Poster W16 - Single-Particle Spectroscopic Hydrodynamic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles

Introduction: Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and developments usually assume that LNPs are homogeneous in population, loading density, and composition. Such intuitive yet unverified perspectives remain due to the lack of suitable tools to characterize these physicochemical properties at single-nanoparticle level. Here, we explore integrating spectroscopy and chromatography as a generalizable strategy to dissect the complexities of multi-component LNP assembly. We characterize a benchmark siRNA LNP formulation with a size distribution ranging from 30 to 200 nm and reveal the existence of seven distinct LNP species, variations in RNA packing densities spanning multiple orders of magnitude among similarly sized LNPs, and size-dependent loading–size correlations.

Materials and

Methods: We used Cylindrical Illumination Confocal Spectroscopy (CICS), a technique recently applied for LNP characterization1 to examine the LNPs with fluorescent labeled siRNA, DSPC lipids, and PEG lipids. The LNPs were injected continuously or in sample plug for Single Nanoparticle Free Solution Hydrodynamic Separation (SN-FSHS)2 in a microfluidic channel. A commonly referenced benchmark LNP formulation with DLin-MC3-DMA as the ionizable lipid was formulated by rapid mixing in a T-junction at pH 4.0 and dialyzed against PBS at pH 7.4.

Results, Conclusions, and Discussions: The platform can report 4-dimensional LNP parameters (Fig. 1a), including the fluorescence of labeled siRNA, PEGylated lipid, and helper lipid detected by the single particle fluorescence (i.e., CICS), plus the size measured by the hydrodynamic chromatography (i.e., SN-FSHS). The 3-color fluorescence was used to distinguish 7 subpopulations within the LNP sample, based on the compositional features. We showed that SN-FSHS can measure LNP size in an unbiased manner and clearly differentiate two populations within a bimodal mixture of LNPs, while the DLS results are masked by the larger LNP population (Fig. 2). By analyzing the LNP content and size at single particle level, we revealed the heterogenous loading-size correlation across LNP size ranges from 30 to 200 nm for 3 tested LNP formulations prepared with different mol% of PEGylated lipid (Fig. 3), that would be otherwise masked by the bulk average method.

The SN-FSHS-CICS platform provides the first integrated characterization tool for simultaneous assessment of population distribution in composition and loading capacity of RNA in LNP formulations. This analysis can provide valuable insights into the heterogeneity of LNP preparations and permit systematic investigations in understanding critical physicochemical parameters in LNP engineering (Fig. 4). The SN-FSHS-CICS platform can guide the formulation optimization and manufacturing processes to achieve greater compositional homogeneity and further improve the performances of LNP delivery systems.

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