Gindy Marian E, DiFelice Katherine, Kumar Varun, Prud'homme Robert K, Celano Robert, Haas R Matthew, Smith Jeffrey S, Boardman David
Department of Pharmaceutical Sciences and ‡Department of RNA Therapeutics, Merck Research Laboratories, Merck and Co., Inc. , West Point, Pennsylvania 19486, United States.
Langmuir. 2014 Apr 29;30(16):4613-22. doi: 10.1021/la500630h. Epub 2014 Apr 9.
Lipid nanoparticles (LNPs) are a leading platform for therapeutic delivery of small interfering RNAs (siRNAs). Optimization of LNPs as therapeutic products is enabled by the development of structure-activity relationships (SAR) linking LNP physiochemical and macromolecular properties to bioperformance. Methods by which LNP properties can be rationally manipulated are thus critical enablers of this fundamental knowledge build. In this work, we present a mechanistic study of LNP self-assembly via a rapid antisolvent precipitation process and identify critical physiochemical and kinetic parameters governing the evolution of LNP three-dimensional macromolecular structure as a biorelevant SAR feature. Using small-angle X-ray scattering, LNPs are shown to undergo a temporal evolution in macromolecular structure during self-assembly, rearranging from initially disordered phases after precipitation into well-ordered structures following a necessary annealing stage of the assembly sequence. The ability of LNPs to undergo structural reorganization is shown to be effected by the chemical nature of the aqueous antisolvent used for precipitation. Antisolvents of varying buffering species differentially influence LNP macromolecular features, revealing a new participatory role of buffer ions in LNP self-assembly. Furthermore, the formation of macromolecular structure in LNPs is shown to improve the efficiency of siRNA encapsulation, thereby offering a simple, nonchemical route for preparation of high-payload LNPs that minimize the dose of lipid excipients. The developed LNP precipitation process and mechanistic understanding of self-assembly are shown to be generalizable, enabling the production of LNPs with a tunable range of macromolecular features, as evidenced by the cubic, hexagonal, and oligo-lamellar phase LNPs exemplarily generated.
脂质纳米颗粒（LNPs）是用于小干扰RNA（siRNAs）治疗递送的领先平台。通过建立将LNP物理化学和大分子性质与生物性能联系起来的构效关系（SAR），可实现将LNPs优化为治疗产品。因此，能够合理调控LNP性质的方法是构建这一基础知识的关键推动因素。在这项工作中，我们通过快速抗溶剂沉淀过程对LNP自组装进行了机理研究，并确定了控制LNP三维大分子结构演变的关键物理化学和动力学参数，将其作为生物相关的SAR特征。使用小角X射线散射表明，LNPs在自组装过程中大分子结构会经历时间演变，从沉淀后最初的无序相重新排列成组装序列中必要的退火阶段后的有序结构。结果表明，LNPs进行结构重组的能力受用于沉淀的水性抗溶剂的化学性质影响。不同缓冲物种的抗溶剂对LNP大分子特征有不同影响，揭示了缓冲离子在LNP自组装中的新参与作用。此外，LNPs中大分子结构的形成提高了siRNA封装效率，从而提供了一种简单的非化学途径来制备高载量LNPs，使脂质辅料的剂量最小化。所开发的LNP沉淀过程和对自组装的机理理解具有通用性，能够生产具有可调范围大分子特征的LNPs，如示例性生成的立方相、六方相和寡层相LNPs所示。
