Lipid Nanoparticles for mRNA Delivery

In recent years, mRNA therapies have emerged as a novel therapeutic approach, demonstrating immense potential in preventing and treating various diseases. The success of COVID-19 vaccines has particularly highlighted the broad application prospects of mRNA technology. However, mRNA itself is unstable and prone to triggering immune responses, making its safe and effective delivery a critical challenge. Lipid nanoparticles (LNPs), the most widely used mRNA delivery system to date, offer excellent safety and scalability. LNPs effectively encapsulate mRNA, protecting it from degradation and facilitating targeted delivery to specific organs and cells in the body. This technology has been employed in clinical research across multiple fields, including cancer immunotherapy, protein replacement therapy, and gene editing.

What is Messenger RNA?

Messenger RNA (mRNA) is a nucleic acid molecule that serves as an intermediary in gene expression, transferring genetic information from DNA to ribosomes in the cytoplasm to guide protein synthesis. Composed of nucleotides, mRNA features a 5' cap structure, an open reading frame (ORF) encoding the target protein, and a 3' polyadenylated tail (poly(A) tail). These structural elements enhance its stability and promote translation. In recent years, mRNA technology has shown tremendous promise in drug discovery, particularly in vaccine development and therapeutic drug production. For instance, mRNA vaccines, such as those for COVID-19, encode specific antigenic proteins and harness the host cell's protein synthesis mechanisms to elicit an immune response. Compared to traditional vaccines, mRNA vaccines are faster to produce, highly programmable, and do not require pathogen cultivation. Moreover, mRNA is being used to develop therapies for gene editing, cancer immunotherapy, and rare diseases. Through optimization of mRNA sequences, delivery systems (e.g., lipid nanoparticles), and translation efficiency, researchers can target specific disease pathways to create safe and effective treatments. The flexibility and adaptability of mRNA establish it as an innovative platform for modern drug discovery.

mRNA Delivery Platform

With rapid advancements in medical technology, mRNA-based drugs, leveraging their unique mechanisms of action, have demonstrated great potential in areas such as vaccine development, cancer treatment, and interventions for genetic disorders. The efficacy of mRNA therapeutics heavily depends on delivery systems that can address stability and efficiency challenges.The pharmacokinetics of mRNA therapy face four major hurdles: (1) mRNA, as a negatively charged macromolecule, cannot readily cross anionic cell membranes or be internalized via endocytosis. (2) mRNA is highly susceptible to degradation by ubiquitous RNases, resulting in a short intracellular half-life. (3) Even when internalized, mRNA may fail to escape endosomes and reach the cytoplasm for translation. (4) The inherent immunogenicity of mRNA can induce toxic immune responses in vivo. While naked mRNA delivery has been used in numerous in vivo studies—especially for vaccines encoding specific antigens—it is insufficient for applications requiring sustained, high-level protein expression. Common mRNA delivery systems currently include protein-mRNA complexes, lipid nanoparticles, polymeric nanoparticles (PNPs), and hybrid carriers combining lipids and polymers.

Fig. 1. Lipid nanoparticles for mRNA drug delivery (Vaccines (Basel). 2023, 11(3): 658).

Lipid-based nanoparticles, including LNPs and liposomes, are extensively employed in nucleic acid delivery. The FDA-approved patisiran—the first RNA-based oligonucleotide drug used to treat transthyretin-mediated amyloidosis (TTR)—utilizes an LNP formulation. In cancer immunotherapy, BioNTech's BNT111, which contains four melanoma-associated antigens (NY-ESO-1, tyrosinase, MAGE A3, and TPTE), uses a liposome-based delivery system and is currently undergoing Phase I-II clinical trials.

Lipid Nanoparticles Definition

Lipid nanoparticles (LNPs) consist of four main components: cationic lipids, phospholipids, cholesterol, and PEG-lipids. The preparation of mRNA-LNPs typically involves dissolving lipids and mRNA separately in ethanol and an acidic aqueous phase, followed by mixing in a microfluidic device at a 1:3 ethanol-to-water volume ratio, resulting in the self-assembly of LNPs. During this process, ionizable cationic lipids are protonated under acidic conditions, carrying a positive charge that electrostatically interacts with the negatively charged mRNA, encapsulating it within the LNPs. The other auxiliary lipids—phospholipids, cholesterol, and PEG-lipids—self-assemble around this core. Subsequently, the pH of the mRNA-LNP solution is neutralized via buffer exchange, forming stable mRNA-LNPs. Optimization strategies for LNPs include designing and screening novel lipid molecules, adjusting the internal lipid composition, and modifying the surface of LNPs.

