DataSheet1_Isolation of goat milk small extracellular vesicles by novel combined bio-physical methodology.PDF

Introduction: Goat milk is notable as a cost-effective source of exosomes, also known as small extracellular vesicles (sEVs). These nanoparticle-like structures are naturally secreted by cells and have emerged as potential diagnostic agents and drug delivery systems, also supported by their proven therapeutic effects. However, the complexity of goat milk and the lack of standardized protocols make it difficult to isolate pure sEVs. This work presents an optimized approach that combines well-established physical isolation methods with the biological treatment of milk with rennet.
Methods: sEVs derived from goat milk were purified using a methodology that combines differential ultracentrifugation, rennet, and size-exclusion chromatography. This novel strategy was compared with two of the main methodologies developed for isolating extracellular vesicles from bovine and human milk by means of physico-chemical characterization of collected vesicles using Transmission Electron Microscopy, Western blot, Bradford Coomassie assay, Dynamic Light Scattering, Nanoparticle Tracking Analysis and Zeta Potential.
Results: Vesicles isolated with the optimized protocol had sEV-like characteristics and high homogeneity, while samples obtained with the previous methods were highly aggregated, with significant residual protein content.
Discussion: This work provides a novel biophysical methodology for isolating highly enriched goat milk sEVs samples with high stability and homogeneity, for their further evaluation in biomedical applications as diagnostic tools or drug delivery systems.

引言：山羊奶作为一种经济高效的外泌体（exosomes）来源而备受关注，外泌体又称为小细胞外囊泡（small extracellular vesicles，sEVs）。这类类纳米级颗粒结构由细胞自然分泌，现已成为极具潜力的诊断试剂与药物递送系统，其经证实的治疗效应进一步为该应用方向提供了支撑。然而，山羊奶成分复杂，且缺乏标准化分离流程，导致难以获取纯净的sEVs。本研究提出一种优化的分离方案，将成熟的物理分离方法与凝乳酶（rennet）对羊奶的生物处理手段相结合。
方法：本研究采用结合差速超速离心、凝乳酶（rennet）与尺寸排阻色谱的方法，对源自山羊奶的sEVs进行纯化。将该新型策略与两种主流的牛、人源细胞外囊泡分离方法进行对比，通过透射电子显微镜（Transmission Electron Microscopy）、蛋白质免疫印迹（Western blot）、考马斯亮蓝 Bradford 法、动态光散射（Dynamic Light Scattering）、纳米颗粒追踪分析（Nanoparticle Tracking Analysis）以及Zeta电位对获取的囊泡进行理化表征。
结果：采用优化方案分离得到的囊泡具有sEVs的典型特征，且均一性较高；而采用传统方法获取的样本则出现严重聚集，且残留蛋白含量显著偏高。
讨论：本研究提出了一种新型生物物理方法，可分离得到高富集度、高稳定性与高均一性的山羊奶sEVs样本，为其后续在生物医学领域作为诊断工具或药物递送系统开展评估提供了坚实基础。