Nucleic Acid Encapsulation in Lipid Nanoparticles

Clarifying Common Questions: Monitoring Nucleic Acid Encapsulation in Lipid Nanoparticles Using ARGEN

Nucleic Acid Encapsulation in Lipid Nanoparticles

 

Nucleic Acid Encapsulation in Lipid Nanoparticles

A lot of our prospective clients have asked questions recently about the capabilities of ARGEN, stemming from a recent webinar hosted by Prof. Wayne Reed which can be viewed here.

So when it comes to evaluating nucleic acid encapsulation and release in lipid nanoparticles (LNPs), we thought it would be helpful to write a short article to clarify some of these common inquiries. We’ll start off with an overview of the underlying light scattering technology behind ARGEN.

Understanding ARGEN and Light Scattering Technology

First things first, ARGEN is a static light scattering (SLS) instrument that detects scattered light intensity changes at a single angle—specifically, 90 degrees from the incident laser beam. This setup is suitable for analyzing Rayleigh scatterers, which are particles small enough to scatter light equally in all directions with no angular dependence. In practical terms, this means ARGEN is well-suited for determining the absolute molar mass (molecular weight) of molecules, polymers, and complexes within a certain size range.

This differs from multi-angle light scattering (MALS) instruments, which detect scattered light intensity changes at multiple angles (with a minimum of three angles relative to the incident laser). MALS is more appropriate for larger particles where angular dependence (Mie scattering effects) becomes significant due to higher molar masses—situations that fall outside the Rayleigh scattering regime. MALS allows for precise determination of absolute molecular weight and the radius of gyration (Rg) of larger species.

It’s important to note that both SLS and MALS measure the average light intensity of all species in solution. They don’t inherently distinguish between different sizes or molecular weights within a mixture. For example, if you’re analyzing a protein sample that is a 50% mix of monomers (100 kDa) and a 50% mix of trimers (300 kDa), the instrument would detect an average molar mass equivalent to dimers (200 kDa). With that said, absolute molar mass experiments are performed at sufficiently low concentrations to mitigate intermolecular interactions. This is why SLS or MALS is often coupled with size-exclusion chromatography (SEC) to separate individual species before analysis.

Addressing Common Questions

Now, let’s dive into some specific questions we have received:

1) Can ARGEN monitor nucleic acid retention or release in lipid nanoparticles?

Absolutely. A virus shares a similar general architecture with an LNP-mRNA complex, which means we can use the same techniques presented in studies involving adeno-associated virus vectors (AAVs) to monitor LNPs. ARGEN can effectively track the solution behavior of LNP-mRNA complexes, including:

  • Aggregation: Observing how LNPs aggregate over time or under stress.
  • Degradation: Monitoring the breakdown of LNP structures.
  • Nucleic Acid Release: Detecting the ejection or separation of mRNA from the lipid nanoparticle capsid.

By measuring changes in light scattering intensity, ARGEN provides insights into the stability and integrity of LNP formulations.

2) What physical properties of LNPs can ARGEN measure?

  • Aggregation Rate (AR): By detecting increases in scattered light intensity, ARGEN can quantify how quickly LNPs aggregate under various conditions.
  • Normalized Molecular Weight (Mw(t)/M₀): This ratio represents the weight-average molecular weight at time t (Mw) compared to the native molecular weight at time zero (M0). It’s useful for monitoring changes due to aggregation or degradation.
  • Time to Dimerization: ARGEN can track the transition from monomers to dimers and higher-order oligomers, providing insights into the kinetics of oligomerization.
  • Arrhenius Plots: By conducting experiments at different temperatures, ARGEN data can be used to create Arrhenius plots, which characterize the temperature dependence of aggregation and degradation rates.
  • Temporal stability at low temperature for shelf-life determination.

3) Can ARGEN determine absolute molecular weight and radius of gyration (Rg) of LNPs?

The short answer is yes, but with some limitations. ARGEN can determine absolute molecular weight outside of the Rayleigh scattering regime, but the accuracy decreases as particle size increases beyond this regime. The key considerations are:

  • Size Relative to Laser Wavelength: The particle diameter should be much smaller than the laser wavelength (660 nm for ARGEN). As particles approach or exceed this size, angular dependence becomes significant due to Mie scattering effects.
  • Error Introduction: Larger particles introduce errors in measurements because they scatter light differently at various angles, which isn’t accounted for in single-angle SLS measurements.
  • Need for MALS: For larger particles where precise determination of molecular weight and Rg is required, multi-angle light scattering (MALS) instruments are more appropriate.

In essence, while ARGEN can provide useful information about larger particles like LNPs, one must be cautious about the limitations and potential errors introduced due to the size of the particles relative to the laser wavelength.

We hope this clarifies some of the common questions out there about using ARGEN for evaluating nucleic acid encapsulation and release in lipid nanoparticles. ARGEN is a versatile instrument that can provide valuable insights into the stability and behavior of LNP-mRNA complexes, albeit within certain size constraints due to its reliance on static light scattering at a single angle.

For more detailed discussions or specific inquiries about your applications, feel free to reach out to us. We are always happy to provide additional information and support to help you make the most of ARGEN in your research and development efforts.

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