Designing and Screening Novel Lipids

Ionizable cationic lipids are key components of LNPs, effectively encapsulating nucleic acids under acidic conditions while reducing toxicity during circulation under physiological conditions. Upon entry into endosomes and lysosomes, where the environmental pH is below the surface pKa, LNPs can regain a positive charge, facilitating endosomal escape and releasing mRNA into the cytoplasm. Researchers often focus on modifying the lipid tail structures, such as altering the number of tails, designing linear or branched configurations, and introducing unsaturated or biodegradable bonds to enhance efficacy or confer specific functions. Currently, high-throughput screening of ionizable lipids has established extensive libraries of novel lipids, with their in vivo effects thoroughly evaluated. Phospholipids, as auxiliary lipids, aid in the formation of lipid nanoparticles and endosomal escape. Liu et al. developed hundreds of ionizable phospholipids termed iPhos, addressing the limitations of traditional phospholipids, such as structural rigidity and limited availability. The incorporation of PEG lipids reduces nanoparticle aggregation, extends circulation time, and helps evade phagocytosis by mononuclear cells. However, PEG can also hinder interactions with target cells and subsequent endosomal escape, leading to reduced transfection efficiency. Adjusting the carbon chain length and molecular weight of PEG lipids can optimize its beneficial effects. Cholesterol contributes to improved stability and membrane fusion of LNPs. Optimizing the structure of cholesterol or incorporating specific cholesterol derivatives can further enhance the delivery efficiency of LNPs, endowing them with unique functionalities.

Adjusting Internal Lipid Composition

The selection of lipid components and their ratios is crucial for LNP-mediated mRNA delivery. While traditional LNPs consist of four components, recent studies have explored the introduction of mixed ionizable lipids to improve delivery efficiency or the addition of a fifth component for tissue-specific delivery. Research has shown that cholesterol and phospholipids are not always essential for LNP function. Novel three-component LNPs, consisting of biodegradable ester-core permanent cationic lipids and PEG-lipid synergies, outperform conventional four- or five-component LNPs in efficacy and lung targeting. Additionally, the N/P molar ratio (N refers to the amine groups of ionizable cationic lipids, and P refers to the phosphate groups of mRNA) significantly affects LNP properties. A lower N/P ratio increases mRNA loading efficiency but may reduce encapsulation efficiency. Studies have also indicated that LNPs with lower N/P ratios exhibit higher protonation levels within the endosomal pH range, promoting endosomal escape and enhancing mRNA expression in vivo.

Surface Modification of LNPs

Surface modification is also a key strategy for optimizing LNPs, particularly by conjugating antibodies or other molecules to LNPs, which can significantly enhance their targeting capabilities. For instance, LNPs conjugated with PECAM-1 antibodies have been developed for lung-targeted delivery, while LNPs modified with CD3, CD4, and CD5 antibodies have been shown to effectively deliver mRNA to T lymphocytes in vivo. Similarly, LNPs modified with c-kit (CD117) antibodies can efficiently deliver RNA to hematopoietic stem and progenitor cells in the body. Overall, antibody modifications hold tremendous potential for improving the delivery efficiency of LNPs.

Lipid Nanoparticles Synthesis

As a key raw material for lipid nanoparticle development, BOC Sciences offers a wide range of high-quality lipids, including cationic lipids, phospholipids, cholesterol, and PEG-lipids. These lipids undergo stringent quality control to ensure purity and performance, making them suitable for mRNA vaccines, gene therapies, and drug delivery applications. BOC Sciences also possesses advanced technical platforms and extensive experience in LNP development, providing end-to-end services from formulation design and process optimization to large-scale production. By optimizing lipid ratios and nanoparticle assembly processes, the company has developed LNP systems with high encapsulation efficiency, exceptional stability, and precise targeting, catering to diverse drug delivery needs.

Targeting Specificity of LNP Delivery

Traditional LNPs administered intravenously primarily accumulate in the liver due to the adsorption of a large amount of apolipoprotein E (ApoE) onto their surface. This promotes hepatocyte uptake via the interaction of ApoE with low-density lipoprotein receptors (LDLR). Current research focuses on designing different ionizable lipids and employing various administration methods to achieve RNA delivery to organs and cells beyond the liver. Polymer lipids, such as 7C1, can efficiently deliver RNA to endothelial cells in multiple organs, including the lungs and bone marrow. Amphiphilic amino lipids like ZA3-Ep10 exhibit lung-targeting properties, while anionic lipids facilitate targeted delivery to the spleen. OF-Deg-Lin LNPs have been shown to enable efficient protein expression in the spleen (>85%) and achieve effective B lymphocyte targeting in vivo (~7%). Lipid compounds containing imidazole groups can deliver mRNA to T lymphocytes, with the lead structure 93-O17S achieving gene recombination in 8.2% of CD4+ and 6.5% of CD8+ splenic T lymphocytes in mice. Additionally, modifications or substitutions of phospholipids and cholesterol in LNP components can alter organ targeting specificity. Systematic screening of phospholipid structures in vitro by Álvarez-Benedicto et al. revealed that zwitterionic phospholipids predominantly facilitate liver-targeted delivery, whereas anionic phospholipids enhance spleen-targeted delivery. Furthermore, replacing cholesterol with 20α-hydroxycholesterol (20α-OH) in LNPs increased mRNA delivery efficiency to endothelial cells and Kupffer cells by fivefold compared to hepatocytes.

