Laboratory of Pharmacology, Deparment of Biology, Faculty of Chemistry and Biology, Center for the Development of Nanoscience and Nanotechnology, University of Santiago de Chile, Santiago, Chile,
Int J Nanomedicine. 2019 Apr 5;14:2433-2447. doi: 10.2147/IJN.S192925. eCollection 2019.
The antitumoral effect of ATP requires its accumulation in the extracellular space to interact with membrane receptors in target cells. We propose the use of albumin nanoparticles (ANPs) coated with erythrocyte membranes (EMs) to load, deliver, release, and enhance the extracellular anticancer activity of ATP.
ANPs were synthesized by desolvation method and optimal values of pH, albumin concentration, and ethanol volume were determined. EMs were derived from erythrocyte lysates and were coated on to ANPs using an extruder. Size was determined by transmission electron microscopy (TEM) and hydrodynamic size and zeta potential were determined by dynamic light scattering. Coating of the ANPs with the EMs was verified by TEM and confocal microscopy. Nanoparticle cell uptake was analyzed by confocal microscopy using HeLa and HEK-293 cell cultures treated with nanoparticles stained with 1,1'-diocta-decyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) for EM-ANPs and Alexa 488 for ANPs. Cell viability was analyzed by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and Annexin V/propidium iodide assays.
Optimal values of ANP preparation were as follows: pH=9, 10 mg/mL albumin concentration, and 2.33±0.04 mL ethanol volume. Size distributions as analyzed by TEM were as follows: ANPs =91.9±4.3 nm and EM-ANPs =98.3±5.1 nm; hydrodynamic sizes: ANPs =180.5±6.8 nm and EM-ANPs =197.8±3.2 nm; and zeta potentials: ANPs =17.8±3.5 mV, ANPs+ATP =-13.60±0.48 and EM-ANPs =-13.7±2.9 mV. The EMs coating the ANPs were observed by TEM and confocal microscopy. A fewer number of internalized EM-ANPs+ATP compared to non-coated ANPs+ATP was observed in HeLa and HEK-293 cells. Cell viability decreased up to 48.6%±2.0% with a concentration of 400 µM ATP after 72 hours of treatment and cell death is caused mainly via apoptosis.
Our current results show that it is possible to obtain nanoparticles from highly biocompatible, biodegradable materials and that their coating with EMs allows the regulation of the internalization process in order to promote extracellular activity of ATP.
ATP 的抗肿瘤作用需要其在细胞外空间积累，以与靶细胞的膜受体相互作用。我们建议使用红细胞膜（EM）包被的白蛋白纳米颗粒（ANPs）来负载、输送、释放和增强 ATP 的细胞外抗癌活性。
通过去溶剂法合成 ANPs，并确定最佳 pH 值、白蛋白浓度和乙醇体积。EM 来源于红细胞裂解物，并使用挤出机涂覆在 ANPs 上。通过透射电子显微镜（TEM）确定粒径，通过动态光散射法确定水动力粒径和 zeta 电位。通过 TEM 和共聚焦显微镜验证 ANPs 与 EM 的包覆。使用 HeLa 和 HEK-293 细胞培养物，用 1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青，4-氯苯磺酸盐（DiD）对 EM-ANPs 进行染色，用 Alexa 488 对 ANPs 进行染色，通过共聚焦显微镜分析纳米颗粒的细胞摄取。通过 [3-(4,5-二甲基噻唑-2-基）-5-(3-羧基甲氧基苯基）-2-(4-磺基苯基）-2H-四唑，内盐（MTS）和 Annexin V/碘化丙啶测定分析细胞活力。
ANP 制备的最佳条件为：pH=9、10mg/mL 白蛋白浓度和 2.33±0.04mL 乙醇体积。TEM 分析的粒径分布如下：ANPs=91.9±4.3nm 和 EM-ANPs=98.3±5.1nm；水动力粒径：ANPs=180.5±6.8nm 和 EM-ANPs=197.8±3.2nm；和 zeta 电位：ANPs=17.8±3.5mV、ANPs+ATP=-13.60±0.48 和 EM-ANPs=-13.7±2.9mV。通过 TEM 和共聚焦显微镜观察到 ANPs 包被的 EM。与未包被的 ANPs+ATP 相比，HeLa 和 HEK-293 细胞中内化的 EM-ANPs+ATP 数量较少。用 400µM ATP 处理 72 小时后，细胞活力下降至 48.6%±2.0%，细胞死亡主要通过凋亡引起。
我们目前的结果表明，有可能从高度生物相容、可生物降解的材料中获得纳米颗粒，并且它们与 EM 的包覆可以调节内化过程，以促进 ATP 的细胞外活性。
