Single-Particle and mRNA-Encapsulation-Rate Analysis of Lipid Nanoparticle (LNP) Formulations

Written by: Yasuma Yoshizumi, Alex Rodriguez, Richard Cuthbert

Introduction

Lipid nanoparticles (LNPs) are generally considered nanoparticles with diameters of approximately 10–1,000 nm. Their ability to encapsulate drugs and active ingredients, deliver them to targeted sites in the body, and release their contents at controlled times has generated significant interest in their use as drug-delivery systems, particularly in the pharmaceutical industry. This is especially important for nucleic-acid-based drugs, whose active ingredients often have poor in vivo stability and therefore require formulation strategies such as LNP encapsulation for practical use.

Table 1 summarizes key characterization parameters for LNPs, liposomes, and other nanoparticle formulations. At present, active-ingredient encapsulation efficiency is typically measured in bulk. However, methods that can assess encapsulation efficiency at the single-particle level, including the proportion of particles that do or do not contain active ingredients, are increasingly needed. Using the ZE5 Cell Analyzer, we demonstrate examples of single-particle LNP detection and mRNA encapsulation-efficiency analysis in a model mRNA formulation.

Table 1. LNP characterization methods.

Materials and Methods

LNPs were formulated by the combination of heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), distearoylphosphatidylcholine (DSPC), cholesterol, and dimyristoyl glycerol conjugated to polyethylene glycol, molecular weight 2,000 (DMG-PEG2000), in the ratio of 50/10/38.5/1.5 and suspended in phosphate-buffered saline (PBS) that had previously been filtered by passing through a 100 nm filter. LNPs and mRNA were combined for analysis as shown in Table 2. All LNPs were pre-labeled by incubation with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD). Additional controls included PBS alone, dye alone, DiD pre-stained LNPs alone, and mRNA with SYTO RNA Select Green. Samples containing mRNA were incubated with RNase at a concentration of 250 μg/mL for 5 minutes at room temperature prior to analysis by flow cytometry (Figure 1). 

Table 2. LNP formulations.

LNP, lipid nanoparticle.

Analysis was performed using a 5-laser ZE5 Cell Analyzer equipped with a small particle detector (SPD) (12004279). The sample flow rate was maintained at 0.1 µL/sec and during analysis the samples were maintained at 25°C. Data analysis was performed using Everest Software and FCS Express (Dotmatics).

Table 3. Reagents used.

DiD, 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine; Ex/Em, excitation/emission; PBS, phosphate buffered saline.

Fig. 1. Sample preparation and staining.

Results

To assess encapsulation rate, samples of mRNA/LNP mixtures and LNP-encapsulated mRNA were incubated with RNase. In order to establish a baseline, the fluorescent signal of SYTO RNA Select Green was measured in LNPs stained with DiD and containing no mRNA (Figure 2A). mRNA-LNP samples showed 93.1% positivity for mRNA content as highlighted by SYTO RNA Select Green fluorescence, which dropped to 74.9% following incubation with RNAse (Figure 2B). In contrast, although 74.4% of LNPs in the mRNA/LNP mixture initially showed positivity for mRNA, this dropped to 7.9% following incubation with RNase (Figure 2C).

Fig. 2. RNA encapsulation efficiency.

Example dot plots showing DiD and SYTO RNA Select Green fluorescence in LNP samples containing no mRNA (A), LNP samples with encapsulated mRNA (B), and LNP samples mixed with mRNA (C). DiD, 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine; LNP, lipid nanoparticle.

Conclusion

By fluorescent staining of both the LNP membrane and mRNA, this analysis enabled flow cytometry-based single-particle detection of LNPs and simultaneous assessment of mRNA encapsulation within individual particles. RNase digestion showed that a high proportion of LNPs in the encapsulated sample remained positive for mRNA content, confirming encapsulation. Whereas in the LNP/mRNA mixture, a large proportion of the mRNA signal was lost following RNase digestion, confirming that mRNA was present on the surface predominantly and therefore not protected from RNase digestion.

These findings demonstrate the potential of this approach for analyzing nucleic-acid-loaded LNP formulations and assessing LNP mRNA drug quality, including mRNA encapsulation efficiency. The ZE5 Cell Analyzer’s 4–37??? temperature-controlled loader may also support formulation-development studies, such as evaluating LNP storage stability.

In summary, the ZE5 Cell Analyzer may provide a useful tool for measuring and analyzing small particles, including LNP-like particles, and for supporting single-particle analysis beyond conventional cell-sized applications.

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