Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) raises questions regarding their biologic functions and their potential theranostic application. Unlike liquid-phase extracellular vesicles (e.g., exosomes), MBV are tightly bound to the ECM, which makes their isolation and harvesting more challenging. The indiscriminate use of different methods to harvest MBV can alter or disrupt their structural and/or functional integrity. The objective of the present study was to compare the effect of various MBV harvesting methods upon yield, purity, and biologic activity. Combinations of four methods to solubilize the ECM (collagenase [COL], liberase [LIB], or proteinase K [PK] and nonenzymatic elution with potassium chloride) and four isolation methods (ultracentrifugation, ultrafiltration [UF], density barrier, and size exclusion chromatography [SEC]) were used to isolate MBV from urinary bladder-derived ECM. All combinations of solubilization and isolation methods allowed for the harvesting of MBV, however, distinct differences were noted. The highest yield, purity, cellular uptake, and biologic activity were seen with MBV isolated by a combination of liberase or collagenase followed by SEC. The combination of proteinase K and UF was shown to have detrimental effects on bioactivity. The results show the importance of selecting appropriate MBV harvesting methods for the characterization and evaluation of MBV and for analysis of their potential theranostic application.

中文翻译：

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基质结合的纳米囊泡：分离方法对产量、纯度和功能的影响

基质结合纳米囊泡 (MBV) 作为细胞外基质 (ECM) 无处不在的成分的鉴定引发了有关其生物学功能和潜在治疗诊断应用的问题。与液相细胞外囊泡（例如外泌体）不同，MBV 与 ECM 紧密结合，这使得它们的分离和收获更具挑战性。不加选择地使用不同的方法来收获 MBV 会改变或破坏其结构和/或功能的完整性。本研究的目的是比较各种 MBV 收获方法对产量、纯度和生物活性的影响。四种溶解 ECM 的方法（胶原酶 [COL]、游离酶 [LIB] 或蛋白酶 K [PK] 和氯化钾非酶洗脱）和四种分离方法（超速离心、超滤 [UF]、密度屏障和尺寸排阻色谱 [SEC]) 用于从膀胱来源的 ECM 中分离 MBV。溶解和分离方法的所有组合都允许收获 MBV，但是，注意到了明显的差异。最高的产量、纯度、细胞摄取和生物活性是通过结合释放酶或胶原酶和 SEC 分离的 MBV 观察到的。蛋白酶 K 和 UF 的组合显示对生物活性具有不利影响。结果显示了选择合适的 MBV 采集方法对 MBV 的表征和评估以及分析其潜在的治疗诊断应用的重要性。溶解和分离方法的所有组合都允许收获 MBV，但是，注意到了明显的差异。最高的产量、纯度、细胞摄取和生物活性是通过结合释放酶或胶原酶和 SEC 分离的 MBV 观察到的。蛋白酶 K 和 UF 的组合显示对生物活性具有不利影响。结果显示了选择合适的 MBV 采集方法对 MBV 的表征和评估以及分析其潜在的治疗诊断应用的重要性。溶解和分离方法的所有组合都允许收获 MBV，但是，注意到了明显的差异。最高的产量、纯度、细胞摄取和生物活性是通过结合释放酶或胶原酶和 SEC 分离的 MBV 观察到的。蛋白酶 K 和 UF 的组合显示对生物活性具有不利影响。结果显示了选择合适的 MBV 采集方法对 MBV 的表征和评估以及分析其潜在的治疗诊断应用的重要性。蛋白酶 K 和 UF 的组合显示对生物活性具有不利影响。结果显示了选择合适的 MBV 采集方法对 MBV 的表征和评估以及分析其潜在的治疗诊断应用的重要性。蛋白酶 K 和 UF 的组合显示对生物活性具有不利影响。结果显示了选择合适的 MBV 采集方法对 MBV 的表征和评估以及分析其潜在的治疗诊断应用的重要性。