What are mRNA-LNPs Used For?

Preventive mRNA Vaccines

mRNA-based therapeutics provide significant advantages, including rapid optimization of specific antigen sequences, the ability to encode multiple proteins and/or subunits, and adjustable immunogenicity. When combined with the cell-specific delivery and immunogenic modulation of LNPs, this approach has demonstrated substantial potential in preventing infectious diseases. During the COVID-19 pandemic, Pfizer-BioNTech's BNT162b2 (Comirnaty) and Moderna's mRNA-1273 (Spikevax) progressed from virus sequencing to FDA emergency use authorization in approximately 11 months, showcasing the rapid translational capability of mRNA-LNP vaccines. Additionally, self-amplifying vaccines, which encode RNA-dependent RNA polymerase to enable RNA amplification, are under development. These vaccines can increase antigen protein expression with a single low-dose injection. Since mRNA vaccines do not produce infectious particles, they avoid the risk of pathogenic transformation seen with live-attenuated and replication-deficient viral vaccines. However, their larger mRNA molecular weight poses delivery challenges.

Cancer mRNA Vaccines

In cancer applications, overcoming the immune suppression of the tumor microenvironment remains a major challenge. This suppression inhibits T-cell infiltration into tumors and leads to T-cell exhaustion. Current research aims to activate cytotoxic T lymphocytes while targeting immune suppression in the tumor microenvironment. One approach involves using LNPs to deliver mRNA encoding tumor antigens or functional proteins such as cytokines and immune checkpoint inhibitors to reshape the tumor microenvironment or enhance immune surveillance. For instance, mRNA-2416 encodes the immune checkpoint regulator OX40L, which promotes T-cell proliferation and provides co-stimulatory survival signals. Another strategy combines mRNA vaccines with therapies designed to counteract the tumor microenvironment. For example, Moderna and Merck Sharp & Dohme developed RNA-4157, which contains 34 neoantigens. Combined with pembrolizumab, it extended recurrence-free survival in high-risk melanoma patients compared to pembrolizumab alone, with controllable safety profiles. In addition, mRNA-LNP systems are advancing the in vivo production of CAR-T cells (chimeric antigen receptor T cells) for cancer therapy. Compared to traditional viral delivery methods for CAR-T preparation, LNPs offer superior payload capacity, safety for repeated dosing, and effective delivery to specific cells.

mRNA-Encoded Protein Therapeutics

The application of mRNA-LNPs has also sparked interest in protein-based and immunomodulatory protein therapies, such as those using antibodies or cytokines. These applications demand higher protein expression levels and, in some cases, lifelong treatment, necessitating reduced immunogenicity. The biocompatibility and biodegradability of LNPs contribute to their lower immunogenicity, and their formulation can be adjusted to suit different biological contexts. For example, MRT5005, an inhalable mRNA therapy for cystic fibrosis (granted FDA approval for clinical development), uses LNPs and demonstrated safety over five consecutive weeks of administration, with no worsening of adverse effects over time. However, certain proteins require additional post-translational modifications to achieve full functionality, which mRNA sequences alone may not provide. Targeted delivery of mRNA to specific organs or cell types remains another challenge. LNP formulations can be optimized through tailored shell components, proportional adjustments, and administration methods to achieve organ- or cell-specific delivery. A collaboration between Neurimmune and Ethris on an inhalable mRNA-encoded monoclonal antibody for COVID-19 demonstrates the cost-effectiveness of mRNA synthesis and the enhanced therapeutic efficacy enabled by LNP inhalation delivery.

In Conclusion

In summary, mRNA-LNPs as therapeutics offer several advantages. They are highly flexible and cost-effective, with both the encapsulated mRNA and the LNP shell capable of being rapidly synthesized and scaled up chemically in vitro. Their in vivo residence time can be controlled through adjustments to the LNP shell or the mRNA structure, enabling biodegradation to be tailored to different biological scenarios. Immunogenicity is adjustable, a feature particularly significant in vaccines for infectious diseases. It is observed that not only do the proteins expressed by mRNA trigger immune responses, but the structure of mRNA itself and various LNP formulations can also induce varying degrees of innate immune reactions. Additionally, LNPs allow for targeted delivery to specific organs and tissues, which can be enhanced through optimization strategies and tailored administration methods.

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Reference

1. Swetha, K. et al. Recent Advances in the Lipid Nanoparticle-Mediated Delivery of mRNA Vaccines. Vaccines (Basel). 2023, 11(3): 658.