Department of Mechanical Engineering, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218, USA.
RASyn, LLC, 700 Main Street, Cambridge, MA, 02139, USA.
Biomed Eng Online. 2024 Nov 21;23(1):116. doi: 10.1186/s12938-024-01311-2.
Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translation of EVs into viable therapeutic delivery vehicles is challenged by lengthy and inefficient processes for cargo loading and pre- and post-loading purification of EVs, resulting in limited quantity and consistency of engineered EVs.
In this work, we develop a fast and streamlined method to load surface protein-specific subpopulations of EVs with miRNA by electroporating EVs, while they are bound to antibody-coated beads. We demonstrate the selection of CD81 EV subpopulation using magnetic microbeads, facilitating rapid EV manipulations, loading, and subsequent purification processes. Our approach shortens the time per post-electroporation EV wash by 20-fold as compared to the gold standard EV washing method, ultracentrifugation, resulting in about 2.5-h less time required to remove unloaded miRNA. In addition, we addressed the challenge of nonspecific binding of cargo molecules due to affinity-based EV selection, lowering the purity of engineered EVs, by implementing innovative strategies, including poly A carrier RNA-mediated blocking and dissociation of residual miRNA and EV-like miRNA aggregates following electroporation.
Our streamlined method integrates magnetic bead-based selection with electroporation, enabling rapid and efficient loading of miRNA into CD81 EVs. This approach not only achieves comparable miRNA loading efficiency to conventional bulk electroporation methods but also concentrates CD81 EVs and allows for simple electroporation parameter adjustment, promising advancements in therapeutic RNA delivery systems with enhanced specificity and reduced toxicity.
细胞外囊泡 (EVs) 作为一种有前途的工具，可用于将治疗剂靶向递送至广泛的疾病。作为源自活细胞的纳米级膜结合颗粒，EVs 具有作为生物分子载体的固有能力。然而，EVs 转化为可行的治疗性递药载体受到载 cargo 装载和 EV 预加载和后加载纯化的冗长且低效的过程的挑战，导致工程化 EVs 的数量和一致性有限。
在这项工作中，我们通过电穿孔开发了一种快速而简化的方法，通过将 EV 与抗体包被的珠结合，将 miRNA 加载到表面蛋白特异性 EV 亚群中。我们使用磁性微珠来选择 CD81 EV 亚群，促进了 EV 的快速操作、装载和随后的纯化过程。与超速离心的金标准 EV 洗涤方法相比，我们的方法将每批电穿孔后 EV 洗涤的时间缩短了 20 倍，从而减少了约 2.5 小时的时间来去除未加载的 miRNA。此外，我们通过实施创新策略解决了由于基于亲和力的 EV 选择而导致货物分子非特异性结合，从而降低了工程化 EV 纯度的问题，包括 poly A 载体 RNA 介导的阻断以及电穿孔后残余 miRNA 和 EV 样 miRNA 聚集体的解离。
我们的简化方法将基于磁性珠的选择与电穿孔相结合，实现了 miRNA 快速有效地装入 CD81 EVs。这种方法不仅达到了与传统批量电穿孔方法相当的 miRNA 装载效率，而且浓缩了 CD81 EVs，并允许简单地调整电穿孔参数，有望在治疗性 RNA 递药系统中取得进展，提高特异性并降低毒性。
