Zetasizer RNA-LNP DLS Size Protocol

by Jonathan Mehtala, Ph.D. | 13 September 2024

This protocol is to measure the hydrodynamic size of RNA-LNPs using dynamic Light scattering (DLS) and the Zetasizer Advanced system.  It is intended to measure RNA-LNP size and stability during formulation development, process development, and between batches.

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About

This protocol is to measure the hydrodynamic size of RNA-LNPs using dynamic Light scattering (DLS) and the Zetasizer Advance system. It is intended to measure RNA-LNP size and stability during formulation development, process development, and between batches.

Materials and Equipment

This back-scatter DLS protocol is compatible with the Zetasizer Pro Blue, Zetasizer Pro Red, Zetasizer Ultra Blue, and Zetasizer Ultra Red using ZS Xplorer software version 3.33 or newer. It is written for use with the 1 mL DTS0012 cuvette, but can be modified for use with the ZEN0040, ZEN2112, and ZSU1020 low volume cuvettes. This protocol can be utilized with the Zetasizer Sample Assistant for an automated workflow. 

Sample Preparation

RNA-LNP samples should be dispersed in an aqueous buffer. Ideal dispersant is water, 0.1X PBS, or 1X PBS, or similar. Buffers that contain sugar additives above 1% will have a different viscosity than water and should ideally be removed prior to DLS analysis. Alternatively, sugars can be diluted out by diluting the sample into a sugar-free buffer. For example, if a buffer that contains 10% sucrose is diluted 100x into 1X PBS, the resulting sample with 0.1% sucrose will have a negligible effect on the sample viscosity.

Protocol

Step 1: Pipette 1 mL RNA-LNP liquid sample into DTS0012 cuvette and cap cuvette.

Step 2: Place capped cuvette into Zetasizer cuvette holder, place thermal cap over cuvette, and close the lid.

Step 3: Configure a Size method with the following parameters:

General settings
  • Sample name: User specifies 
  • Cell: DTS0012 (Note: Select ZEN0040, ZEN2112, or ZSU1020 cells, if used) 
  • Material: Liposome (Refractive Index 1.45, Abs 0.001) 
  • Dispersant: Water (R.I. 1.33, Viscosity 0.8872 mPa.s at 25 °C) 
  • Project: User specifies
Method Builder
  • Size
  • Number of repeats: 3 or 5
Properties
  • Temperature: 25 °C 
  • Return to default temperature: yes 
  • Equilibration Time (s): 60
Data Processing
  • Analysis Model: General Purpose 
  • Size display limit mode: Automatic 
  • Size threshold mode: Automatic
Post analysis settings
  • Auto size average: User preference
Advanced Settings
  • Angle of detection: Backscatter 
  • Positioning method: Measure at optimal position 
  • Attenuation: Automatic 
  • Measurement process: Automatic 
  • User pause after sub-runs: No 
  • Optical Filter: No 
  • Filter Pause Between repeats (s): 0

Note:  Once configured, methods can be saved as .zskd files.

Step 4: Click Green “Play button” To start measurement.

Step 5: Configure a statistics report with the following parameters:

  • Z-Average (nm)
  • Polydispersity Index (PI)
  • Peak 1 Mean by Intensity (nm)
  • Peak 1 Area by Intensity (%)
  • Run Retention (%)
  • Attenuator
  • Intercept
  • Derived Mean Count Rate (kcps)

Example:

[Figure 1 AN240913-zetasizer-rna-lnp-protocol.jpg] Figure 1 AN240913-zetasizer-rna-lnp-protocol.jpg

Appendix

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