Messenger RNA (mRNA) based systems have recently attracted considerable attention for their use in the delivery of functional antibodies and therapeutic proteins. Both viral and non-viral vectors, including liposomes, polymeric nanoparticles, and mRNA-protein complexes, have been studied in clinic. Significant advances have already been made. Yet there is still room for improving delivery efficiency and maximizing the therapeutic windows of mRNA therapeutics. These are relevant in their application for a variety of conditions to “mRNA drug delivery”.
Both Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) as performed by Malvern Panalytical’s Zetasizer range are proving to be particularly adept at determining the optimal size, zeta potential, and overall stability of mRNA-based systems. Benefits of Zetasizer systems include but are not limited to:
- accurate and reliable results with the real ease of use
- patented Non-Invasive Backscatter (NIBS) optics to ensure exceptional performance
- rapid sample measurement with little/no dilution
- confidence in results from unique data quality test and automated ‘expert advice’ system
- operator independence ensured by highly automated analysis
- automation of temperature trends.
Example 1: Self-assembled Messenger RNA Nanoparticles (mRNA-NPs) for efficient gene expression
Hyejin Kim et al  created a one-step process to synthesize self-assembled mRNA-NPs packed with multiple repeats. The process required minimal amounts of plasmid DNA to produce RNA transcripts through rolling circle transcription (RCT). The team confirmed that cells were transfected with mRNA-NPs encoded with green fluorescent protein (GFP) and would therefore be an effective platform for gene delivery. They used a Malvern Zetasizer Nano ZS90 to determine the optimal size of mRNA-NPs generated by varying the concentrations of plasmid DNA through RCT. Consequently, they determined that the threshold level of template DNA needed to produce the nanoparticles was between 0.05 nM and 0.1 nM.
They further used DLS to confirm that the self-assembled mRNA-NPs were relatively stable in serum-containing media. mRNA-NPs were treated with 2% or 10% nuclease-containing fetal bovine serum for 5 min or 1 hour, or were left untreated as a control. Self-assembled mRNA-NPs remained relatively stable in all cases, whereas free or capped-mRNA was degraded by the serum.
The team treated mRNA-NPs with the transfection reagent TransIT-X2 to deliver them into cells. ELS was used to determine the effectiveness of this treatment and the subsequent efficiency of the surface modification. mRNA-NPs were easily coated with a positively charged transfection reagent because of the high charge density. There was a significant change in the charge (-19 mV for mRNA-NP to +11.4 mV for TransIT-X2 coated mRNA-NP). Subsequently, DLS confirmed that there was a 5 nm increase in mRNA-NP size after treatment with TransIT-X2. Light scattering confirmed stability and surface modification in this mRNA drug delivery example.
Example 2: Optimization of lipid-like nanoparticles for mRNA delivery in vivo
Bin Li et al  designed a new class of N1, N3, N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide-derived lipid-like nanoparticles (TT-LLNs) for mRNA delivery. They studied variations of the N value by changing the length of the molecule from N =1 through 8. They went on investigate the effects of PEGylation on the stability, particle size, and mRNA delivery efficiency of these nanoparticles.
The authors used a Malvern Zetasizer Nano ZS to measure the particle size and zeta potential of the TT-LLNs. Results showed that the presence of DMG-PEG2000 was negatively correlated with delivery efficiency and particle size. The higher the relative concentration of DMG-PEG2000, the lower the luciferase expression and the smaller the particles. The team also used their Zetasizer system to examine the effects of helper lipids on delivery efficiency, particle size and zeta potential.
They analyzed the delivery efficiency of their TT-LLNs by studying their uptake in Hep3B cells (a human hepatoma cell line). Studies showed that TT3-LLNs were optimal for the transfection of mRNA to Hep3B cells (a human hepatoma cell line). The group further studied the optimization of delivery efficiency by variation of particle sizes and formulation zeta potential. In this study, light scattering supported optimizing mRNA drug delivery development.
These are just a few examples of similarly important work being performed in numerous laboratories globally, using Malvern Panalytical Zetasizer systems. You may also like previous posts on siRNA & Bioopolymers and Lipidoid nanoparticles for siRNA delivery. Learn more about the Zetasizer product range, including our flagship new Zetasizer Pro and Zetasizer Ultra.
 Kim, H. et al. Self-assembled Messenger RNA Nanoparticles (mRNA-NPs) for Efficient Gene Expression. Sci. Rep. 5, 12737
 Li, B. et al. An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo. Nano Lett. 2015, 15(12), 8099