Institute of Inorganic Chemistry , Graz University of Technology , Graz 8010 , Austria.
Center for Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia , Pisa 56127 , Italy.
ACS Appl Mater Interfaces. 2018 Jul 11;10(27):22951-22962. doi: 10.1021/acsami.8b04962. Epub 2018 Jun 26.
More than 20 years after its approval by the Food and Drug Administration (FDA), liposomal doxorubicin (DOX) is still the drug of choice for the treatment of breast cancer and other conditions such as ovarian cancer and multiple myeloma. Yet, despite the efforts, liposomal DOX did not satisfy expectations at the clinical level. When liposomal drugs enter a physiological environment, their surface gets coated by a dynamic biomolecular corona (BC). The BC changes liposome's synthetic identity, providing it with a new one, referred to as "biological identity" (size, aggregation state, and BC composition). Today, the concept is emerging that specific BCs may determine either success (e.g., stealth effect and accumulation at the target site) or failure (e.g., rapid blood clearance and off-target interactions) of liposomal drugs. To get a comprehensive investigation of liposome synthetic identity, biological identity, and cellular response as a function of human plasma (HP) concentration, here we used a straightforward combination of quantitative analytical and imaging tools, including dynamic light scattering, microelectrophoresis, synchrotron small-angle X-ray scattering, transmission electron microscopy (TEM), fluorescence lifetime imaging microscopy (FLIM), nano-liquid chromatography tandem mass spectrometry/mass spectrometry (nano-LC-MS/MS), confocal microscopy, flow cytometry, and cell viability assays. Doxoves was selected as a reference. Following exposure to HP, Doxoves was surrounded by a complex BC that changed liposome's synthetic identity. Observations made with nano-LC-MS/MS revealed that the BC of Doxoves did not evolve as a function of HP concentration and was poorly enriched of typical "opsonins" (complement proteins, immunoglobulins, etc.). This provides a possible explanation for the prolonged blood circulation of liposomal DOX. On the other hand, flow cytometry showed that protein binding reduced the internalization of DOX in MCF7 and MDA-MB-435S human breast carcinoma. Combining FLIM and TEM experiments, we clarified that reduction in DOX intracellular content was likely due to the frequent rupture of the liposome membrane and consequent leakage of the cargo. In light of reported results, we are prompted to speculate that a detailed understanding of BC formation, composition, and effects on liposome stability and uptake is an indispensable task of future research in the field, especially along the way to clinical translation of liposomal drugs.
多柔比星脂质体（DOX）在获得美国食品和药物管理局（FDA）批准 20 多年后，仍然是治疗乳腺癌和卵巢癌、多发性骨髓瘤等疾病的首选药物。然而，尽管付出了努力，多柔比星脂质体在临床水平上仍未达到预期效果。当脂质体药物进入生理环境时，其表面会被一层动态的生物分子冠（BC）所覆盖。BC 改变了脂质体的合成身份，赋予其新的“生物学身份”（大小、聚集状态和 BC 组成）。如今，一种新的概念正在出现，即特定的 BC 可能决定脂质体药物的成败（例如，隐身效应和在靶部位的积累）。为了全面研究脂质体的合成身份、生物学身份和细胞反应与人类血浆（HP）浓度的关系，我们在这里使用了定量分析和成像工具的直接组合，包括动态光散射、微电泳、同步加速器小角 X 射线散射、透射电子显微镜（TEM）、荧光寿命成像显微镜（FLIM）、纳米液相色谱串联质谱/质谱（nano-LC-MS/MS）、共聚焦显微镜、流式细胞术和细胞活力测定。Doxoves 被选为参比药物。在暴露于 HP 后，Doxoves 被一层复杂的 BC 所包围，这改变了脂质体的合成身份。通过 nano-LC-MS/MS 观察到，Doxoves 的 BC 不会随 HP 浓度的变化而演变，并且很少富含典型的“调理素”（补体蛋白、免疫球蛋白等）。这为脂质体 DOX 的延长血液循环提供了一个可能的解释。另一方面，流式细胞术显示，蛋白结合减少了 MCF7 和 MDA-MB-435S 人乳腺癌细胞对 DOX 的内化。结合 FLIM 和 TEM 实验，我们澄清了 DOX 细胞内含量的减少很可能是由于脂质体膜频繁破裂和随后货物泄漏所致。根据报告的结果，我们推测，详细了解 BC 的形成、组成以及对脂质体稳定性和摄取的影响，是该领域未来研究的一项不可或缺的任务，特别是在脂质体药物向临床转化的过程中。
