Role of biomimetic nanomaterials made from glioma cell- derived extracellular vesicles in targeted delivery of STAT3-siRNA.

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.目的: 胶质瘤是最常见的颅内原发性肿瘤，目前仍无理想的治疗手段，基因治疗作为胶质瘤治疗的新方法之一近年来备受关注，但因缺少有效的递送载体，从而使其在胶质瘤治疗中的应用仍十分有限。本研究旨在探讨用胶质瘤细胞来源胞外囊泡(extracellular vessel，EV)制作的仿生纳米材料靶向递送基因药物STAT3-siRNA来治疗胶质瘤的可行性。方法: 首先通过超速离心法提取胶质瘤细胞U251来源的EV(U251 glioma cells derived EV，EVU251)，采用纳米粒子跟踪分析法对其粒径分布进行表征分析，透射电镜进行形貌分析，蛋白质印迹法验证其表面特征蛋白的表达；使用膜染料试剂盒PKH67将EVU251标记后，通过细胞摄取实验验证其靶向U251的归巢能力；采用低渗法去除EVU251的内容物，并通过微孔膜制备EVU251囊泡(EVU251 memberane，EVMU251)；采用电转染法将EVMU251中载入靶向信号转导与转录活化因子3(signal transducers and activators of transcription 3，STAT3)的小干扰RNA(small interfering RNA，siRNA)(STAT3-siRNA)制成仿生纳米材料EVMU251@STAT3-siRNA；使用real-time PCR对成功包载的siRNA进行定量分析，并计算包载率及载药率；采用荧光染料CY5标记siRNA后，通过细胞摄取实验评价仿生纳米材料EVMU251@CY5-siRNA靶向U251细胞的能力，再结合绿色溶酶体示踪剂评价该仿生纳米材料的溶酶体逃逸能力；最后通过体外细胞实验分别检测EVMU251@STAT3-siRNA敲低STAT3 mRNA表达水平以及选择性杀伤U251的能力。结果: EVU251粒径分布范围为50~200 nm，呈天然的圆盘形态，并且在其膜上检测到EV标志性蛋白质的表达。细胞摄取实验证明其具有靶向U251的归巢能力。将EVU251制备为EVMU251@STAT3-siRNA后，粒径变为(177.9±5.0) nm，siRNA包载率为(33.5±2.2)%。细胞摄取实验证明其仍具有靶向U251细胞的能力，且能成功避开溶酶体降解而将siRNA递送至胞质。体外细胞实验证明EVMU251@STAT3-siRNA能有效敲低U251细胞STAT3基因的表达，并能对U251细胞产生选择性杀伤。结论: 用EVU251制作的仿生纳米材料EVMU251@STAT3-siRNA能选择性敲低U251细胞STAT3基因的表达，并能产生选择性杀伤作用，可为胶质瘤的基因治疗提供新思路。.