Department of Bioengineering, School of Engineering, Santa Clara University, Santa Clara, CA 95053, USA.
Department of Bioengineering and Tsinghua-Berkeley Shenzhen Institute, University of California at Berkeley, Berkeley, CA 94720, USA.
Int J Nanomedicine. 2019 May 9;14:3413-3425. doi: 10.2147/IJN.S196975. eCollection 2019.
Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.
外泌体是普遍存在的天然分泌的稳定纳米囊泡，可以对其进行工程改造，以靶向并递送新型治疗药物来治疗多种人类疾病。我们通过在细胞衍生的纳米囊泡表面展示人 TNF 受体-1（hTNFR1）的 TNFα 结合域，将其设计为通过拮抗主要促炎细胞因子肿瘤坏死因子α（TNFα）来治疗炎症的诱饵。使用稳定转染的 HEK293 细胞，通过在外泌体的外表面展示 hTNFR1 的 TNFα 结合域，来产生诱饵外泌体。我们开发了一种有效的方法，基于连续离心、超滤和沉淀，从条件培养基中纯化工程化的外泌体。我们使用免疫定量、纳米颗粒跟踪分析和共聚焦显微镜来表征诱饵外泌体，以确认它们保留了天然产生的外泌体的正确方向、大小和形状。我们使用炎症报告细胞系证明了工程化的诱饵外泌体特异性拮抗 TNFα 的活性。在人细胞中产生的诱饵外泌体作为一种新型生物试剂，可拮抗 TNFα 介导的炎症信号转导。
