Invitrogen™ Vivofectamine™ VF233 IM LNP Composition in Ethanol

To measure delivery efficiency following lipid nanoparticle (LNP) administration, a reporter system such as FLuc mRNA (firefly luciferase mRNA) is first utilized within the LNPs. After administering the LNPs containing FLuc mRNA to the animals, in vivo imaging systems (IVIS) are employed to quantify bioluminescence.

For practical applications, such as protein or antigen expression, assays are conducted to measure the levels of expressed protein or immunoglobulin G (IgG) against the antigen, alongside analyses of other relevant parameters. This comprehensive approach provides insights into the delivery efficiency and functional outcomes of the administered LNPs.

The desirable range of polydispersity index (PDI) for lipid nanoparticles (LNPs) is typically below 0.2. A PDI value closer to 0 indicates a more uniform particle size distribution, which is critical for ensuring consistent delivery, stability, and performance of the LNP formulation.

The desirable range of particle size for lipid nanoparticles (LNPs) typically falls between 50 to 200 nanometers (nm). This size range is optimal for efficient delivery and cellular uptake of therapeutic agents, such as mRNA or siRNA. Smaller particles (within 60-120 nm) generally have better circulation times and avoid rapid clearance by the immune system, while larger particles (closer to 150 nm) can enhance payload delivery but might risk more rapid clearance or potential aggregation. However, the exact desirable size can vary depending on the specific therapeutic application and delivery route. For example: For intravenous administration, smaller LNPs (60-120 nm) are often preferred to optimize circulation time and minimize immune detection and for local or intramuscular delivery, slightly larger particles may be more acceptable.

The desirable encapsulation efficiency (EE) for lipid nanoparticles (LNPs) typically ranges from 75% to 95% or higher, depending on the application. A high EE is crucial for maximizing the payload delivered by the LNPs, ensuring that a substantial portion of the nucleic acids (mRNA, siRNA, etc.) or drugs is successfully encapsulated within the nanoparticle.

Encapsulation efficiency (EE) of lipid nanoparticles (LNPs) is measured by determining the proportion of the nucleic acid payload successfully incorporated into the nanoparticles compared to the total amount initially used in the formulation. RiboGreen Assay (Cat. No. R11490, https://www.thermofisher.com/order/catalog/product/R11490) is very commonly used to measure the encapsulation efficiency.

Ionizable lipids are increasingly utilized in lipid nanoparticle (LNP) formulations due to their unique properties that enhance the delivery of nucleic acids such as mRNA and DNA. They differ significantly from traditional cationic lipids in terms of their charge characteristics and functional roles in drug delivery systems. The key advantages of ionizable lipids are: 1) pH sensitivity - Neutral at physiological pH, becoming positively charged in acidic environments (like endosomes), which aids in endosomal escape. 2) Enhanced delivery - Effectively encapsulate nucleic acids with lower toxicity, improving stability and efficacy, and 3) Versatility - Structural diversity allows for optimization in various therapeutic applications. On the other hand, cationic lipids are permanently charged and this leads to higher cytotoxicity and mechanistically, it primarily relies on electrostatic interactions with nucleic acids which limits pH-dependent endosomal release.

Instrument-driven controlled processes produce highest quality LNPs. Instruments based on microfluidic mixing, impingement jet mixing (IJM), or T-mixers are typically used. Another option would be to utilize our Vivofectamine services (www.thermofisher.com/vivofectamine) to formulate LNPs encapsulating your nucleic acid. Lastly, you can also adopt a manual pipette-based mixing process (Wang et al., Nature Protocols, 2023 (https://www.nature.com/articles/s41596-022-00755-x.pdf), but it will lead to higher variability in particle characteristics compared to formulation instruments and could affect in vivo activity.

Lipid nanoparticles (LNPs) are the most advanced non-viral delivery system. They protect fragile nucleic acid molecules from degradation, enabling efficient cellular uptake and cytoplasmic delivery. Additionally, LNPs can be engineered for targeted delivery to specific tissues or cells, enhancing therapeutic precision.

As non-viral vectors, LNPs are well-tolerated by biological systems and can be optimized to minimize immune responses. A key advantage is their scalability: LNPs can be produced on a large scale through synthetic manufacturing processes, eliminating the need for cell culture or bioprocessing. This makes them both cost-effective and efficient, contributing to a cleaner, more streamlined production system.

1 mL of Vivofectamine VF232 Liver LNP Composition in Ethanol reagent can encapsulate up to 0.6 mg mRNA at reactive amine to phosphate (N:P) ratio of 4:1 (yield is dependent on the formulation process and varies from 50-80%). Assuming 100% yield, 1 mL of Vivofectamine VF232 Liver LNP Composition in Ethanol reagent allows administration to approximately 30 mice at 1 mg/kg dose. We have tested compatibility with RNA payload ranging from 23 bp to 6,000 bp for encapsulation using Vivofectamine VF232 Liver LNP Composition in Ethanol reagent.

In theory, lipid nanoparticles (LNPs) utilize charge-based interactions between positively charged ionizable lipids at lower pH and negatively charged nucleic acids. This allows LNPs to encapsulate various types of nucleic acids, including mRNA, mRNA/sgRNA, siRNA, miRNA, ASO, and pDNA. However, depending on the formulation process and the size of the payload, some optimization of the reactive amine to phosphate (N:P) ratio may be required to enhance encapsulation efficiency.

No, Vivofectamine VF232 Liver LNP Composition in Ethanol reagent does not provide a path to clinical/commercial licensing and GMP lipid supply. Even if you are in the discovery/preclinical stage with a goal of developing a drug, we encourage you to start with Vivofectamine Drug Developers portfolio (www.thermofisher.com/vivofectamine) instead as soon as possible for consistency in clinical translation.