血小板膜仿生纳米靶向探针的制备及体外寻靶实验研究
Preparation and in vitro targeting experiment of platelet membrane biomimetic targeting nanoprobes
周佳 1余才贵 1姜楠 1郭瑞强 1曹省1
作者信息
- 1. 430060 武汉市,武汉大学人民医院超声影像科 心血管病湖北省重点实验室 武汉大学心血管病研究所
- 折叠
摘要
目的 制备血小板膜仿生纳米靶向探针(PLT-RAP@NPs),探讨其在体外的免疫逃逸、靶向和黏附能力,以及联合超声靶向微泡释放技术(UTMD)后的载药释放情况.方法 采用单乳化-溶剂挥发法合成纳米探针RAP@NPs,梯度离心-反复冻融法提取血小板膜囊泡,超声震荡法制备PLT-RAP@NPs,检测其粒径、表面电位及稳定性,观察其微观形态,计算其包封率和载药率,确定雷帕霉素(RAP)负载的最佳方案.DiI荧光染料分别标记聚乳酸-羟基乙酸共聚物(PLGA,DiI@PLGA组)和PLT@PLGA(PLT-DiI@PLGA组),用于模拟RAP@NPs、PLT-RAP@NPs进行体外寻靶实验的荧光强度检测;将其分别与巨噬细胞、泡沫细胞和内皮细胞共孵育2h,观察DiI@PLGA组与PLT-DiI@PLGA组橙红色荧光强度,分析各细胞对DiI@PLGA和PLT-DiI@PLGA的吞噬、摄取和黏附能力.细胞增殖-毒性实验观察不同浓度RAP(3.00 µg/ml、6.25 µg/ml、12.50 µg/ml、25.00 µg/ml、50.00 µg/ml、100.00 µg/ml)的游离RAP、RAP@NPs和PLT-RAP@NPs对细胞增殖活性的影响.体外药物控释实验观察PLT-RAP@NPs联合UTMD后载药释放效果.结果 本实验制备的PLT-RAP@NPs呈球形,大小均匀,表面覆盖薄膜,"核-壳"结构清晰,平均粒径(286.83±5.25)nm,平均表面电位-(15.60±5.04)mV.当100 mg PLGA负载3 mg RAP时,包封率为60.35%,载药率为2.18%.体外寻靶实验结果显示,巨噬细胞与纳米靶向探针共孵育2h后,DiI@PLGA组橙红色荧光强度约为PLT-DiI@PLGA组3倍;泡沫细胞与靶向纳米探针共孵育2h后,PLT-DiI@PLGA组橙红色荧光强度约为DiI@PLGA组4倍;内皮细胞与纳米靶向探针共孵育2h后,PLT-DiI@PLGA组橙红色荧光强度较DiI@PLGA组增强.细胞增殖-毒性实验结果显示,随着RAP浓度增高,游离RAP中细胞增殖活性下降,而RAP@NPs和PLT-RAP@NPs中细胞增殖活性变化较小.体外药物控释实验结果显示,RAP@NPs和PLT-RAP@NPs中RAP均缓慢释放,72 h积累药物释放量分别为42.12%和33.74%,联合UTMD后积累药物释放量分别提升至75.57%和67.54%.结论 成功制备PLT-RAP@NPs,其可抑制巨噬细胞吞噬、增强泡沫细胞摄取和提高内皮细胞黏附,从而实现免疫逃逸及靶向能力,联合UTMD可进行RAP靶向控释.
Abstract
Objective To prepare platelet membrane biomimetic targeting nanoprobes(PLT-RAP@NPs),and to explore its immune escape,targeting and adhesion ability in vitro,as well as drug delivery after combined with ultrasound-targeted microbubble destruction(UTMD).Methods A nanoprobe RAP@NPs were synthesized by single emulsification-solvent evaporation technique.Platelet membrane vesicles extracted by density-gradient centrifugation and repeated freeze-thaw cycles.PLT-RAP@NPs were prepared by ultrasonic oscillation method.The particle size,surface potential and stability were detected,the microscopic morphology was observed,the encapsulation efficiency and drug loading efficiency of nanoprobes were calculated to determine the best drug-loading protocol for rapamysin(RAP).DiI dye was encapsulated into PLGA and PLT@PLGA to form DiI@PLGA group and PLT-DiI@PLGA group instead of the RAP@NPs and PLT-RAP@NPs,which were used to simulate RAP@NPs and PLT-RAP@NPs for fluorescence detection in in vitro targeting experiments.They were co-incubated with macrophages,foam cellsand endothelial cells,endothelial cells for 2 h in vitro,respectively.The orange-red fluorescence intensity of DiI@PLGA group and PLT-DiI@PLGA group were observed.The phagocytosis,uptake and adhesion ability of these cells to the DiI@PLGA and PLT-DiI@PLGA were observed and analyzed.For evaluating cell proliferation and cytotoxicity,the cell viability at different concentration of RAP(3.00µg/ml,6.25µg/ml,12.50µg/ml,25.00µg/ml,50.00µg/ml,100.00µg/ml)in free RAP,RAP@NPs and PLT-RAP@NPs were observed.The drug release of PLT-RAP@NPs combined with UTMD in vitro were evaluated.Results PLT-RAP@NPs were spherical,and showed a uniform size and clear core-shell structure.The surface of the shell was coated with a film.The particle size and surface potential were(286.83±5.25)nm and-(15.60±5.04)mV,respectively.When 100 mg PLGA was loaded with 3 mg RAP,the encapsulation efficiency and drug loading efficiency were 60.35%and 2.18%,respectively.The results of in vitro targeting experiment showed that after macrophages were co-incubated with DiI@PLGA and PLT-DiI@PLGA for 2 h,the orange-red fluorescence intensity of the DiI@PLGA group was approximately 3 times higher than that of the PLT-DiI@PLGA group,while the opposite result was obtained when the foam cells were co-incubated with the NPs.The orange-red fluorescence intensity of the PLT-DiI@PLGA group was approximately 4 times higher than that of the DiI@PLGA group.And the orange-red fluorescence intensity of the PLT-DiI@PLGA group was enhanced compared with DiI@PLGA group after endothelial cells co-incubated with the NPs.With the increase of the concentration of RAP,the cell viability of the free RAP gradually decreased,while the cell proliferation was less affected in RAP@NPs and PLT-RAP@NPs.Slow release of RAP from RAP@NPs and PLT-RAP@NPs were shown,the release percentage were 42.12%and 33.74%at 72 h,and were raised to 75.57%and 67.54%after combined with UTMD,respectively.Conclusion The successfully prepared PLT-RAP@NPs can inhibit macrophage phagocytosis,enhance foam cell uptake and improve endothelial cell adhesion,so as to achieve immune escape and targeting ability.Combined with UTMD,RAP targeted controlled release can be realized.
关键词
血小板膜仿生纳米靶向探针/雷帕霉素/超声靶向微泡释放技术/动脉粥样硬化Key words
Platelet membrane biomimetic targeting nanoprobes/Rapamycin/Ultrasound-targeted microbubble destruction/Atherosclerosis引用本文复制引用
基金项目
国家自然科学基金(81901757)
国家自然科学基金(82102045)
出版年
2024
NETL
NSTL
万方数据