Kang Jia-Jhen, Huang Zhih-Chen, Tang Li-Wen, Su Chun-Jen, Gao Hua-De, Lee Hsien-Ming, Jeng U-Ser
National Synchrotron Radiation Research Center Hsinchu 300092 Taiwan.
Institute of Chemistry Academia Sinica Taipei 115024 Taiwan.
J Appl Crystallogr. 2025 May 29;58(Pt 3):897-908. doi: 10.1107/S1600576725003577. eCollection 2025 Jun 1.
Poly(ethyl-ene glycol)-grafted (PEGylated) liposomes receive increasingly more attention due to their practical applications in delivering vaccines, nutrients and drug molecules such as doxorubicin (DOX). PEGylated liposomes have been well documented for their capability in carrying DOX as rod-like crystallites enclosed inside the unilamellar vesicles. This study addresses the previously unresolved question of whether DOX intercalates into liposome bilayers by employing simultaneous small- and wide-angle X-ray scattering (SWAXS), complemented by an integrated asymmetric flow field-flow fractionation system coupled with multi-angle light scattering, dynamic light scattering and refractive index detection. The DOX-loaded PEGylated liposomes used are composed of phosphatidylcholine (:0 PC) lipids, with different lipid chain lengths = 18, 20 and 22, and a fixed molar ratio of lipid:cholesterol:DSPE-PEG2000 of 45:50:5. SWAXS analysis reveals that rod-like DOX nanocrystallites-approximately 70-95 nm in length and 14 nm in diameter-are encapsulated within the PEGylated liposomes across all three lipid types, with each exhibiting distinct membrane structural responses to DOX incorporation. Notably, 22:0 PC liposomes demonstrate significant DOX-induced disruption of lipid chain packing, accompanied by enhanced alignment of phosphate headgroups in the outer leaflet. Consistently, cryo-EM imaging reveals pronounced faceted membrane morphologies in DOX-loaded 22:0 PC liposomes. This faceting phenomenon is attributed to the accumulation of DOX within the excess hydro-phobic core regions created by the extended aliphatic chains beyond the cholesterol saturation limit. These DOX-enriched domains locally stiffen the membrane, promoting the formation of rigid, faceted structures.
聚（乙二醇）接枝（聚乙二醇化）脂质体因其在递送疫苗、营养物质和药物分子（如阿霉素（DOX））方面的实际应用而受到越来越多的关注。聚乙二醇化脂质体因其能够将DOX作为包裹在单层囊泡内的棒状微晶进行运载而被充分记录。本研究通过同时使用小角和广角X射线散射（SWAXS）来解决之前未解决的DOX是否插入脂质体双层的问题，并辅以集成的不对称流场-流分馏系统，该系统结合了多角度光散射、动态光散射和折射率检测。所使用的负载DOX的聚乙二醇化脂质体由磷脂酰胆碱（:0 PC）脂质组成，具有不同的脂链长度 = 18、20和22，以及脂质:胆固醇:DSPE-PEG2000的固定摩尔比为45:50:5。SWAXS分析表明，在所有三种脂质类型的聚乙二醇化脂质体内都包裹着长度约为70 - 95 nm、直径为14 nm的棒状DOX纳米微晶，每种脂质体对DOX掺入都表现出不同的膜结构响应。值得注意的是，22:0 PC脂质体显示出DOX诱导的脂质链堆积的显著破坏，同时伴随着外层小叶中磷酸头部基团排列的增强。一致地，冷冻电镜成像揭示了负载DOX的22:0 PC脂质体中明显的多面膜形态。这种多面现象归因于DOX在由超过胆固醇饱和极限的延长脂肪链产生的过量疏水核心区域内的积累。这些富含DOX的区域使膜局部变硬，促进了刚性多面结构的形成。
