犬红细胞膜仿生纳米载体的制备和体外毒性及靶向初探  

Preparation,toxicity in vitro and preliminary targeting of canine red blood cell membrane biomimetic nanocarriers

作　　者：林子俊 马月飞 裴世敏 LIN Zijun;MA Yuefei;PEI Shimin(College of Tropical Crops,Hainan University,Haikou 570228,China)

机构地区：[1]海南大学热带农林学院,海口570228

出　　处：《黑龙江畜牧兽医》2024年第11期101-106,112,121,共8页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了提高纳米颗粒的安全性和靶向性,试验采用低渗处理法制备犬红细胞膜(RBCM),双乳法制备聚乳酸-羟基乙酸共聚物纳米颗粒(BLANK-PLGA),并用脂质体挤出器挤出法制备犬红细胞膜囊泡(RBCM-NVE)和犬红细胞膜仿生纳米载体(PLGA-RBCM);采用透射电镜、动态光散射技术对不同纳米颗粒的结构、大小和电位进行表征鉴定;通过稳定性试验考察不同纳米颗粒的粒径及聚集性变化;采用SDS-PAGE和Western-blot法验证挤出前后细胞CD47膜蛋白表达情况;通过犬乳腺肿瘤细胞(CIPp)摄取BLANK-PLGA和PLGA-RBCM研究纳米颗粒的同源靶向性;通过体外试验探究BLANK-PLGA和RBCM-NVE纳米颗粒的细胞毒性。结果表明:BLANK-PLGA呈球形结构,RBCM-NVE呈杯状囊泡结构,PLGA-RBCM呈明显的“核-壳”结构;BLANK-PLGA平均粒径为(305.17±2.59)nm,聚合物分散性指数(polymer dispersity index, PDI)为0.10±0.01,平均电位为(-3.86±0.59)mV;RBCM-NVE平均粒径为(200.50±0.26)nm, PDI为0.22±0.02,平均电位为(-9.60±0.88)mV;PLGA-RBCM平均粒径为(274.16±2.11)nm, PDI为0.18±0.03,平均电位为(-6.03±0.64)mV;第0~25天,在PBS中PLGA-RBCM粒径保持在300 nm以下,PDI为0.2左右,随着时间推移,BLANK-PLGA的粒径与PDI均明显增大,粒径大小不均;BLANK-PLGA和PLGA-RBCM在胎牛血清中4 h内吸光度没有较大变化;RBCM在通过脂质体挤出器挤出前后均保持相同的蛋白条带并且均表达CD47膜蛋白;CIPp细胞对PLGA-RBCM的摄取量极显著多于对BLANK-PLGA(P<0.01);BLANK-PLGA和RBCM-NVE与CIPp细胞共孵育6,12,24 h后细胞活力差异不显著(P>0.05)。说明成功制备的PLGA-RBCM具有良好的稳定性、安全性及对同源细胞的靶向性。In order to improve the safety and targeting of nanoparticles,canine red blood cell membrane(RBCM)was prepared by hypotonic treatment,PLGA nanoparticles(BLANK-PLGA)were prepared by double emulsion method,red blood cell membrane vesicles(RBCM-NVE)and canine red blood cell membrane biomimetic nanocarriers(PLGA-RBCM)were prepared by"top-down"extrusion method.The structure,size and potential of different nanoparticles were characterized by transmission electron microscopy and dynamic light scattering techniques.The changes in particle size and aggregation of different nanoparticles were investigated by stability experiments.The cell membrane surface proteins were verified by SDS-PAGE and Western-blot.The homologous targeting of different nanoparticles was studied by cellular uptake.The cytotoxic effects of different nanoparticles were studied by in vitro experiments.The results showed that BLANK-PLGA had a spherical structure,RBCM-NVE exhibited a cup-shaped vesicle structure,and PLGA-RBCM had an obvious“core-shell”structure.The average particle size of BLANK-PLGA was(305.17±2.59)nm;the Polymer Dispersity Index(PDI)was 0.10±0.01,and the average potential was(-3.86±0.59)mV.RBCM-NVE had an average particle size of(200.50±0.26)nm,a PDI of 0.22±0.02,and an average potential of(-9.60±0.88)mV.PLGA-RBCM had an average particle size of(274.16±2.11)nm,a PDI of 0.18±0.03,and the average potential of(-6.03±0.64)mV.The PLGA-RBCM particle size in PBS stayed below 300 nm from day O to day 25,and its PDI was approximately O.2.BLANK-PLGA particle size and PDI both dramatically increased over time,and the particle size distribution was not uniform.BLANK-PLGA and PLGA-RBCM did not undergo a stronger change in absorbance in FBS over a period of 4 h.Canine red blood cell membranes maintained the same protein bands before and after extrusion through the liposome extruder and expressed CD47 membrane proteins,which did not cause a lot of protein loss.PLGA-RBCM was successfully taken up by canine mammary tumor CIPp cells with ho

关 键 词：犬红细胞膜仿生纳米载体 PLGA纳米颗粒 结构表征 安全性 靶向性 

分 类 号：S829.2[农业科学—畜牧学] Q811.7[农业科学—畜牧兽医]