RGD修饰载阿霉素固体脂质纳米粒的制备及体外转运能力观察

Preparation of arginine-glycine-aspartate(RGD) peptide-modified doxorubicin-loaded solid lipid nanoparticles and their transport ability in vitro

目的 制备精氨酸—甘氨酸—天冬氨酸肽(RGD)修饰的载阿霉素固体脂质纳米粒(RGD-DOX-SLNs),观察其在肠道M细胞模型中的转运能力。方法 采用不同乳化剂配方,以乳化溶剂挥发法制备固体脂质纳米粒(SLNs),通过检测SLNs的稳定性,选取较优乳化剂配方进行阿霉素包载;制备载阿霉素的SLNs(DOX-SLNs),测定粒径、表面电位及包封率,选择最优乳化剂配方,对DOX-SLNs表面进行RGD修饰,制备RGD-DOX-SLNs;以空白SLNs作为对照。使用透射电镜观察RGD-DOX-SLNs和DOX-SLNs的形态,采用CCK-8法测算与载药SLNs共培养的Caco-2细胞及293T细胞存活率以评价载体安全性。建立滤泡相关上皮单层细胞模型,分别加入含有阿霉素单药、DOX-SLNs或RGD-DOX-SLNs的HBSS液,采用荧光分光光度法测定转运小室下侧的阿霉素浓度,共聚焦显微镜观察荧光,评价RGD-DOX-SLNs和DOX-SLNs的药物转运能力。结果 选择最优乳化剂配方为1%吐温与1%泊洛沙姆188体积比2∶1,DOX-SLNs粒径、表面电位及包封率分别为(147.4±11.2)nm、(-20.0±2.3)mV、80.9%±2.8%,RGD-DOX-SLNs分别为(153.1±6.2)nm、(-22.0±1.3)mV、81.3%±0.8%。透射电镜下RGD-DOX-SLNs和DOXSLNs形态圆整,RGD-DOX-SLNs表面略粗糙。RGD-DOX-SLNs和DOX-SLNs在0~1 000μg/mL浓度范围内安全性较好。阿霉素单药组、DOX-SLNs组及RGD-DOX-SLNs组的转运量依次增高(P均<0.05);阿霉素单药组红色荧光微弱,DOX-SLNs组X-Y、Y-Z、X-Z三个切面均可见红色荧光,RGD-DOX-SLNs组红色荧光强度较DOX-SLNs增强。结论 成功制备RGD-DOX-SLNs,较DOX-SLNs对阿霉素的体外转运能力更高。

Objective To prepare doxorubicin-loaded solid lipid nanoparticles(DOX-SLNs) modified by arginineglycine-aspartate(RGD) peptide and to investigate their transport ability in the intestinal M cell model.Methods The emulsion-solvent evaporation method was used to prepare SLNs.The type and proportion of emulsifier were investigated.The optimal formulation was selected by comparing the particle size and zeta potential.Then,DOX-SLNs were prepared and the particle size,zeta potential and entrapment efficiency were investigated.The surface of DOX-SLNs was modified by RGD peptide to prepare RGD-DOX-SLNs,and the blank SLNs were taken as the controls.The morphology of RGDDOX-SLNs and DOX-SLNs was observed by transmission electron microscopy(TEM).The survival rate of Caco-2 cells and 293T cells co-cultured with SLNs was measured by CCK-8 method to evaluate the safety of the carrier.FAE(follicleassociated epithelium) monolayer model was established,and was added with HBSS solution containing DOX,DOXSLNs,and RGD-DOX-SLNs.The concentration of DOX in the lower side of the transport chamber was determined by fluorescence spectrophotometry,and the fluorescence was observed by confocal microscope to evaluate the drug transport capacity of RGD-DOX-SLNs and DOX-SLNs.Results The optimal emulsifier formula was selected successfully,which was 1% tween and 1% Poloxamer,and the volume ratio was 2:1.The particle size,zeta potential and entrapment efficiency of DOX-SLNs were(147.4 ± 11.2) nm,(-20.0 ± 2.3) mV,and 80.9%±2.8%,respectively.The particle size,potential and entrapment efficiency of RGD-DOX-SLNs was(153.1 ± 6.2) nm,(-22.0 ± 1.3) mV,and 81.3%±0.8%,respectively.TEM results showed that the morphology of RGD-DOX-SLNs and DOX-SLNs was round,and the surface was slightly rough after RGD modification.RGD-DOX-SLNs and DOX-SLNs were safe in the range of 0-1000 μg/mL.The transport abilities increased in the DOX group,DOX-SLNs group,and RGD-DOX-SLNs group gradually(all P<0.05).The red fluorescence was weak in the DOX group,the red fluorescence was observed in the X-Y,Y-Z and X-Z sections in the DOX-SLNs group;the red fluorescence intensity in the RGD-DOX-SLNs group was stronger than that in the DOX-SLNs group.Conclusion RGD-DOX-SLNs were successfully prepared,and their in vitro transport ability of DOX was higher in comparison with that of DOX-SLNs.