RNA-based therapeutics are one of the most attractive classes of drugs for treating a variety of diseases. To function in vivo, RNA requires safe, effective and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and RNA release. Lipid nanoparticles (LNPs) have been extensively studied as non-viral vectors for the delivery of RNA to target cells because of their relatively easy and scalable manufacturing processes.

RNA Delivery: Challenges and Limitations

RNAs are always subjected to both systemic and cellular barriers that hinder their access to intracellular targets. Firstly, RNAs are highly susceptible to the destruction by nucleases or hydrolases in blood or body fluids, and rapid clearance by the kidney. Secondly, RNAs with the physicochemical properties of hydrophilic, negatively charged and high molecular weight is difficult to cross cell membrane into cytoplasm. Thirdly, after cell internalization, internalized RNAs were often trafficked through early/late endosomes and acidic lysosomes microenvironment and destroyed by enzyme degradation, which limits integral RNAs to exert gene effects.

Fig. 1 Extracellular and intracellular barriers for in vivo delivery of RNAs using non-viral vectors.

Therefore, for efficient delivery of RNAs, especially systemic delivery, one of the most attractive approaches is the development of non-viral vectors to overcome the above hurdles. The ideal delivery system for RNAs should:

• Have efficient encapsulation and protection on RNAs from nuclease-based degradation during systematic delivery;
• Prolong blood circulation time, prevent rapid clearance by kidney and phagocytosis by liver or spleen;
• Enhance targeted tissue/organ penetration and accumulation;
• Facilitate targeted cell internalization;
• Avoid lysosomal degradation during intracellular trafficking pathway;
• Enhance release of RNAs in the cytoplasm to exert gene effects.

Potential of Lipid Nanoparticles in RNA Delivery

So far, several RNA delivery systems have been developed to overcome the limitations of in vivo RNA drugs application, such as lipid nanoparticles, liposomes, polymeric nanoparticles, exosome and virus-like particles (VLP), etc. As the RNA delivery system evolved from viral vectors to nonviral vectors, the use of nano-sized carriers as the intracellular delivery system has drawn much attention. Such nanoparticles would enable an encapsulation of the loading cargo, which endows a protective effect to the particles, to avoid degradation from the harsh intracellular condition. Moreover, with appropriate chemical functionalizations, the nanoparticle itself may facilitate the intracellular approaching process, including cellular uptake, endosomal escape, and localization.

LNPs are capable of encapsulating and delivering therapeutic agents to specific locations in the body and releasing their contents at the desired time, thus providing a valuable platform for the treatment of a variety of diseases.

Composition of LNPs

Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a homogeneous lipid core. Typically, LNPs consist of a cationic/ionizable lipid and helper lipids such as phospholipids, cholesterol, and/or PEGylated lipids.

Fig. 2 Schematic representation of mRNA lipid nanoparticles. (Yan, Y. et al., 2022)

• Cationic lipid

Cationic lipids are amphiphilic molecules, consisting of a positively charged polar head group, and a hydrophobic tail domain, that in aqueous solution spontaneously self-assemble into higher order aggregates. Thanks to their cationic amino groups, they can electrostatically interact with the negatively charged phosphate groups of mRNA molecules and allow their entrapment in a lipid-based nanoparticle.

Fig. 2 Chemical structure of the major cationic lipids utilized for mRNA delivery. (Guevara, M. L. et al.,2020)

• Lonizable lipids and lipid-like polymers

A second generation of transfecting lipids was developed due to the necessity of novel delivery systems for siRNA molecules with improved safety profile and able to accumulate more efficiently at the target site, avoiding sequestration by blood-filtering organs like liver and spleen, a phenomenal frequently observed mostly with positively charged nanoparticles.

Fig. 2 Chemical structure of the most common ionizable lipids and lipidoids used for mRNA delivery. (Guevara, M. L. et al.,2020)

• Helper lipids and stealth lipids

In addition to charged or ionizable materials, lipid-based nano-formulations typically comprise supplementary components including cholesterol, for improving nanoparticle's stability; helper lipid, such as DSPC and DOPE, to facilitate the maintenance of the lipid bilayer structure and to promote endosomal release; and a PEG-conjugated lipids to prevent opsonization by serum proteins, thus enhancing the circulation time of nanoparticles.

Fig. 3 Chemical structure of the most common helper and stealth lipids employed for the preparation of formulated mRNA. (Guevara, M. L. et al., 2020)

Current Developments and Innovations in Lipid Nanoparticles for RNA Therapeutics

Lipid nanoparticles (LNPs) have become a prominent delivery system for RNA therapeutics, such as small interfering RNA (siRNA) and messenger RNA (mRNA), due to their ability to protect and efficiently deliver the RNA molecules to target cells. Several advancements have been made in recent years to improve the stability, encapsulation efficiency, cellular uptake, and surface properties of LNPs, enhancing their effectiveness as RNA delivery vehicles.

Improved stability

Traditional LNPs were prone to instability, leading to premature degradation and loss of RNA payload during storage or circulation in the body. To improve stability, researchers have incorporated more stable lipid components, such as cholesterol, into LNP formulations. These modifications have significantly increased the shelf-life and in vivo stability of LNPs, ensuring the sustained release of RNA therapeutics.

Increased encapsulation efficiencies

Efficient encapsulation of RNA molecules within LNPs is crucial for successful delivery. Researchers have developed novel techniques to improve encapsulation efficiencies. For instance, the use of ionizable cationic lipids has enabled efficient encapsulation of negatively charged RNA molecules. Additionally, the incorporation of fatty acid-conjugated lipids and other lipid modifications has further improved the capacity of LNPs to encapsulate and protect RNA payloads, leading to higher delivery efficiencies.

Increased cellular uptake

Enhanced cellular uptake is another critical aspect of LNP development. Researchers have focused on optimizing LNP formulations to facilitate efficient intracellular delivery of RNA payloads. The surface charge of LNPs plays a significant role in cellular uptake. By fine-tuning the charge via modifications of lipid composition or inclusion of targeting ligands, researchers have significantly increased the cellular uptake of LNPs in specific cell types.

Tailoring the surface properties of LNPs

Another exciting development in LNP-based RNA therapy is the use of modified RNA molecules, such as nucleoside-modified mRNA or chemically modified siRNA. These modifications can improve RNA stability, reduce immunogenicity, and enhance therapeutic activity, thereby increasing the overall efficacy of LNP-based therapy.