Lipid nanoparticles (LNPs) have the potential to revolutionize modern medicine. This technology has great potential to go beyond infectious diseases to reach targets that were once thought to be undruggable, and to an almost endless range of diseases. But the field is still in its infancy, and there are still many challenges to overcome to unlock the full potential of LNPs.
Uses of LNPS.
Over the years, scientists have demonstrated the potential of nucleic acids in simplified cell models and culture flasks. However, the application of these proof-of-concept experiments in real-life situations is more challenging, as administering these in living organisms increases their complexity. LNPs enable the administration of these ** in a way that both prevents them from degrading in vivo and delivers them to the target.
LNPs greatly increase the chances of finding druggable targets. With LNPs, it is possible to move beyond traditional small molecules** to nucleic acid-based ones**. With the right design, it is possible to design LNPs for a wide range of payloads for vaccination, protein substitution, and gene knockout.
Limitations of LNPS.
mRNA-based COVID-19 vaccines simplify the application of LNP in some ways. These vaccines are given infrequently, and booster shots are given as soon as the original protection begins to wane, and intramuscularly is convenient. While the transient nature of the payload helps make mRNA-based COVID-19 vaccines possible by strongly stimulating the immune response, the transient nature of traditional mRNA-LNPs becomes a limiting factor when the persistence of the product is required.
Therefore, other methods are needed to achieve effective protein substitution. Theoretically, frequent administration of LNPs may lead to immune response and particle inactivation, although self-amplifying RNA (SARNA) may help extend the time between doses2. Not only does SARNA prolong the duration of protein expression, but it also allows the use of lower doses of RNA, thereby reducing adverse effects. Delivering gene editing tools such as CRISPR Cas9 and associated guide RNA via LNPs ensures the persistence of the change, but there are also ethical considerations. Researchers are actively investigating how to alter the lipid composition of LNPs and add specific targeting ligands to address these issues, but much remains to be done1,3,4.
Advances in the safety and efficacy of LNP.
As with conventional medicines, LNPs must be formulated in such a way that the benefits outweigh the harms. LNPs must be stable during storage and transport in vivo and avoid detection by the immune system in order to reach their targets of action. That is, the LNPs must have a sufficiently long in vivo half-life, but they must be formulated to quickly and completely eliminate the nucleic acid payload and avoid the production of toxic metabolites after the delivery of the nucleic acid payload.
LNPs consist of a lipid mixture of ionizable cationic lipids, auxiliary 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 woven into LNP tolerance, immunogenicity, and cytotoxicity**5.
The addition of multiple environmental response properties to ionizable cationic lipids greatly improves the biocompatibility and biodegradability of these lipids6,8. Ionizable cationic lipids contain a transient positive charge obtained at low pH, which is used to not only encapsulate the nucleic acid payload, but also deliver the nucleic acid payload into the cell and facilitate endoplasmid escape with minimal toxicity. Ester or amide linkers can be cleaved by endogenous enzymes, facilitating the degradation of these lipids upon cellular uptake, while the addition of bioreducible disulfide bonds helps facilitate the release of nucleic acid cargo in the cytoplasm7,8.
However, some ionizable cationic lipids have immunostimulatory effects and can induce immune activation themselves1. While this can act as an adjuvant and be beneficial in some ways such as vaccines, in some other indications, this effect is less than ideal. The immune response stimulated by repeated administration of LNPs inhibits protein translation over time, which is a significant hurdle to overcome in the large-scale application of LNPs for protein replacement**.
The use of polyethylene glycol lipids also raises concerns about immunogenicity1,9. PEGylated lipids can prevent the loosening and clearance of the immune system, making LNP a ** preparation, thereby protecting LNP from being rapidly eliminated in the systemic circulation. They extend the half-life of LNPs and help ensure their safe delivery to target cells. However, PEGylated lipids cause an inflammatory response and hinder the absorption of particles, a phenomenon known as the "PEG dilemma"9. PEGylated lipids may be immunogenic, leading to PEGylated hypersensitivity and anti-PEGIGM and IgG production, which is contrary to the intent of PEGylated lipids and accelerates blood clearance of LNPs1. PEGylated lipids are also large and bulky molecules, and while their purpose is to prevent the immune system from taking up particles, they may also inhibit the uptake of particles during delivery, thereby limiting drug delivery to target cells10.
