Lipid nanoparticles (LNPs) are at the forefront of modern drug delivery systems, offering a versatile and efficient method to transport therapeutic agents like mRNA, siRNA, and other drugs to targeted cells. Their successful application in COVID-19 vaccines has highlighted their potential in pharmaceutical development. Here’s a detailed checklist to guide you through the formulation, design, and characterization of LNPs.

1. Lipid Selection and Proportions

To create effective LNPs, four primary types of lipids are required:

1. Ionizable Lipids (35-50%): These are crucial for binding and releasing the RNA or other cargo within the cell. Examples include ALC-0315, cKK-E12, SM-102, and Dlin-MC3-DMA.
2. PEGylated Lipids (0.5-3%): Small amounts of PEGylated lipids such as ALC-0159, DSPE-mPEG, and DMG-mPEG enhance the LNP’s circulatory half-life in the body.
3. Cholesterol (40-50%): This structural “helper” lipid improves efficacy by promoting membrane fusion and endosomal escape.
4. Neutral Phospholipids (~10%): Synthetic phospholipids like DSPC serve as structural "helper" lipids to facilitate cell binding.

2. Formulation Design Considerations

Formulating LNPs involves several critical considerations to ensure safety, efficacy, and suitability for their intended application

Administration Route

• Oral: LNPs should withstand stomach acidity and enzymatic degradation, with a size and surface coating that enhance intestinal absorption.
• Intravenous (IV): Requires stability in the bloodstream, avoiding rapid clearance by the immune system.
• Intramuscular (IM): Essential for vaccines, ensuring efficient uptake by muscle cells and antigen-presenting cells.
• Subcutaneous (SC): Formulated for sustained release and minimal local irritation.
• Topical: Needs to penetrate the skin’s stratum corneum while remaining stable and non-irritating.
• Pulmonary: Designed for deep lung penetration and stability in the respiratory tract.

Stability

• Physical Stability: Maintain size, shape, and uniformity over time.
• Chemical Stability: Ensure resistance to degradation of active ingredients and lipids.
• Shelf-Life: Techniques like lyophilization can extend shelf-life, requiring careful formulation.

Release Profile

• Controlled Release: Designed to release the active ingredient at a desired rate.
• Triggering Mechanisms: Release in response to specific stimuli like pH or temperature.

Targeting Capabilities

• Passive Targeting: Leverages natural LNP distribution based on size and surface properties.
• Active Targeting: Involves adding ligands to the LNP surface to bind specific receptors on target cells.

Biocompatibility and Safety

• Toxicity: Components must be non-toxic at the intended dose.
• Immunogenicity: Avoid unintended immune responses through careful lipid selection.
• Biodegradability: Components should be biodegradable to prevent accumulation in the body.

Manufacturing and Scalability

• Process Parameters: Consistent production requires control of mixing speed, temperature, and pH.
• Cost: Economically viable materials and manufacturing processes are essential.
• Regulatory Compliance: Adherence to Good Manufacturing Practices (GMP) and regulatory requirements is mandatory.

3. Formulation Techniques

Several methods are employed to formulate LNPs, each offering unique advantages:

• Solvent-Based Emulsification: Involves dissolving lipids and drugs in organic solvents before emulsifying into an aqueous phase. Examples include thin layer and nanoprecipitation.
• Nonsolvent-Based Emulsification: Melts solid lipids and mixes them with surfactants without toxic solvents. Utilized in Moderna and Pfizer COVID-19 vaccines.
• Microfluidic Mixing: Employs controlled flow conditions for precise particle size and high encapsulation efficiency. Used in the formulation of Onpattro, an FDA-approved LNP-based therapy.

4. Characterization of LNPs

Assessing the following parameters is crucial for effective LNP formulation:

• Size: Influences biodistribution and cellular uptake.
• Polydispersity Index (PDI): Indicates size distribution uniformity.
• Charge: Affects stability and cellular interaction.
• Morphology: Impacts stability and drug release.
• Encapsulation Efficiency (EE): Measures drug encapsulation efficiency.
• Stability: Assesses shelf life and therapeutic efficacy.

5. In Vitro Cell Experiments

Conducting in vitro cell experiments is essential for evaluating the efficacy and safety of LNP formulations.

References

1. Wiley Online Library: LNPs in Drug Delivery
2. NCBI: Cholesterol's Role in LNP Stability
3. EU NCL Assay Cascade: EU NCL
4. US NCL Assay Cascade: US NCL

Useful Links

• Buffer Preparations: AAT Bioquest
• Phase Transition Temperatures: Avanti Lipids
• LNP Protocol: Echelon