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RNA Research and Splicing / RNA Interference and Gene Delivery · Journal article
The Journal of Immunology · July 28, 2026
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This is a proof-of-concept optimization of SYTO 9 Green staining combined with CytoFLEX nano flow cytometry to differentiate loaded and empty lipid nanoparticles in vitro. The method discriminated between loaded and empty LNPs with reported minimal background staining in empty controls, but the study lacks validation against established reference methods or independent replication.
Single-laboratory protocol optimization study. In vitro lipid nanoparticle samples (Cytiva-sourced LNP1/LNP2 and UBC-sourced siRNA-LNPs) with and without nucleic acid cargo. Intervention: SYTO 9 Green staining and flow cytometric analysis using CytoFLEX nano Flow Cytometer. Not stated.
Loaded LNPs exhibited strong fluorescence signals with positive staining efficiency peaking at optimal dye concentrations Empty LNPs consistently showed minimal background staining (≤1% positive events) Dye titration range tested: 25 µM down to 3.125 µM SYTO 9 Green
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This methodological development may support LNP manufacturing quality control and characterization workflows, but clinicians and researchers should await independent validation and comparison to existing encapsulation assessment methods before adoption in regulatory or clinical contexts.
A methodological study optimizing a flow cytometric protocol for LNP characterization with no clinical or in vivo validation; demonstrates technical feasibility but lacks independent replication or validation against established standards.
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This methodological development may support LNP manufacturing quality control and characterization workflows, but clinicians and researchers should await independent validation and comparison to existing encapsulation assessment methods before adoption in regulatory or clinical contexts.
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Abstract Introduction Lipid nanoparticles (LNPs) have emerged as nanoscale carriers for therapeutic agents, including mRNA vaccines and gene therapies. Accurate methods for characterizing nucleic acid encapsulation within these particles are essential for LNP development and quality control. This study presents an optimized flow cytometric protocol that uses SYTO 9 Green, a nucleic acid-binding dye, to differentiate and quantify nucleic acid-loaded versus empty LNPs with high sensitivity using the CytoFLEX nano Flow Cytometer. Methods We optimized a SYTO 9 Green staining protocol and applied it to empty and loaded LNP samples (Cytiva-sourced LNP1/LNP2 and UBC-sourced siRNA-LNPs). A sequential gating strategy was used to ensure single-particle detection by excluding debris and doublets. Dye titration experiments (ranging from 25 µM down to 3.125 µM SYTO 9 Green) were performed, along with critical controls, including unstained LNPs and SYTO 9 Green dye-only samples. Results Our results demonstrate that SYTO 9 Green staining enabled clear, concentration-dependent discrimination between loaded and empty LNPs. Loaded LNPs exhibited strong fluorescence signals, with positive staining efficiency peaking at optimal dye concentrations. Staining efficiency varies across concentrations, reflecting a complex interplay between dye-to-target binding, nucleic acid content, and potential quenching effects. In contrast, empty LNPs consistently showed minimal background staining ( 1% positive events). Unstained LNPs and dye-only controls presented negligible positive events, confirming that the observed signals are attributable to nucleic acid targets. Conclusion These findings highlight the combined use of the CytoFLEX nano Flow Cytometer and optimized SYTO 9 Green staining as a sensitive, robust approach for precise, multiparametric characterization of nucleic acid encapsulation in LNPs. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)
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