Khalin Igor, Adarsh Nagappanpillai, Schifferer Martina, Wehn Antonia, Boide-Trujillo Valeria J, Mamrak Uta, Shrouder Joshua, Misgeld Thomas, Filser Severin, Klymchenko Andrey S, Plesnila Nikolaus
Institute for Stroke and Dementia Research (ISD), LMU University Hospital, LMU Munich, Munich 81377, Germany.
Normandie University, UNICAEN, INSERM UMR-S U1237, Physiopathology and Imaging of Neurological Disorders (PhIND), GIP Cyceron, Institute Blood and Brain @ Caen-Normandie (BB@C), Caen 14000, France.
ACS Nano. 2025 Apr 22;19(15):14780-14794. doi: 10.1021/acsnano.4c17011. Epub 2025 Apr 3.
Nanotechnology holds great promise for improving the delivery of therapeutics to the brain. However, current approaches often operate at the organ or tissue level and are limited by the lack of tools to dynamically monitor cargo delivery in vivo. We have developed highly fluorescent lipid nanodroplets (LNDs) that enable tracking of nanocarrier behavior at the subcellular level while also carrying a Förster resonance energy transfer (FRET)-based drug delivery detection system (FedEcs) capable of monitoring cargo release in vivo. Using two-photon microscopy, we demonstrate that circulating LNDs in naïve mouse brain vasculature exhibit 3D real-time FRET changes, showing size-dependent stability over 2 h in blood circulation. Further, in the Nanostroke model, dynamic intravital two-photon imaging revealed that LNDs accumulated within cerebral postischemic microthrombi, where they released their cargo significantly faster than in normal blood circulation. Furthermore, the blood-brain barrier (BBB) became permeable at the microclot sites thereby allowing accumulated FedEcs-LNDs to cross the BBB and deliver their cargo to the brain parenchyma. This microthrombi-associated translocation was confirmed at the ultrastructural level via volume-correlative light-electron microscopy. Consequently, FedEcs represents an advanced tool to quantitatively study the biodistribution and cargo release of nanocarriers at high resolution in real-time. By enabling us to resolve passive targeting mechanisms poststroke, specifically, accumulation, degradation, and extravasation via poststroke microthrombi, this system could significantly enhance the translational validation of nanocarriers for future treatments of brain diseases.
纳米技术在改善治疗药物向大脑的递送方面具有巨大潜力。然而，目前的方法通常在器官或组织水平上操作，并且受到缺乏在体内动态监测药物递送工具的限制。我们开发了高荧光脂质纳米液滴（LNDs），它能够在亚细胞水平追踪纳米载体的行为，同时还携带一种基于Förster共振能量转移（FRET）的药物递送检测系统（FedEcs），能够监测体内药物释放。使用双光子显微镜，我们证明在未处理的小鼠脑血管中循环的LNDs表现出三维实时FRET变化，在血液循环中显示出超过2小时的尺寸依赖性稳定性。此外，在纳米中风模型中，动态活体双光子成像显示LNDs在脑缺血后微血栓内积累，在那里它们释放药物的速度明显快于正常血液循环。此外，血脑屏障（BBB）在微血栓部位变得通透，从而使积累的FedEcs-LNDs能够穿过血脑屏障并将其药物递送至脑实质。通过体积相关光电子显微镜在超微结构水平证实了这种与微血栓相关的转运。因此，FedEcs是一种先进的工具，可实时高分辨率定量研究纳米载体的生物分布和药物释放。通过使我们能够解析中风后的被动靶向机制，特别是通过中风后微血栓的积累、降解和外渗，该系统可以显著增强纳米载体用于未来脑部疾病治疗的转化验证。
