血小板膜仿生纳米靶向探针PLT-RAP@NPs的制备及体外寻靶实验研究

目的：构建血小板膜仿生纳米靶向探针PLT-RAP@NPs,并探索其在体外的免疫逃逸、靶向及黏附能力以及联合UTMD后的载药释放。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,利用超声震荡法制备血小板膜仿生纳米探针PLT-RAP@NPs。通过动态光散射技术检测纳米探针的粒径大小、电位及稳定性;透射电镜观察微观形态;高效液相色谱法检测包封率和载药率,确定RAP负载的最佳方案。封装DiI荧光染料形成DiI@PLGA、PLT-DiI@PLGA,模拟RAP@NPs、PLT-RAP@NPs进行荧光检测。将其分别与RAW264.7巨噬细胞、泡沫细胞和内皮细胞共孵育,通过荧光显微镜分析细胞对纳米靶向探针的吞噬、摄取和黏附情况。CCK8法评价不同RAP浓度的纳米探针对细胞活性影响。体外透析法和高效液相色谱法评估PLT-RAP@NPs联合UTMD后载药控释效果。结果：PLT-RAP@NPs粒径大小均匀、呈球形,表面覆盖薄膜、“核-壳”结构清晰。当100 mg PLGA负载3 mg RAP时,药物包载率较高为60.35 %,载药率为2.18 %。巨噬细胞与纳米靶向探针共孵育2 h 后,DiI@PLGA组橙红色荧光强度比PLT-DiI@PLGA组高;泡沫细胞与靶向纳米探针共孵育2 h后,则PLT-DiI@PLGA组荧光强度高。TNF-α刺激血管内皮细胞上调表达vWF后与纳米靶向探针共孵育,PLT-DiI@PLGA组橙红色荧光与vWF绿色荧光共定位结合明显,而DiI@PLGA组未见明显结合。细胞增殖-毒性实验结果显示游离RAP组浓度增高,细胞活性下降,而PLT-RAP@NPs组细胞活性无明显影响。体外药物释放实验结果显示RAP@NPs组和PLT-RAP@NPs组RAP均缓慢释放,72 h释放量分别为42.12 %和33.74 %。联合UTMD后,RAP释放量明显提升至75.57 %和67.54 %。结论：成功制备的血小板膜仿生纳米探针PLT-RAP@NPs通过血小板膜的生物学功能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。

Preparation and in vitro targeted experiment of platelet membrane bionic nanoprobe PLT-RAP@NPs

Objective To construct a platelet membrane bionic nanoprobe targeting PLT-RAP@NPs, and explore its immune escape, targeting and adhesion ability in vitro, as well as drug delivery after combination with UTMD.Methods RAP was loaded onto polymer PLGA by single emulsification-solvent evaporation technique, and a nanoprobe RAP@NPs was synthesized. Platelet membrane vesicles extracted by density-gradient centrifugation and repeated freeze-thaw cycles were used to prepare platelet membrane-coated biomimetic nanoprobe PLT-RAP@NPs by ultrasonic oscillation method. The particle size, potential and stability of nanoprobe were detected by dynamic light scattering technique. The microscopic morphology of the nanoprobe was observed by transmission electron microscopy. High-performance liquid chromatography was used to detect the encapsulation efficiency and drug loading efficiency of RAP in various proportions of nanoprobe to determine the best drug-loading protocol for RAP. DiI dye was encapsulated into PLGA to form DiI@PLGA and PLT-DiI@PLGA instead of the RAP@NPs and PLT-RAP@NPs for fluorescence detection, and then the NPs were co-incubated with RAW264.7 macrophages, foam cells, and endothelial cells in vitro. The phagocytosis and adhesion of these cells to the NPs were observed and analyzed by fluorescence microscope. For evaluating cell proliferation and cytotoxicity, CCK8 was used to evaluate the cell viability. In vitro dialysis and high-performance liquid chromatography were used to evaluate RAP release of PLT-RAP@NPs combined with UTMD.Results PLT-RAP@NPs were transparent and spherical, and showed a uniform size and clear core-shell structure. The surface of the core was coated with a film. The drug encapsulation efficiency reached the highest when 100 mg PLGA and 3 mg RAP entered the organic solvent; the encapsulation and drug loading efficiency were 60.35% and 2.18%, respectively. After RAW264.7 cells were co-incubated with DiI@PLGA and PLT-DiI@PLGA separately for 2 h, the orange-red fluorescence intensity of the DiI@PLGA group was significantly 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. After TNF-α stimulated HUVEC up-regulated the expression of von Willebrand factor (vWF) and then co-incubated with the NPs for 2 h, a significant overlap between orange-red and green fluorescence in the PLT-DiI@PLGA group was shown, while no significant overlap was displayed in the DiI@PLGA group. With the increase of RAP concentration, the cell viability of the free RAP group gradually decreased, while the cell proliferation was less affected in PLT-RAP@NPs group. Slow release of RAP in RAP@NPs and PLT-RAP@NPs was shown, the release percentage was 42.12 % and 33.74 % at 72 h, and were raised to 75.57 % and 67.54 % after combination with UTMD, respectively.Conclusions The successfully prepared platelet membrane bionic nanoprobe PLT-RAP@NPs can inhibit macrophage phagocytosis, enhance foam cell uptake and improve endothelial cell adhesion through the biological function of platelet membrane, so as to achieve immune escape and targeting ability. Combined with UTMD, RAP controlled release of targeted can be realized.