Maji Dolonchampa, Lu Jin, Sarder Pinaki, Schmieder Anne H, Cui Grace, Yang Xiaoxia, Pan Dipanjan, Lew Matthew D, Achilefu Samuel, Lanza Gregory M
Optical Radiology Lab, Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Department of Biomedical Engineering, Washington University in St. Louis, MO 63130, USA.
Precis Nanomed. 2018 Jul;1(2):128-145. doi: 10.33218/prnano1(2).180724.1. Epub 2018 Jun 30.
While the efficacy of Sn-2 phosphatidylcholine prodrugs incorporated into targeted, non-pegylated lipid-encapsulated nanoparticles was demonstrated in prior preclinical studies, the microscopic details of cell prodrug internalization and trafficking events are unknown. Classic fluorescence microscopy, fluorescence lifetime imaging microscopy, and single-molecule super-resolution microscopy were used to investigate the cellular handling of doxorubicin-prodrug and AlexaFluor™-488-prodrug. Sn-2 phosphatidylcholine prodrugs delivered by hemifusion of nanoparticle and cell phospholipid membranes functioned as phosphatidylcholine mimics, circumventing the challenges of endosome sequestration and release. Phosphatidylcholine prodrugs in the outer cell membrane leaflet translocated to the inner membrane leaflet by ATP-dependent and ATP-independent mechanisms and distributed broadly within the cytosolic membranes over the next 12 h. A portion of the phosphatidylcholine prodrug populated vesicle membranes trafficked to the perinuclear Golgi/ER region, where the drug was enzymatically liberated and activated. Native doxorubicin entered the cells, passed rapidly to the nucleus, and bound to dsDNA, whereas DOX was first enzymatically liberated from DOX-prodrug within the cytosol, particularly in the perinuclear region, before binding nuclear dsDNA. Much of DOX-prodrug was initially retained within intracellular membranes. anti-proliferation effectiveness of the two drug delivery approaches was equivalent at 48 h, suggesting that residual intracellular DOX-prodrug may constitute a slow-release drug reservoir that enhances effectiveness. We have demonstrated that Sn-2 phosphatidylcholine prodrugs function as phosphatidylcholine mimics following reported pathways of phosphatidylcholine distribution and metabolism. Drug complexed to the Sn-2 fatty acid is enzymatically liberated and reactivated over many hours, which may enhance efficacy overtime.
虽然在先前的临床前研究中已证明，掺入靶向、非聚乙二醇化脂质包裹纳米颗粒中的Sn-2磷脂酰胆碱前药具有疗效，但细胞前药内化和运输事件的微观细节尚不清楚。使用经典荧光显微镜、荧光寿命成像显微镜和单分子超分辨率显微镜来研究阿霉素前药和AlexaFluor™-488前药的细胞处理过程。通过纳米颗粒与细胞磷脂膜的半融合递送的Sn-2磷脂酰胆碱前药起到磷脂酰胆碱模拟物的作用，规避了内体隔离和释放的挑战。外细胞膜小叶中的磷脂酰胆碱前药通过ATP依赖和ATP非依赖机制转运到内膜小叶，并在接下来的12小时内在胞质膜中广泛分布。一部分填充有磷脂酰胆碱前药的囊泡膜运输到核周高尔基体/内质网区域，在那里药物被酶解并激活。天然阿霉素进入细胞，迅速进入细胞核并与双链DNA结合，而阿霉素首先在胞质溶胶中，特别是在核周区域从阿霉素前药中酶解出来，然后再结合核双链DNA。许多阿霉素前药最初保留在细胞内膜中。两种药物递送方法在48小时时的抗增殖效果相当，这表明残留的细胞内阿霉素前药可能构成一个缓释药物库，从而提高疗效。我们已经证明，Sn-2磷脂酰胆碱前药在遵循报道的磷脂酰胆碱分布和代谢途径后起到磷脂酰胆碱模拟物的作用。与Sn-2脂肪酸复合的药物在数小时内被酶解并重新激活，这可能会随着时间的推移提高疗效。
