Facile Synthesis of Phosphatidyl Saccharides for Preparation of Anionic Nanoliposomes with Enhanced Stability

Physical stability during storage and against processing such as dehyration/rehydration are the cornerstone in designing delivery vehicles. In this work, mono-, di- and tri-saccharides were enzymatically conjugated to phosphatidyl group through a facile approach namely phospholipase D (PLD) mediated transphosphatidylation in a biphasic reaction system. The purified products were structurally identified and the connectivities of carbohydrate to phosphatidyl moiety precisely mapped by 1H, 31P, 13C NMR pulse sequences and LC-ESI-FTMS. The synthetic phosphatidyl saccharides were employed as the sole biomimetic component for preparation of nanoliposomes. It was found that the critical micelle concentration (CMC) of phosphatidyl saccharides increases as more bulky sugar moiety (mono- to tri-) is introduced. Phosphatidyl di-saccharide had the largest membrane curvature. In comparison to the zwitterionic phosphatidylcholine liposome, all phosphatidyl saccharides liposomes are anionic and demonstrated significantly enhanced stability during storage. According to the confocal laser scan microscopy (CLSM) and atom force microscopy (AFM) analyses, the nanoliposomes formed by the synthetic phosphatidyl saccharides also show excellent stability against dehydration/rehydration process in which most of the liposomal structures remained intact. The abundance hydroxyl groups in the saccharide moieties might provide sufficient H-bondings for stabilization. This work demonstrated the synthesized phosphatidyl saccharides are capable of functioning as enzymatically liable materials which can form stable nanoliposomes without addition of stabilizing excipients.

储存过程中以及脱水/复水等加工过程中的物理稳定性是设计递送载体的核心基石。本研究通过一种简便方法，即磷脂酶D（phospholipase D，PLD）介导的转磷脂酰化反应，在双相反应体系中将单糖、二糖与三糖酶促结合至磷脂酰基团。通过1H、31P、13C核磁共振（Nuclear Magnetic Resonance，NMR）脉冲序列以及液相色谱-电喷雾电离-傅里叶变换质谱（LC-ESI-FTMS）对纯化产物进行结构鉴定，并精准绘制碳水化合物与磷脂酰基团的连接图谱。本研究将合成的磷脂酰糖类作为唯一的仿生组分用于制备纳米脂质体。研究发现，随着引入的糖基团体积增大（从单糖到三糖），磷脂酰糖类的临界胶束浓度（critical micelle concentration，CMC）逐渐升高。二糖磷脂酰化产物具有最大的膜曲率。与两性离子型的磷脂酰胆碱脂质体相比，所有磷脂酰糖类脂质体均带负电，且在储存过程中展现出显著提升的稳定性。通过共聚焦激光扫描显微镜（confocal laser scanning microscopy，CLSM）与原子力显微镜（atomic force microscopy，AFM）分析可知，由合成磷脂酰糖类制备的纳米脂质体同样在脱水/复水过程中表现出优异的稳定性，绝大多数脂质体结构得以完整保留。糖类基团中丰富的羟基可为脂质体稳定提供充足的氢键作用。本研究证实，合成的磷脂酰糖类可作为酶促可降解材料，无需添加稳定赋形剂即可制备出稳定的纳米脂质体。