Nucleic Acid Delivery systems

Recently, lipid nanoparticles (LNPs) have been used to deliver mRNA capable of fighting SARS-CoV-2, as well as CRISPR/Cas-9 gene editing technologies. Not only do delivery systems change, but their individual components are constantly evolving to meet current challenges and demands. To date, LNPs have primarily been formulated using the following components: Cationic Lipids, Phospholipids, PEG (Polymer) - Lipid Conjugates, Cholesterol.

   Considerations for future cationic lipids

In LNP formulations, cationic ionizable lipids are used to complex negatively charged mRNA. Aside from playing a major role in the LNP formulation itself, cationic lipids also serve a major role in the biological administration of mRNA to target cells. The RNA-loaded LNP fuses with the cell membrane and is then delivered into the cytosol. To be able to play these roles efficiently, a cationic ionizable lipid must be engineered with a suitable apparent acid dissociation constant (pKa). The apparent pKa of a cationic ionizable lipid is the likely pKa at the LNP surface. Currently, the cationic ionizable lipids in FDA-approved therapeutics all have an apparent pKa between 6-7. This is crucial for the cationic ionizable lipid to maintain a neutral charge while in systemic circulation (pH above the pKa of the lipid, pH ~7.5), as well as its ability to become positively charged in the endosome (pH ~6.5) and facilitate membrane fusion and subsequent cytosolic release.5

As crucial components in LNPs, new cationic ionizable lipids for nucleic acid delivery are being investigated to optimize mRNA complexation, endosomal membrane fusion and cargo release into the cytosol.

   PEGylated lipids and the future of stealth agents in LNPs

Historically, polyethylene glycol (PEG)-lipids have been the polymer-lipid conjugate of choice for LNP formulations. PEG-lipids are used to prevent proteins from binding to LNPs and increase systemic circulation times, mediate indirect targeting capabilities of LNPs, promote LNP self-assembly, and control LNP size and stability. The ability of LNPs formulated with PEG-lipids to bypass the reticulo-endothelial clearance system and stay in systemic circulation gives them their nickname - “stealth agents”.

As great as PEG-lipids are, they also present a few challenges. These challenges are commonly referred to as the “PEG dilemma”:

• Steric hindrance of long PEG chains hinders the ability of a cell to uptake the therapeutic
• PEGylation also hinders endosomal escape of nanoparticles, leading to decreased activity of the delivery system
• Repeated administration of PEGylated systems can result in a phenomenon called accelerated blood clearance (ABC)

Other alternatives to PEG-lipids in LNPs are being explored, and recent work has identified polysarcosine (pSar)-lipids as a promising alternative. pSar-lipids, a polymer-lipid conjugate based on the amino acid sarcosine, have demonstrated similar stealth properties to PEG-lipids. Previous studies have also shown polysarcosine lipids to induce a lower immunogenic response than PEG-lipids when administered to rabbits, zebrafish embryos, and mice. And most recently, pSar-lipids were evaluated in mRNA-LNPs and displayed high RNA transfection ability and an improved safety profile. Avanti is now offering pSar-lipids with varying polymeric chain lengths and lipid chain lengths for your PEG-free mRNA delivery research!

   Cholesterol and its structural analogs

Another major component of LNPs, making up roughly 35-45% of the final formulation, is cholesterol. Cholesterol works as a stability enhancer by filling gaps in the lipid layer and assists in the transfection of RNA. Cholesterol must be present to have an effective lipid-based delivery system but using too much cholesterol can prevent particle formation.

Since cholesterol makes up such a large portion of an LNP formulation, it makes sense that current research efforts would attempt to find more effective alternatives. With so many naturally occurring analogs of cholesterol available, evaluating the substitution of cholesterol with its structural analogs was a great starting point for a collaboration between Moderna and Oregon State University. They evaluated how three groups of steroids, 9,10-secosteroids, C-24 alkyl, and pentacyclic steroids, affected the particle size, mRNA encapsulation efficiency, and transfection efficiency of the resultant LNP formulations.

Of the evaluated cholesterol analogs, β-sitosterol proved to be head and shoulders above the rest. β-sitosterol LNPs had a comparable particle size (diameter ~100 nm) and encapsulation efficiency (~95% using 200 ng mRNA) as the cholesterol control. Where the β-sitosterol-substituted LNPs stood out was their transfection efficiency which was several-fold higher than the cholesterol control. So, comparable size and encapsulation efficiency, coupled with markedly improved transfection efficiency makes β-sitosterol an intriguing alternative to cholesterol in future RNA-LNPs. Avanti now has β-sitosterol (synthetic from plant sterol) available in the research catalog.

LNPs are being evaluated in research labs across the globe as therapeutic delivery systems to diagnose, prevent, or fight a number of diseases. As you encounter new delivery challenges, Avanti will be here to offer innovative lipids to tackle those challenges head on. If you have a product-specific question or need to discuss a custom project with one of our experts, get in touch with us.