隐丹参酮靶向脂质体的构建及其体外抗脑胶质瘤考察

Construction of targeted cryptotanshinone liposomes and research on its in vitro anti-glioma effect

隐丹参酮(cryptotanshinone,CPT)具有抗脑胶质瘤活性,但其存在溶解度低、肿瘤渗透性弱等问题,因此设计具有深度穿透、精准靶向的纳米给药系统迫在眉睫。本文采用乳化蒸发法制备了tLyp-1修饰的隐丹参酮脂质体(tLyp-1 modified liposomes loaded with CPT,tLipo/CPT),用脂质体负载CPT,并在其表面修饰具有靶向肿瘤新生血管和肿瘤细胞膜神经毡蛋白受体(neuropilin receptors,NRP)的穿膜肽tLyp-1,使CPT能够精准靶向脑胶质瘤并准确释放。通过粒径、多分散系数(polymer dispersity index,PDI)、胞内荧光参数、透射电镜扫描等确定tLipo/CPT的粒径大小为(162.2±14.6)nm,PDI为0.24±0.03;tLyp-1肽修饰的最佳摩尔比例为0.5%;靶向脂质体呈光滑圆整的球形。高效液相色谱法定量测定其包封率为(70.06±7.22)%;靶向脂质体(liposomes modified with tLyp-1 peptide,tLipo)相较于脂质体(liposomes not modified with tLyp-1,Lipo)能够被GL261细胞更多地摄取,tLipo组胞内荧光强度较Lipo组增加40%,进一步确认GL261细胞对tLipo/CPT的摄取为通过神经毡蛋白受体1(NRP-1)介导的内吞途径;MTT实验表明tLipo/CPT可显著抑制脑胶质瘤GL261细胞的增殖,其半数抑制浓度(half maximal inhibitory concentration,IC_(50))为5.70μmol·L^(-1);tLipo/CPT可跨越体外血脑屏障(blood-brain barrier,BBB)模型;此外,体内荧光成像实验显示,尾静脉注射DiR标记的tLipo后,0.5 h在小鼠脑部即可观察到荧光分布,且24 h后脑部仍存在荧光,进一步确定tLipo/CPT可穿透BBB,且其可通过抑制脑胶质瘤GL261细胞的增殖发挥抗脑胶质瘤作用。动物福利和实验过程均遵循南京中医药大学动物伦理委员会的规定。

Cryptotanshinone(CPT),an active ingredient with the inhibitory effect on brain glioma cells,is trapped with poor solubility and low tumor permeability.Therefore,it is urgent to design nano drug delivery systems characterized with deep penetration and accurate targeting.In the present study,tLyp-1 modified liposomes loaded with CPT(tLipo/CPT)was prepared by emulsion solvent evaporation method.Peptide tLyp-1 which targeting tumor angiogenesis and neuropilin receptors(NRP)was modified on surface of CPT liposomes,with the aim of active targeting brain glioma cells and further release CPT precisely.The size and polymer dispersity index(PDI)of tLipo/CPT were(162.2±14.6)nm and 0.24±0.03.The optimal molar ratio of tLyp-1 modified on CPT liposomes was 0.5%determined by intracellular fluorescence parameters.The morphology displayed a smooth sphericity structure as determined by transmission electron microscope.Efficiency of CPT encapsulated in tLipo/CPT was detected by high performance liquid chromatography.The encapsulation efficiency of CPT was(70.06±7.22)%.Liposomes modified with tLyp-1 peptide(tLipo)were internalized more than liposomes not modified with tLyp-1(Lipo)by GL261 cells.Fluorescence intensity of tLipo in GL261 cells increased 40%than that of Lipo.Furthermore,we proved that the intake of tLipo/CPT in GL261 cells was mediated by NRP-1 receptor.MTT analysis indicated that tLipo/CPT significantly inhibit the proliferation of GL261 cells.The half maximal inhibitory concentration(IC_(50))was 5.70μmol·L^(-1).In vitro blood-brain barrier(BBB)model experiment indicated that tLipo/CPT could penetration across BBB.Moreover,in vivo fluorescence biodistribution study indicated tail vein injection of DiR labeled tLipo after 0.5 h,DiR fluorescence could be observed in the brain of mice.Even after 24 h,DiR fluorescence still was observed in the brain.Our research certified that tLipo/CPT can penetrate the BBB and show effect of anti-glioma by inhibiting the proliferation of GL261 cells.The animal experiment was carried out in accordance with protocol evaluated and approved by the Ethics Committee of Nanjing University of Chinese Medicine.