What are Lipid Nanoparticles and Their Applications?

Naked DNA or RNA is easily degraded by nucleases in body fluids and is difficult to accumulate in target tissues. The immune system can also recognize and degrade exogenous nucleic acids to trigger an immune response. The biggest problem with DNA- or RNA-based gene therapies is drug delivery. To achieve safe and effective nucleic acid delivery, scientists have developed lipid nanoparticles to protect nucleic acids from degradation, maximize delivery to target cells, and reduce exposure of nucleic acids to off-target cells. Lipid nanoparticles, as an important component of COVID-19 mRNA vaccines, play a key role in effectively protecting and transporting mRNA into cells.

Liposomes: The Earliest Lipid Nanoparticles

The term liposome emerged in the 1960s, when it was discovered that enclosed lipid bilayer vesicles could form spontaneously in water. Liposomes are nanocarriers composed of one or several lipid bilayers ranging in size from 20 nM to 1000 nM. Hydrophilic drugs can be enclosed in the aqueous interior regions of liposomes, while hydrophobic drugs can be encapsulated in the hydrocarbon chain regions of the lipid bilayer. Therefore, liposomes have been extensively studied for drug delivery.

Fig. 1. Schematic representation of different types of liposomal drug delivery systems. (A) Traditional liposomes; (B) PEGylated liposomes; (C) Ligand-targeted liposomes; (D) Theranostic liposomes (Front Pharmacol. 2018, 6(9): 80).

Liposomes stabilize therapeutic compounds and overcome barriers to cellular and tissue uptake, improving targeting of compounds to disease sites and thereby reducing accumulation in non-target organs. Depending on the route of administration and site of disease, combining liposomes with different delivery platforms can further improve the delivery of encapsulated compounds.

Lipid Nanoparticle Structure

Lipid nanoparticles are spherical vesicles composed of one (monolayer) or multiple (multilayer) phospholipid bilayers. They are usually composed of four components: cationic lipids, helper lipids, cholesterol and PEG-lipids (Fig. 2).

Fig. 2. Schematic representation of lipid nanoparticles containing mRNA (Nat Rev Mater. 2017, 2: 17056).

Cationic Lipids

The earliest lipid nanoparticles used cationic lipids, which readily bind to negatively charged nucleic acids. However, this cationic lipid-based delivery system is toxic and immunogenic both in vivo and in vitro. DOTAP and DOTMA, for example, can be neutralized by negatively charged serum proteins, resulting in toxicity and reduced efficacy.

As an alternative, ionizable cationic lipids have been developed, which reduce the toxicity caused by cationic lipid nanoparticles while retaining efficient transfection properties. The ionizable tertiary amine moiety of ionizable cationic lipids such as DLin-KC2-DMA and D-Lin-MC3-DMA has a net positive charge at acidic pH but remains neutral in the blood circulation system (pH 7.4) sex. This pH sensitivity facilitates the delivery of nucleic acid drugs in the body because lipid nanoparticles that remain neutral interact less with the anionic membranes of blood cells, thereby improving the biocompatibility of lipid nanoparticles. Normally, cells take up lipid nanoparticles through endocytosis. When lipid nanoparticles enter the endosome with low pH value, the ionizable cationic lipid is protonated and becomes positively charged, which promotes the instability of the endosomal membrane, causes the membrane to rupture, and the lipid nanoparticles undergo endosomal escape. Currently, there are five major ionizable lipid types widely used for RNA delivery, including branched-tail lipids, biodegradable lipids, polymeric lipids, multi-tail lipids, and unsaturated lipids.

PEGylated Lipids

PEGylated lipids are another important component of lipid nanoparticles. PEGylated lipids, such as DMG-PEG 2000 and DSPE-MPEG-2000, are composed of hydrophilic PEG bound to hydrophobic alkyl chains via phosphate, glycerol, or other linkers. PEGylated lipids are located on the surface of lipid nanoparticles, with lipid domains buried deep within the particles and PEG domains protruding from the surface.

Fig. 3. Effect of PEG lipid content on lipid nanoparticle morphology and size (2019, 11(45): 21733-21739).

PEGylated lipids can prolong the circulation time of liposomes because PEG forms a steric barrier that prevents binding of plasma proteins that would otherwise lead to their rapid clearance by reticuloendothelial cells. Additionally, PEGylated lipids can control the size of nanoparticles. This is because the low pH and ethanol environment will promote aggregation and fusion of lipid nanoparticles during the manufacturing process. The steric barrier of PEGylated lipids prevents this and helps produce a uniform population of particles with narrow polydispersity and small particle size (typically 50-100 nM). Formulations completely lacking PEG lipids produced unstable, polydisperse lipid nanoparticles.

Cholesterol

Cholesterol is hydrophobic and rigid, filling the gaps between lipids within the liposome membrane and promoting vesicle stability. The molecular geometry of cholesterol derivatives can further influence the delivery efficacy and biodistribution of lipid nanoparticles. Auxiliary lipids are mostly phospholipids, such as DSPC and DOPE, which promote cellular uptake and endosomal release by promoting fusion with cell and endosomal membranes.

Lipid Nanoparticles in Drug Delivery

It is known that more than 40% of small molecule drugs used to treat cancer have low solubility in water, and liposomes as drug delivery systems can encapsulate these drugs and improve their water solubility. Liposome-encapsulated drugs can reduce the toxicity of drugs to normal tissues and prolong the residence time of drugs. Many lipid nanoparticle drug formulations have been widely used in many clinical trials as delivery systems for anticancer, anti-inflammatory, antibiotic, antifungal, anesthetic, and other drugs and gene therapies, especially in the field of delivering nucleic acid drugs.

Fig. 4. COVID-19 mRNA vaccine lipid nanoparticles (ACS Nano. 2021, 15(11): 16982-17015).

For example, the first approved liposomal drug, Doxil, a lipid nanoparticle formulation of the antitumor drug doxorubicin (liposomal doxorubicin nano-drug), uses nanoparticles to extend circulation time in human plasma while reducing the cardiotoxicity of doxorubicin. Second, the nucleic acid drug Patisiran, a lipid nanoparticle-loaded siRNA drug that reduces transthyretin formation in the liver, was recently approved by the FDA for the treatment of hereditary transthyretin-mediated amyloidosis. It is the first approved lipid nanoparticle preparation nucleic acid drug and is regarded as an important milestone in the development of nucleic acid therapy. Additionally, the latest successful application of lipid nanoparticles is as a delivery vehicle for two COVID-19 mRNA vaccines.

Lipid Nanoparticle Development Services

BOC Sciences is a leading provider of chemical products and services, including expertise in lipid nanoparticles. We have a wide range of lipid materials and can design and optimize formulations based on desired properties and applications. In addition, our R&D scientists have expertise in controlling the size of lipid nanoparticles, which is critical to their stability, drug-loading capacity, and targeted delivery. We can optimize formulation parameters to achieve the particle size distribution required by our customers.

References

1. Robson, A.L. et al. Advantages and limitations of current imaging techniques for characterizing liposome morphology. Front Pharmacol. 2018, 6(9): 80.
2. Hajj, K. et al. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat Rev Mater. 2017, 2: 17056.
3. Kulkarni, J.A. et al. On the role of helper lipids in lipid nanoparticle formulations of siRNA. Nanoscale. 2019, 11(45): 21733-21739.
4. Tenchov, R. et al. Lipid nanoparticles-from liposomes to mrna vaccine delivery, a landscape of research diversity and advancement. ACS Nano. 2021, 15(11): 16982-17015.