%,and Caco-2cells had better absorption of T&PTCPL.Conclusion The Box-Behnken design is suitable for optimizing the formulation of T&PTCPL,and the observed responses are in close agreement with the predicted values of the mathematic models;Moreover,T&PTCPL shows a better sustained release effect in vitro release,which promots the absorption of tilianin in Caco-2cells and suggests that T&PTCPL may have protective effect on myocardial ischemia reperfusion injury.