LNPs were formulated as previously described4 (link),5 (link),27 (link), with the lipid components (SM-102, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-2000 at molar ratios of 50:10:38.5:1.5) being rapidly mixed with an aqueous buffer solution containing ABE8.8 or SpRY-ABE8.8 mRNA and either PAH1 gRNA or PAH2 gRNA in a 1:1 ratio by weight in 25 mM sodium acetate (pH 4.0), with an N:P ratio of 5.6. The resulting LNP formulations were subsequently dialyzed against sucrose-containing buffer, concentrated using Amicon Ultra-15 mL Centrifugal Filter Units (Millipore Sigma), sterile-filtered using 0.2-μm filters, and frozen until use. The LNPs underwent quality control for particle size (Z-Ave, hydrodynamic diameter), polydispersity index as determined by dynamic light scattering (Malvern NanoZS Zetasizer) and total RNA encapsulation as measured by the Quant-iT Ribogreen Assay (Thermo Fisher Scientific).
Protocol detail
Lipid Nanoparticle Formulation and Characterization
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Corresponding Organization : California University of Pennsylvania
Other organizations : Agilent Technologies (Germany), Children's Hospital of Philadelphia
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Variable analysis
independent variables
- Lipid components (SM-102, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-2000) at molar ratios of 50:10:38.5:1.5
- MRNA (ABE8.8 or SpRY-ABE8.8)
- GRNA (PAH1 or PAH2) in a 1:1 ratio by weight
- Particle size (Z-Ave, hydrodynamic diameter)
- Polydispersity index
- Total RNA encapsulation
- Rapid mixing of lipid components with aqueous buffer solution
- N:P ratio of 5.6
- Dialysis against sucrose-containing buffer
- Concentration using Amicon Ultra-15 mL Centrifugal Filter Units
- Sterile filtration using 0.2-μm filters
- Freezing until use
Annotations
Based on most similar protocols
Optimize N:P ratio of 5.6 to ensure efficient encapsulation of mRNA and gRNA (Input protocol)
Use Quant-iT Ribogreen Assay to measure total RNA encapsulation, with 90-100% encapsulation reported (Protocol 1)
Analyze particle size using dynamic light scattering, with reported Z-Ave hydrodynamic diameters of 69-89 nm and polydispersity index <0.21 (Protocol 1)
Dialyze LNP formulations against sucrose-containing buffer to improve stability (Input protocol, Protocols 1-4)
Concentrate LNP formulations using Amicon Ultra centrifugal filters to achieve desired final concentration (Input protocol, Protocols 1-4)
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