Klein Philipp Michael, Wagner Ernst
Pharmaceutical Biotechnology, Center for System-based Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany.
Nanosystems Initiative Munich, Munich, Germany.
Methods Mol Biol. 2019;2036:141-164. doi: 10.1007/978-1-4939-9670-4_8.
Lipopolyplexes present well-established nucleic acid carriers assembled from sequence-defined cationic lipo-oligomers and DNA or RNA. They can be equipped with additional surface functionality, like shielding and targeting, in a stepwise assembly method using click chemistry. Here, we describe the synthesis of the required compounds, an azide-bearing lipo-oligomer structure and dibenzocyclooctyne (DBCO) click agents as well as the assembly of the compounds with siRNA into a surface-functionalized formulation. Both the lipo-oligomer and the DBCO-equipped shielding and targeting agents are produced by solid-phase synthesis (SPS). This enables for precise variation of all functional units, like variation in the amount of DBCO attachment sites or polyethylene glycol (PEG) length. Special cleavage conditions with only 5% trifluoroacetic acid (TFA) must be applied for the synthesis of the shielding and targeting agents due to acid lability of the DBCO unit. The two-step lipopolyplex assembly technique allows for separate optimization of the core and the shell of the formulation.
脂质多聚体是由序列确定的阳离子脂质低聚物与DNA或RNA组装而成的成熟核酸载体。通过点击化学的逐步组装方法，它们可以具备额外的表面功能，如屏蔽和靶向功能。在此，我们描述了所需化合物的合成，一种含叠氮基的脂质低聚物结构和二苯并环辛炔（DBCO）点击试剂，以及将这些化合物与小干扰RNA（siRNA）组装成表面功能化制剂的过程。脂质低聚物以及配备DBCO的屏蔽和靶向试剂均通过固相合成（SPS）制备。这使得所有功能单元能够精确变化，例如DBCO连接位点数量或聚乙二醇（PEG）长度的变化。由于DBCO单元对酸不稳定，在合成屏蔽和靶向试剂时必须采用仅含5%三氟乙酸（TFA）的特殊裂解条件。两步脂质多聚体组装技术允许分别优化制剂的核心和外壳。
