Lipid nanoparticles (LNPs) are primed to revolutionize modern medicine. This technology has immense possibilities to evolve beyond infectious disease to reach targets once considered undruggable and treat a near infinite number of conditions. But the field is still in its infancy, and there are a number of challenges yet to overcome to unlock the full potential of LNPs. 

  

 

This article was featured in  Cayman Currents Issue 36: Lipid Nanoparticles

 

 

The Utility of LNPs

Scientists have demonstrated the potential of nucleic acid-based therapies in simplified cellular models and culture flasks for many years. However, the real-life application of these proof-of-concept experiments has been much more challenging because of the added complexity that comes with administering these therapies in a living organism with its biological intricacies. LNPs made it possible to administer these therapies in a way that protected them from degradation in the body and delivered them to targeted sites. 

LNPs have significantly expanded the opportunity for druggable targets. With LNPs, it is now possible to move beyond traditional small molecules into nucleic acid-based therapies. With the right design, it should be possible to engineer LNPs to suit any number of payloads for vaccination, protein replacement therapy, and therapeutic gene knockdown.

The Limitations of LNPs 

mRNA-based COVID-19 vaccines were, in some ways, a simplified application of LNPs. These vaccines were administered infrequently, with booster shots given once the original protection began to wear off, and by way of easily administered intramuscular injection. While the short-lived nature of the payload helped to make the mRNA-based COVID-19 vaccines possible through strong stimulation of the immune response, the transient nature of traditional mRNA-LNPs is a limitation when persistence of the product is necessary. 

Alternative methods are required to achieve effective protein replacement. Frequent dosing of LNPs could, in theory, result in immune responses and inactivation of the particles, though self-amplifying RNA (saRNA) may help to lengthen the time between administrations.²saRNA not only increases the duration of protein expression, but also offers the ability to use lower doses of RNA, which reduces adverse effects. Delivery by LNPs of gene editing tools such as CRISPR/Cas9 and associated guide RNAs ensure the permanence of the therapeutic change, but also present with ethical considerations. Researchers are actively pursuing modifications to the lipid composition of LNPs as well as addition of specific targeting ligands to address these concerns, but much work remains to be done.^1,3,4

Advancements in LNP Safety & Efficacy 

As with traditional drugs, LNPs must be formulated such that the benefits outweigh the risks. LNPs must be stable, both during storage and transit throughout the body, and avoid immune detection to reach their target site of action. That is, they must have a sufficiently long  in vivohalf-life yet be formulated to have rapid and complete elimination that avoids the production of toxic metabolites after delivery of the nucleic acid payload. 

LNPs are formed from lipid mixtures of ionizable cationic lipids, helper lipids, sterol lipids, and PEGylated lipids. Ionizable cationic lipids are the main drivers of nucleic acid payload encapsulation and LNP efficacy, but they are also intertwined with LNP tolerability, immunogenicity, and cellular toxicity.⁵

The addition of multiple environmentally responsive features in ionizable cationic lipids has greatly improved the biocompatibility and biodegradability of these lipids.^6-8 Ionizable cationic lipids contain a transient positive charge that is acquired at low pH. This can be leveraged to not only encapsulate nucleic acid payloads, but to deliver them to cells and promote endosomal escape with minimal toxicity. Ester or amide linker groups can be cleaved by endogenous enzymes, promoting the degradation of these lipids upon cellular uptake, and incorporating bioreducible disulfide bonds can help encourage the release of the nucleic acid cargo in the cytosol.^7,8

 

Related Literature: Read more on how to tune ionizable cationic lipid design for efficacy and safety

 

However, some ionizable cationic lipids are immunostimulatory, and can themselves induce immune activation.¹ While this may act as an adjuvant effect and is beneficial for certain therapeutic modalities such as vaccines, there are other indications where this is less than desirable. The immune response stimulated by repeat dosing of LNPs can  suppress protein translation over time, representing a significant barrier to overcome in order to apply LNPs on a larger scale to protein replacement therapies.  

Concerns over immunogenicity also arise from the use of PEGylated lipids.^1,9 PEGylated lipids help protect the LNP from rapid elimination in systemic circulation by preventing opsonization and clearance by the immune system, making them act as stealth agents. They improve the LNP half-life and help ensure its safe delivery to the target cell. However, concerns over inflammatory responses and the hindrance of particle uptake arise from PEGylated lipids, a phenomenon known as the "PEG dilemma".⁹ PEGylated lipids can be immunogenic, leading to PEGylated hypersensitivities and the production of anti-PEG IgM and IgG, which, counter-productive to the intent of PEGylated lipids, accelerates blood clearance of LNPs.¹ PEGylated lipids are also big, bulky molecules, and while intended to prevent particle uptake by the immune system, it may also inhibit particle uptake during delivery, limiting delivery of the therapeutic cargo to target cells.¹⁰

Researchers have been exploring new technologies with reduced immunogenicity and comparable stealth activities in LNP formulations. Polysarcosine (pSAR) is a synthetic polymer based on an endogenous amino acid with promising potential to act as a substitute for PEGylated lipids in LNPs.¹¹ LNPs formulated using pSAR as a substitute for PEGylated lipids have been shown to have high transfection efficiencies yet with reduced immunogenicity. Hence, pSAR-functionalized LNPs may offer opportunities to boost LNP potency without a corresponding increase in side effects. 

