Kalinec Gilda M, Cohn Whitaker, Whitelegge Julian P, Faull Kym F, Kalinec Federico
1 Department of Head and Neck Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
2 Pasarow Mass Spectrometry Laboratory, Jane and Terry Semel Institute for Neuroscience and Human Behavior and Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Ann Otol Rhinol Laryngol. 2019 Jun;128(6_suppl):52S-60S. doi: 10.1177/0003489419836226.
Isolate, purify, and characterize extracellular vesicles (EVs) obtained from auditory HEI-OC1 cells, and evaluate their suitability for intracochlear transport and delivery of pharmacological drugs and/or pro-resolution mediators of acute inflammatory processes.
HEI-OC1 EVs were isolated and purified using the exoEasy Maxi Kit, and their size was evaluated by nanoparticle tracking techniques. Bottom-up proteomics of the EVs, either freshly obtained or stored for up to 4 months at -20°C, was performed by LC-ESI-MS/MS. LC-ESI-MS/MS-MRM was used to measure the loading of dexamethasone inside EVs following co-incubation at room temperature for 1 hour with and without 5 minutes sonication.
Routinely, we were able to obtain purified fractions of >2 × 10 EVs/mL, with diameters varying between 50 and 800 nm. Bottom-up proteomics showed that among the most abundant EVs proteins, 19.2% were cytoplasmic, 17.2% were membrane localized, 12.3% were cytosolic, and 14.6% were nucleolar. No significant differences between fresh and stored EVs were detected. Importantly, co-incubation of HEI-OC1 EVs (1 × 10 EVs/mL) with dexamethasone (10 mM) resulted in the incorporation of 10.1 ± 1.9 nM dexamethasone per milliliter of EVs suspension.
Altogether, the results suggest that EVs from HEI-OC1 cells could be advantageously used as biological nanocarriers for the delivery of specific molecules and pharmacological drugs into the inner ear.
分离、纯化并鉴定从听觉HEI-OC1细胞获得的细胞外囊泡（EVs），并评估其在耳蜗内运输以及递送药物和/或急性炎症过程促消退介质的适用性。
使用外泌体提取试剂盒（exoEasy Maxi Kit）分离和纯化HEI-OC1 EVs，通过纳米颗粒追踪技术评估其大小。对新鲜获得的或在-20°C下储存长达4个月的EVs进行自下而上的蛋白质组学分析，采用液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）。在室温下共同孵育1小时，有或无5分钟超声处理后，使用LC-ESI-MS/MS多反应监测（MRM）来测量EVs内的地塞米松负载量。
通常，我们能够获得>2×10个EVs/mL的纯化级分，直径在50至800 nm之间变化。自下而上的蛋白质组学分析表明，在最丰富的EVs蛋白质中，19.2%为细胞质蛋白，17.2%为膜定位蛋白，12.3%为胞质蛋白，14.6%为核仁蛋白。未检测到新鲜和储存的EVs之间存在显著差异。重要的是，HEI-OC1 EVs（1×10个EVs/mL）与地塞米松（10 mM）共同孵育导致每毫升EVs悬浮液中掺入10.1±1.9 nM地塞米松。
总之，结果表明来自HEI-OC1细胞的EVs可有利地用作生物纳米载体，用于将特定分子和药物递送至内耳。
