Department of Pharmaceutics and Biopharmaceutics, Johannes Gutenberg University Mainz , 55099 Mainz, Germany.
BioNTech RNA Pharmaceuticals , 55131 Mainz, Germany.
Mol Pharm. 2018 Feb 5;15(2):642-651. doi: 10.1021/acs.molpharmaceut.7b01022. Epub 2018 Jan 18.
Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.
将高分子量信使 RNA（mRNA）插入溶致脂质相中，作为 mRNA 控释制剂的模型系统进行研究。低比例的 1,2-二油酰基-3-三甲铵丙烷（DOTAP）被用作将 mRNA 加载到层状脂质基质中的锚点。在存在越来越多的 mRNA 和阳离子脂质的情况下，在水相中将两性离子脂质分散，研究了其分子组织作为 mRNA 和阳离子脂质分数的函数。从物理化学特性清楚地证明了阳离子脂质和 mRNA 的插入。通过小角 X 射线散射（SAXS）测量确定的脂质双层的 d 间距对插入的 DOTAP 和 mRNA 的量敏感地响应。得出了一个关于 mRNA 在脂质基质中插入的简明模型，表明 mRNA 被容纳在脂质双层之间的水层中。根据 DOTAP 和 mRNA 分数，在该水层中存在不同的过量水。包括游离和结合的 mRNA、zeta 电位和量热数据在内的进一步物理化学特性的结果与这一假设一致。这些浓缩的脂质/mRNA 制剂的结构在稀释后得以保持。使用编码荧光素酶的 mRNA 的 C2C12 鼠成肌细胞进行细胞培养实验，证明了插入的 mRNA 的功能。所描述的脂质相作为 mRNA 的载体可适用于不同的局部给药途径，其中需要控制释放动力学和释放的 mRNA 的形式（结合或游离）。
