Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Central South University, Changsha 410083.
Hunan Provincial Key Laboratory of New Drug Preparation Research and Development, Changsha Medical College, Changsha 410219.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2022 Dec 28;47(12):1646-1654. doi: 10.11817/j.issn.1672-7347.2022.210764.
Glioma is the most common primary intracranial tumor and there is still no ideal treatment at present. Gene therapy, as one of the new methods for treating glioma, has attracted attention in recent years. But its application in treating glioma is very limited due to lack of effective delivery vectors. This study aims to investigate the feasibility of biomimetic nanomaterials made from glioma cells-derived extracellular vesicles (EV) for targeted delivery of signal transducers and activators of transcription 3 (STAT3)-small interfering RNA (siRNA) in treating glioma.
First, U251 glioma cells-derived extracellular vessel (EVU251) was extracted by ultra-centrifugal method. Nanoparticle tracking analysis was used to characterize the particle size distribution, the transmission electron microscope was used to analyze the morphology, and Western blotting was used to verify the expression of srface characteristic protein. The homing ability was verified by cell uptake assay after labeling EVU251 with membrane dye kit PKH67; the EVU251 contents were removed by a low permeability method and then EVMU251 was prepared through a microporous membrane. Finally, the biomimetic nanomaterials EVMU251@STAT3-siRNA were prepared by loading STAT3-SiRNA with electro-dyeing method. The real-time quantitative PCR was used to quantify the successful encapsulation of siRNA, and the encapsulation and drug loading rate was calculated; then Cy5-labeled siRNA was used to evaluate the ability of biomimetic nanomaterials (EVMU251@CY5-siRNA) to target U251 cells. Lysosomal escape ability of the biomimetic nanomaterial was evaluated by lysosomal dye lyso-tracker green. At last, the ability of EVMU251@STAT3-siRNA to knock down STAT3 gene and selective killing of U251 cells was detected by cell experiments in vitro.
The size of EVU251 ranged from 50 nm to 200 nm with a natural disc shape. The expression of extracellular vesicle marker proteins could be detected on the membrane of EVU251. The cell uptake assay demonstrated that it had homing ability to target U251 cells. After EVU251 was prepared as EVMU251@STAT3-siRNA, the particle size was (177.9±5.0) nm, the siRNA loading rate was (33.5±2.2)% and the drug loading rate was (3.24±0.21)%. The biomimetic nanomaterial EVMU251@STAT3-siRNA still had the ability to target U251 cells and successfully deliver siRNA to the cytoplasm without lysosomal degradation. The EVMU251@STAT3-siRNA can effectively knock down the expression of STAT3 gene and produce selective killing ability in U251 cells.
The biomimetic nanomaterials EVMU251@STAT3-siRNA made from glioma U251 cells-derived extracellular vesicles can knock down STAT3 gene of U251 cells and produce selective killing effect, which can provide a new idea for the treatment of glioma.
脑胶质瘤是最常见的原发性颅内肿瘤，目前仍没有理想的治疗方法。基因治疗作为治疗脑胶质瘤的新方法之一，近年来受到关注。但由于缺乏有效的递送载体，其在治疗脑胶质瘤中的应用受到很大限制。本研究旨在探讨利用脑胶质瘤细胞衍生的细胞外囊泡（EV）制备仿生纳米材料，用于靶向递送达拉斯转录因子 3（STAT3）-小干扰 RNA（siRNA），治疗脑胶质瘤的可行性。
首先采用超速离心法提取 U251 脑胶质瘤细胞衍生的细胞外囊泡（EVU251）。采用纳米颗粒跟踪分析技术对其粒径分布进行表征，采用透射电子显微镜分析其形态，采用 Western blot 验证其表面特征蛋白的表达。通过用膜染料试剂盒 PKH67 标记 EVU251 验证其归巢能力；采用低通透性方法去除 EVU251 内容物，然后通过微孔膜制备 EVMU251。最后，采用电染色法将 STAT3-siRNA 载入仿生纳米材料 EVMU251@STAT3-siRNA。采用实时定量 PCR 定量检测 siRNA 的成功包封，并计算包封率和载药率；然后用 Cy5 标记的 siRNA 评价仿生纳米材料（EVMU251@CY5-siRNA）靶向 U251 细胞的能力。采用溶酶体染料 lyso-tracker green 评价仿生纳米材料的溶酶体逃逸能力。最后，通过体外细胞实验检测 EVMU251@STAT3-siRNA 敲低 STAT3 基因和选择性杀伤 U251 细胞的能力。
EVU251 的大小范围为 50nm 至 200nm，呈天然盘状。可以检测到 EVU251 膜上存在细胞外囊泡标记蛋白的表达。细胞摄取实验表明其具有靶向 U251 细胞的归巢能力。将 EVU251 制备成 EVMU251@STAT3-siRNA 后，粒径为（177.9±5.0）nm，siRNA 载药率为（33.5±2.2）%，载药率为（3.24±0.21）%。仿生纳米材料 EVMU251@STAT3-siRNA 仍具有靶向 U251 细胞的能力，并能将 siRNA 有效递送至细胞质，而不被溶酶体降解。EVMU251@STAT3-siRNA 可有效敲低 U251 细胞中 STAT3 基因的表达，并在 U251 细胞中产生选择性杀伤作用。
由脑胶质瘤 U251 细胞衍生的细胞外囊泡制备的仿生纳米材料 EVMU251@STAT3-siRNA 可敲低 U251 细胞的 STAT3 基因并产生选择性杀伤作用，为脑胶质瘤的治疗提供了新的思路。
