携单抗载增效剂超声纳米微泡制备实验的探索与研究  

Exploration and study on preparation experiment of ultrasonic nanobubbles carrying monoclonal antibody and loaded with synergist

作　　者：王婧茹 红华 张惠卿 王占忠 梁丹艳 刘倩 孙冉 WANG Jing-ru;HONG Hua;ZHANG Hui-qing;WANG Zhan-zhong;LIANG Dan-yan;LIU Qian;SUN Ran(Department of Medical Ultrasonic,Inner Mongolia Autonomous Region People’s Hospital,Hohhot 010017,Inner Mongolia,China)

机构地区：[1]内蒙古自治区人民医院超声医学科,内蒙古呼和浩特010017

出　　处：《生物医学工程与临床》2023年第5期567-574,共8页Biomedical Engineering and Clinical Medicine

基　　金：内蒙古自治区自然科学基金面上项目(2021MS08107,2022MS08072,2020MS08206)。

摘　　要：目的超声纳米微泡造影剂可携带药物在实现精准成像的同时定向到达目标微环境实现靶向给药。探索制备携单抗超声纳米微泡造影剂的实验条件及验证方式。方法通过乳化法制备超声纳米微泡并用链霉亲和素对其进行修饰;再通过生物素-亲和素系统与被生物素化的帕博利珠单抗(PD-1单抗)进行连接,得到携PD-1单抗载增效剂超声纳米粒;探索高效率的实验条件及验证方式;对实验制得超声纳米微泡的基本性质和实验表征分别进行检测与分析;对纳米微泡进行体外相变及超声显像效果的初步验证。结果成功制备出以高分子聚合物聚乳酸羟基乙酸包载液态氟碳和血卟啉单甲醚、同时用PD-1单抗修饰其表面的携单抗载增效剂超声纳米微泡;血卟啉单甲醚与聚乳酸羟基乙酸的比例在1∶25左右时饱和状态良好,血卟啉单甲醚的包封率为40.4%;吗啉乙磺酸缓冲溶液(MES Buffer)pH=7.4时血卟啉单甲醚的包封状态及聚乳酸羟基乙酸壳膜与链霉亲和素的结合效率可以取得较好的平衡;纳米微泡及链霉亲和素修饰后的生物素化单抗在激光共聚焦显微镜下分别表现出红色和绿色荧光,叠加融合后呈现黄色荧光,间接表明携PD-1单抗载增效剂纳米微泡制备成功;纳米微泡复溶后水溶性及分散度较好,基于其较稳定的物理性质,4℃条件下可短期保存;纳米微泡在扫描电子显微镜下表现为表面光整的类球形结构,大小整体较均匀,局部可见小范围粘连聚集现象;透射电子显微镜下纳米微泡呈内部黑色的类球形结构;动态激光散射仪测得纳米微泡平均粒径为(322.29±83.56)nm,Zeta电位为(-2.41±1.75)mV,聚合物分散指数约为0.287。纳米微泡在40℃左右时开始出现相变表现,一定温度范围(40℃~50℃)内有较好的超声显像效果,但分散性欠佳。结论成功制备出携单抗载增效剂超声纳米微泡;纳米微泡整体大小较均匀,局�Objective Ultrasound nanobubble contrast agent can carry the drug for accurate imaging,and simultaneously reach target microenvironment to achieve targeted drug delivery.To explore the experimental conditions and verification methods for the preparation of monoclonal antibody-loaded ultrasound nanobubble contrast agents.Methods The ultrasonic nanobubbles were prepared by emulsification method and modified by Streptavidin.Then,the biotin-avidin system was linked biotinylated pabolizumab(PD-1 monoclonal antibody)to obtain PD-1 monoclonal antibody-loaded synergist-loaded ultrasound nanoparticles.The highly efficient experimental conditions and verification methods were explored,the basic properties and experimental characterization of ultrasonic nanobubbles were detected and analyzed respectively.The phase transition and ultrasonic imaging effect of nanobubbles were preliminarily verified in vitro.Results The ultrasound nanobubbles carrying monoclonal antibody-loaded synergist were successfully prepared with liquid fluorocarbon,hematoporphyrin monomethyl ether encapsulated in poly(lactic-co-glycolic acid)and surface modified PD-1 monoclonal antibody.The ratio of hematoporphyrin monomethyl ether to polylactic acid glycolic acid was about 1∶25,and encapsulation efficiency of hematoporphyrin monomethyl ether was 40.4%.The encapsulation state of hematoporphyrin monomethyl ether and binding efficiency of polylactic acid glycolic acid shell membrane to streptavidin were well balanced in MES Buffer(pH=7.4).The nanobubbles and biotinylated monoclonal antibodies modified by Streptavidin displayed red and green fluorescence under laser confocal microscope,and yellow fluorescence after superposition and fusion,which indirectly indicated that nanobubbles carrying PD-1 monoclonal antibody were successfully prepared.The water solubility and dispersion of nanobubbles after re-dissolution were better,which was short-term preserved at 4°C based on their stable physical properties.The nanobubbles exhibited spherical like structure

关 键 词：超声纳米微泡 微泡造影剂 单抗 增效剂 液态氟碳相变 

分 类 号：R318.1[医药卫生—生物医学工程]