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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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.
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.
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.
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 |
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| Method Builder |
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| Properties |
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| Data Processing |
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| Post analysis settings |
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| Advanced Settings |
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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:
Example:
![[Figure 1 AN240913-zetasizer-rna-lnp-protocol.jpg] Figure 1 AN240913-zetasizer-rna-lnp-protocol.jpg](https://dam.malvernpanalytical.com/7cae4b34-de63-4b1e-ab57-b1f000bcf1d4/Figure%201%20AN240913-zetasizer-rna-lnp-protocol_Original%20file.jpg)