Researchers have been exploring new techniques to reduce immunogenicity and have similar activity in LNP preparations. Polysarcosine (PSAR) is a synthetic polymer based on endogenous amino acids that is expected to replace PEGylated lipids in LNPs11. LNPs formulated with PSAR instead of PEGylated lipids have been shown to have high transfection efficiency and low immunogenicity. Therefore, it is possible for PSAR functionalized LNPS to increase the potency of LNP without a corresponding increase in ***.
Target selectivity for LNP surface modification.
The development of LNPs that selectively target tissue, cell, and even subcellular-specific sites is the focus of many R&D efforts. Targeting not only expands the range of applications for LNPs, but also reduces unwanted applications like traditional small molecules
Taking advantage of the passive delivery capabilities conferred by basic biology, some targets are relatively easy to enter, while others are more difficult and require a well-thought-out design of active targeting mechanisms. The structure of certain organs favors the accumulation of LNPs. Due to their small size, LNPs can easily pass through epithelial palisade organs and or organs that receive a high proportion of cardiac output, such as the liver, spleen, and lungs12-14. Some researchers have found ways to alter the lipid composition of LNPs to further direct LNPs to these organs. Tailor the net surface charge of LNPs for tissue nourishment (LNPs with positive, neutral, and negative net surface charges target the lungs, liver, and spleen, respectively). The addition of a fifth lipid, the lipid sort molecule, to a traditional four-component LNP mixture can further modulate LNP delivery using a selective organ-targeting (SORT) approach15,16. However, there are also many diseases in areas other than the liver, spleen, and lungs. To push LNPs to the forefront of medicine, strategies must be developed to deliver LNPs to any site.
Due to the presence of the blood-brain barrier, diseases that affect the brain are difficult**. Despite their small size, LNPs do not passively cross the blood-brain barrier. Instead of struggling with biology, some researchers have found clever ways to harness it. Qiaobing Xu's lab learned that neurotransmitters are endogenous molecules, some of which can cross the blood-brain barrier, and thus synthesized novel lipids based on tryptamine, a functional group common to many neurotransmitters17. These neurotransmitter-derived lipids, known as NT-like lipids, allow otherwise impermeable LNP preparations to cross the blood-brain barrier.
Antibody-mediated delivery can also be used to achieve specific cell targeting. Decorating the surface of LNPs with antibodies targeting specific receptors is an interesting strategy for directing LNPs to target cells. However, chemical conjugation between proteins and lipids is difficult, and it is challenging to anchor antibodies to the LNP surface and maintain their functional localization18. Targeting platforms that anchor secondary SCFV targeting (Asset) molecules can address these issues. Lipidized single-stranded variable fragments (SCFVs) easily bind to LNPs. They recognize and bind to the Fc region of the antibody, ensuring that the FAB of the antibody binds to the ligand. Using this approach, a large number of antibody-antigen pairings can be utilized, and it may prove to be a versatile platform for targeted cell-specific delivery.
Lipid considerations for the future.
One of the biggest unanswered questions in LNP formulations is how to increase LNP potency without producing too much immunogenicity. The identification of novel lipids is the subject of many R&D efforts. A large number of ionizable cationic lipids are available and are constantly increasing. Modification of polar head groups, linker groups, and/or hydrophobic tails of ionizable cationic lipids will continue to improve the encapsulation and transfection efficiency of LNPs without compromising biocompatibility or biodegradability.
Researchers may find it beneficial to properly design new lipids. Some researchers may take inspiration from endogenous compounds, as in the case of developing PSAR functionalized LNPs and NT lipids. Toxicity can be avoided by using LNP components constructed from endogenous building blocks because they are more readily biodegradable or metabolized into endogenous compounds that are more hydrophilic and least toxic. In fact, some researchers have begun to explore exosomes—"lipid nanoparticles in nature"—as a more biomimetic nucleic acid delivery pathway19. Others want to use the properties of existing compounds to develop new lipids. This approach has been used to develop new lipids with special adjuvant activity using the structure of known toll receptor-like (TLR) agonists. In fact, the addition of adjuvanted lipids to traditional four-component LNPs can enhance the cellular immune response of SARS-CoV-2 mRNA vaccines, and they are well tolerated in mice20.
We are at the beginning of a new era where LNP technology has evolved to the point where it is possible to achieve what was once just a hypothetical. Continued progress in this area may require the integration of multiple approaches to address the current limitations of LNP, as well as collaborative R&D efforts that draw on the broad expertise of various scientific disciplines to bring LNP to the forefront of modern medicine.
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