Encapsulation Efficiency (RiboGreen® / PicoGreen® Assay)
Percentage of RNA or DNA successfully encapsulated within the nanocarrier.
To determine the percentage of nucleic acid (RNA or DNA) successfully encapsulated within the nanocarrier, distinguishing between encapsulated and free/unentrapped nucleic acid. RiboGreen® is highly sensitive for RNA (including mRNA, siRNA); PicoGreen® is specific and sensitive for double-stranded DNA. This is crucial for optimising formulation parameters (e.g., lipid ratio, N/P ratio) and ensuring batch-to-batch consistency.
RiboGreen® and PicoGreen® are fluorescent dyes that bind nucleic acids and exhibit >1000-fold fluorescence enhancement.
Free nucleic acid is separated from nanoparticles (via centrifugation, filtration or dialysis), then quantified.
Total RNA/DNA is measured after disrupting the nanoparticles.
Standard protocol
Step 1 — Prepare standard curve
- 1
Dilute the mRNA standard in TE buffer to 0, 0.1, 0.5, 1, 2, 5 and 10 ng/μL.
- 2
Mix 2 μL of each standard with 98 μL TE buffer in a well.
- 3
Add 100 μL of 1:200 diluted RiboGreen® reagent (in TE).
- 4
Incubate 5–10 min at RT, protected from light.
- 5
Measure fluorescence (Ex 485 nm, Em 520 nm).
- 6
Plot fluorescence vs. concentration to generate the standard curve.
Step 2 — Measure free (unencapsulated) RNA
- 1
Dilute the LNP sample 10–100× in TE buffer (to stay within the linear range).
- 2
Load 100–200 μL onto a 0.22 μm centrifugal filter.
- 3
Centrifuge (e.g., 14,000 × g, 30 min, 4 °C).
- 4
Collect the filtrate (contains free RNA) and dilute appropriately.
- 5
Mix 2 μL filtrate + 98 μL TE + 100 μL RiboGreen® working solution.
- 6
Incubate, read fluorescence and use the standard curve to calculate free RNA.
Step 3 — Measure total RNA
- 1
Take a separate aliquot of the LNP sample.
- 2
Disrupt nanoparticles
- 1% (v/v) Triton™ X-100, or
- 1% SDS, or
- 0.5 M NaOH (for lipid systems).
- 3
Incubate 10–15 min at RT, then dilute appropriately (to avoid interference).
- 4
Add RiboGreen® reagent, measure fluorescence and determine total RNA from the standard curve.
EE% = (1 − Free RNA ÷ Total RNA) × 100
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