Target Selectivity with LNP Surface Modifications 

The development of LNPs that selectively target tissue-, cell-, and even subcellular-specific sites is a focus point of many R&D efforts. Not only will targeted therapies expand the utility of LNP applications, but much like traditional small molecule therapies, it will also serve to reduce unwanted side effects. 

Some target sites of action are relatively easy to access by leveraging passive delivery endowed by basic biology, whereas others are more difficult and require thoughtfully designed active targeting mechanisms. The architecture of certain organs favors the accumulation of LNPs. Because of their small size, LNPs pass easily through organs with fenestrated epithelium and/or receive a high proportion of cardiac output, like the liver, spleen, and lungs.^12-14 Some researchers have found means to alter the LNP lipid composition to further direct LNPs to these organs. Tailoring the net surface charge of the LNP to achieve tissue tropism (LNPs with net positive, neutral, and negative surfaces charges can be targeted to the lungs, liver, and spleen, respectively). Using a selective organ targeting (SORT) approach, which includes a fifth lipid, a lipid SORT molecule, in the traditional four-component LNP mixture, may further tune LNP delivery.^15,16 However, there are plenty of diseases that arise in locations outside the liver, spleen, and lungs. To bring LNPs to the forefront of medicine requires strategies to achieve LNP delivery to any site.

 

Related Literature: Read more on the heads and tails of lipid-based drug delivery

 

Conditions that affect the brain are difficult to treat because of the blood-brain barrier. Despite their small size, LNPs do not passively cross the blood-brain barrier. Rather than fighting against biology, some researchers have found ingenious ways to leverage it. Armed with the knowledge that neurotransmitters are endogenous molecules, some which can cross the blood-brain barrier, Qiaobing Xu's lab synthesized novel lipids based on tryptamine, a functional group shared by many neurotransmitters.¹⁷ These neurotransmitter-derived lipidoids (termed NT-lipidoids) could permit passage across the blood-brain barrier of otherwise brain-impermeable LNP formulations.  

Antibody-mediated delivery could also be used to achieve cell-specific targeting. Decorating the surface of LNPs with antibodies that target certain receptors is an intriguing strategy to direct LNPs to target cells. However, chemical conjugation between proteins and lipids is difficult, and it is challenging to anchor antibodies to LNP surfaces in a way that retains the functional orientation of the antibodies.¹⁸ A targeting platform that harnesses an anchored secondary scFv enabling targeting (ASSET) moiety can be used to circumvent these issues. The lipidated single-chain variable fragments (scFvs) are readily incorporated into LNPs. They recognize and bind to the Fc region of antibodies, ensuring the Fab arms of the antibody are accessible for ligand binding. There is a large repertoire of antibody-antigen pairs to tap using this approach, and it could prove to be a versatile platform for targeted cell-specific delivery.

Considerations for Future Lipids 

One of the biggest outstanding questions in LNP formulations is how to boost LNP potency without excessive immunogenicity. The identification of novel lipids is the subject of many R&D efforts. A large collection of ionizable cationic lipids is now available, and the availability and number of them continues to grow. Modifications to the polar head group, linker group, and/or hydrophobic tails of ionizable cationic lipids will continue to refine the encapsulation and transfection efficiencies of LNPs without compromising biocompatibility or biodegradability.

Researchers may find it advantageous to look towards the rational design of new lipids. Some may use inspiration from endogenous compounds, much like how pSAR-functionalized LNPs and NT-lipidoids were developed. Using LNP components built from endogenous building blocks could potentially avoid toxicity by being more readily biodegradable or metabolized into endogenous compounds with improved hydrophilicity and minimal toxicity. Indeed, some researchers have begun to explore exosomes, "nature's lipid nanoparticles", as a more biomimetic route for the delivery of nucleic acid therapies.¹⁹ Still others could look to harness the properties of existing compounds to develop new lipids. Such an approach has been taken to develop new lipids with specific adjuvant activity by leveraging the structure of known toll-like receptor (TLR) agonists. Indeed, the inclusion of adjuvant lipidoids into the traditional four-component LNP boosted cellular immune responses of a SARS-CoV-2 mRNA vaccine and were well-tolerated in mice.²⁰  

We are at the beginning of a new era where LNP technology has advanced to the point where it is possible that therapies once only hypothesized can be actualized. To continue to make headway in this field will likely involve the integration of multiple approaches to address the current limitations of LNPs and collaborative R&D efforts to draw from the extensive expertise across scientific disciplines to bring LNPs to the forefront of modern medicine. 

Cayman Helps Make Research Possible

Cayman Chemical has been supplying high-purity lipids to the scientific community for over 40 years, and our industry-leading expertise in lipid chemistry, synthesis, and purification is supported by state-of-the-art analytical equipment. 

We are committed to offering an unprecedented portfolio of LNP research tools. We offer an impressive collection of ready-to-use lipids for LNPs and research-ready LNPs to screen different LNP compositions along with in-house lipid synthesis, screening, and LNP formulation services. 

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