What can electrophoretic light scattering tell you about LNP samples and when should you use it?

Introduction

Following a recent publicationⁱ investigating the characterization of viral and non-viral drug delivery vectors, this application note takes a close look at how researchers used the Zetasizer Advance Ultra to measure the zeta potential of two mRNA-LNP samples.

What are lipid nanoparticles?

Lipid nanoparticles (LNPs) are the most common non-viral vector used in gene therapy and mRNA-based vaccines. Following decades of research and development their acceptance as a drug delivery vector has been accelerated by the approval of multiple mRNA-LNPs COVID-19 vaccines. LNPs are composed of at least four components: 

1. phospholipids that can be neutral, cationic, anionic, or zwitterionic 
2. cholesterol and other naturally occurring hydrophobic molecules
3. a therapeutic transgene, typically nucleic acid biopolymers like DNA or RNA
4. lipid-anchored polyethylene glycol (PEG), which “hides” the LNP surface from immune detection, slowing removal by the body

Figure 1: The basic structure and compositions of the mRNA-LNP characterized in this study.

Why measure the zeta potential of LNP formulations?

Zeta potential is a charge property that predicts nanoparticle stability and correlates to other properties. It is used in drug development to compare stability between buffer formulations, lipid composition, and for basic nanomaterials. 

Charged particles exert an electrostatic inter-particle repulsive force that increases colloidal stability. In the absence of a strong stabilizing electrostatic force, it is common to add steric stabilizers in the form of non-ionic surfactants such as PEG to the liquid formulation or particle surface.  For pharmaceutical drug products, poor stability can result in undesired outcomes, such as short shelf-life, loss of efficacy, and increased safety risksⁱⁱ.

Methods for analyzing zeta potential

Two mRNA-LNP formulations were used, LNP1 and LNP2ⁱⁱⁱ. Electrophoretic light scattering (ELS) measurements of the lipid nanoparticles samples were measured using a Zetasizer Ultra Red instrument equipped with a He-Ne laser at a wavelength of 633 nm and a maximum power of 10 mW. Amounts of 700 µL of the samples were introduced into a folded capillary cell, DTS1070^iv, using the Diffusion Barrier method (see below). The duration of 35 zeta potential measurements was manually set up, with 10 sub-runs per measurement.

pH titrations of the mRNA-LNP formulations, prepared in 10 mM NaCl (10 mL sample volume), were performed with the multipurpose titrator 3 (MPT3) accessory, degasser and Zetasizer Ultra Red at a temperature of 25°C.
 

Results

The zeta potential for LNP formulation samples in PBS were mRNA-LNP1: -19.5 mV and mRNA-LNP2: -6.62 mVBoth measurements showed excellent repeatability with standard deviations of only 1.32 mV and 1.47 mV, respectively (Table 1).

Table 1: Zeta potential mean values (in mV) and Z-average diameters (in nm) for the mRNA-LNP1 and mRNA-LNP2 samples measured in PBS using the diffusion barrier method

The MPT-3 auto titrator was used to measure zeta potential over a pH range of 2.5 to 9.5.  At high and low pH in 10 mM NaCl, the zeta potential for mRNA-LNP1 and mRNA-LNP2 were virtually identical, at approximately +19 mV and -35 mV, respectively.  However, the isoelectric points (pI) of the two samples were different, with mRNA-LNP1 having a pI at pH 5.89 and mRNA-LNP2 at pH 5.21 (Figure 2).

Figure 2. A plot of the zeta potential values (in mV) as a function of pH for mRNA-LNP1 and mRNA-LNP2 samples prepared in 10 nM NaCl.

Conclusion

Zeta potential is often regarded as a charge property related only to the particle. However, many factors influence the zeta potential of the particle and the buffer conditions. Examples of LNP properties that influence zeta potential are the type and amount of charged lipids, charge density, surface PEGylation, and nucleic acid cargo located either inside or on the LNP surface. Examples of buffer properties that can influence zeta potential are ionic strength, salt type and amount, pH, and other additives such as sugars, amino acids, and non-ionic surfactants.

At low and high pH, both mRNA-LNP samples had approximately the same zeta potential. However, In the middle pH ranges, mRNA-LNP1 was consistently more negatively charged than mRNA-LNP2. Under identical buffer conditions, the cause of this charge difference can only be due to differences in either the lipid composition or mRNA.  Most of the charge associated with a mRNA-LNP will be in the phosphate backbone of the nucleic acid cargo or the charged headgroups of the phospholipids, either as cationic, anionic, or zwitterionic functional groups.

These results predict that mRNA-LNP1 is more stable and/or contains a larger mRNA therapeutic payload, both of which indicate that it is a superior drug candidate to mRNA-LNP2.

Further reading

• What can differential scanning calorimetry tell you about LNP samples and when should you use it: read the App Note >>
  
• What can nanoparticle tracking analysis tell you about LNP samples and when should you use it: read the App Note >>
• Watch webinars from the Vector Analytics Masterclass series on-demand: discover more here >>
  
• Read the full peer-reviewed paper on which this application note is based Read more >>
  
• To read about zeta potential measurements read this technical note >>
  
• Learn more about Zeta potential
  
• Learn more about Electrophoretic light scattering (ELS)
• Learn more about how zeta potential measurements are made in this technical note >> 

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References

ⁱ  N. Markova, et al. Vaccines 10, 49 (2022).  
ⁱⁱ   Sanyal, G., Särnefält, A. & Kumar, A. Considerations for bioanalytical characterization and batch release of COVID-19 vaccines. npj Vaccines 6, 53 (2021). https://doi.org/10.1038/s41541-021-00317-4 .
ⁱⁱⁱ mRNA-LNPs were provided by SINTEF Industri (Department of Biotechnology and Nanomedicine, Trondheim, Norway), and made as described in detail elsewhere. In short, two formulations of mRNA-LNPs were synthetized using Nanoassembler (Precision Nanosystems) with stock lipid solutions of cholesterol, D SPC, and PEG2000-DMG (from Avanti Polar) at 10 mg/mL and of MC3 and SM102 (from Organix) at 20 mg/mL, respectively called LNP1 and LNP2. FLuc CleanCap® FLuc mRNA (5moU) used to formulate the LNPs in this study was purchased from Tebu-Bio (Roskilde, Denmark).
^iv  DTS1070, Malvern Panalytical Ltd., Malvern, UK.
^v The Diffusion Barrier Technique for Accurate and Reproducible Protein Mobility Measurement .