Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of “fractions” was observed across the gradient, from the less dense topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the topmost fraction compared to the lower one, indicating some degree of heterogeneity, while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers, and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their subpopulations.

中文翻译：

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使用蔗糖密度梯度超速离心对 mRNA-脂质纳米颗粒的异质性进行分析表征

合理的设计和稳健的配方工艺对于脂质纳米颗粒 (LNP) 最佳地递送 mRNA 至关重要。 mRNA-LNP 不同程度的异质性会影响其生物物理和功能特性。鉴于 mRNA-LNP 的高度复杂性，开发全面的正交分析技术以更好地理解这些配方至关重要。为此，我们开发了一种强大的超速离心方法，用于基于密度的 mRNA-LNP 亚群分离。使用两种可电离脂质 A 和 B 以及两种辅助脂质 1,2-二油酰-sn-甘油-3-磷酸乙醇胺 (DOPE) 和 1,2-二芥酰基合成了四种封装具有不同功能的人促红细胞生成素 (hEPO) 的 LNP 制剂。- sn -甘油-3-磷酸乙醇胺 (DEPE)，以及胆固醇和 DMG-PEG-2K。在蔗糖梯度上进行超速离心后，在整个梯度上观察到明显的“级分”模式，从密度较低的顶部级分到密度越来越大的底部级分，收集这些级分用于综合分析。亲本 LNP，A-DOPE 和 B-DOPE，被分解为三个基于密度的级分，每个级分在静脉内和肌内给药途径后的 hEPO 表达显着不同。亲本 B-DEPE LNP 分解为两个基于密度的级分，与较低级分相比，大部分有效负载和脂质含量归因于最上面的级分，表明存在一定程度的异质性，而亲本 A-DEPE LNP 显示出显着的同质性，如通过三个基于密度的级分之间可比较的体内效力、脂质数量和颗粒计数来表明。这项研究首次展示了基于密度梯度的超速离心 (DGC) 的应用，以对亲本 mRNA-LNP 及其亚群的生物学性能和生物物理特征的异质性进行头对头比较。