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客户 发表外泌体相关文章采用我司巯基磁珠Journal of Extracellular Vesicles(SCI 影响因子19.6)

2021-11-8 22:15:33点击:

Chaoxiang Chen, Mengdi Sun, Jialin Wang, Liyun Su, Junjie Lin, Xiaomei Yan. Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour, Journal of Extracellular Vesicles,Volume10, Issue13:e12163, First published: 31 October 2021 https://doi.org/10.1002/jev2.12163


Abstract
Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named “Sonication and Extrusion-assisted Active Loading” (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin-loaded milk-derived EVs (Dox-mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10-fold enhancement of drug encapsulation efficiency compared with passive loading. Single-particle analysis by nano-flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox-mEVs which would otherwise be overlooked by bulk-based approaches. Correlation analysis between doxorubicin auto-fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid-enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid-probe- and immuno-mediated magnetic isolation techniques to selectively remove the contaminants of non-lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL-prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as-developed cargo-loading approach and nano-flow cytometry-based characterization method will provide an instructive insight in the development of EV-based delivery systems.


摘要

细胞外囊泡(EVs)在作为纳米载体用于药物递送方面表现出独特的优势,但货物封装效率远非预期,尤其是对于亲水性化疗药物。此外,EV 的内在异质性使得评估药物包封行为变得困难。受脂质体纳米药物主动载药策略的启发,我们报告了一种名为“超声和挤出辅助主动载药”(SEAL)的方法的开发,用于有效和稳定地包封 EVs。使用加载多柔比星的乳源性 EVs (Dox-mEVs) 作为模型系统,应用超声处理暂时渗透膜,促进硫酸铵溶液流入管腔,以建立主动加载所必需的跨膜离子梯度。除了挤压以缩小大 mEV 的尺寸、均匀粒径和重塑非球形或多层囊泡,与被动加载相比,SEAL 显示药物封装效率提高了约 10 倍。纳米流式细胞术的单粒子分析被进一步用于揭示 Dox-mEV 的异质封装行为,否则这些行为会被基于批量的方法所忽视。阿霉素自发荧光与亲脂性染料 DiD 的荧光之间的相关性分析表明,只有脂质包裹的颗粒是可主动加载的。同时,免疫荧光分析显示,超过 85% 的酪蛋白阳性颗粒不含阿霉素。这些发现进一步激发了脂质探针和免疫介导的磁隔离技术的发展,以分别选择性地去除非脂质封闭颗粒和酪蛋白组件的污染物。最后,细胞内评估证实了 SEAL 制备的 mEV 制剂的优越性能,并证明了封装异质性对治疗结果的影响。开发的货物装载方法和基于纳米流式细胞术的表征方法将为基于 EV 的递送系统的开发提供有益的见解。


在该论文种,我公司研发的PuriMag Si-SH磁珠被用来固定化Casei抗体,然后用于清除所分离的外囊泡种不纯的casein聚集体。

5.8 Immuno-magnetic isolation technique for the removal of casein assemblies

Briefly, casein antibody (Bioss Antibodies) was maleimide-activated by sulfo-SMCC (Sigma-Aldrich) and conjugated to the thiol magnetic beads (MBs, PuriMag) according to the manufacturer's instructions. The unconjugated antibody was removed by washing with PBS for three times. For the removal of the casein assemblies, the LPMIT-purified Dox-mEV samples were mixed with the antibody conjugated MBs for 1 h at RT. The mixtures were then placed on a magnet for 1 min and the supernatant was transferred to a second tube of antibody conjugated MBs for another round of separation. The IMIT process was repeated three times for the effective removal of casein assemblies.