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

細胞外囊泡（EVs）在作為納米載體用于藥物遞送方面表現出獨特的優勢，但貨物封裝效率遠非預期，尤其是對于親水性化療藥物。此外，EV 的內在異質性使得評估藥物包封行為變得困難。受脂質體納米藥物主動載藥策略的啟發，我們報告了一種名為“超聲和擠出輔助主動載藥”（SEAL）的方法的開發，用于有效和穩定地包封 EVs。使用加載多柔比星的乳源性 EVs (Dox-mEVs) 作為模型系統，應用超聲處理暫時滲透膜，促進硫酸銨溶液流入管腔，以建立主動加載所必需的跨膜離子梯度。除了擠壓以縮小大 mEV 的尺寸、均勻粒徑和重塑非球形或多層囊泡，與被動加載相比，SEAL 顯示藥物封裝效率提高了約 10 倍。納米流式細胞術的單粒子分析被進一步用于揭示 Dox-mEV 的異質封裝行為，否則這些行為會被基于批量的方法所忽視。阿霉素自發熒光與親脂性染料 DiD 的熒光之間的相關性分析表明，只有脂質包裹的顆粒是可主動加載的。同時，免疫熒光分析顯示，超過 85% 的酪蛋白陽性顆粒不含阿霉素。這些發現進一步激發了脂質探針和免疫介導的磁隔離技術的發展，以分別選擇性地去除非脂質封閉顆粒和酪蛋白組件的污染物。最后，細胞內評估證實了 SEAL 制備的 mEV 制劑的優越性能，并證明了封裝異質性對治療結果的影響。開發的貨物裝載方法和基于納米流式細胞術的表征方法將為基于 EV 的遞送系統的開發提供有益的見解。

在該論文種，我公司研發的PuriMag Si-SH磁珠被用來固定化Casei抗體，然后用于清除所分離的外囊泡種不純的casein聚集體。

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.