Lipid nanoparticles (LNPs) are the leading platform for mRNA delivery, with their in vivo performance governed by lipid composition and colloidal stability. While anionic helper lipids can bias LNP expression toward the spleen, weak RNA-lipid interactions during purification often induce nanoparticle rearrangement and reduced activity. These stability limitations effectively narrow the accessible formulation design and screening space, leaving large regions of anionic compositional space underexplored. Here, we extend our cleavable crosslinking strategy to stabilize anionic LNPs without replacing the primary lipid constituents of the parent LNP formulation. By tuning the lengths of the cholesterol-derived acid-cleavable crosslinker and PEG-diamine, we achieved balanced structural stability. The optimized crosslinked formulation exhibited a significant increase in splenic mRNA expression at 12 h compared to the uncrosslinked LNPs. Notably, 33.2% of CD45+ tdTomato+ cells in the spleen were identified as T cells. Mechanistic analyses suggest that controlled mRNA release and altered intracellular processing contribute to the improved transfection efficiency. Together, these findings define a tunable crosslinking window that expands the accessible design landscape for tissue- and cell-specific mRNA delivery.
Lipid nanoparticles (LNPs) have represented a leading platform for mRNA delivery and therapeutics. While extensive studies have focused on optimizing ionizable lipids, the specific roles of helper lipids such as cholesterol, phospholipid, and polyethylene glycol lipid (PEG-lipid) remain relatively poorly understood. To elucidate the distinct contributions of individual lipid components, we reengineer LNP formulations by selectively removing each lipid component, especially helper lipids, to systematically analyze their effects on physicochemical properties, in vivo mRNA delivery, and inflammatory responses. Results reveal that ionizable lipid plays a critical role in mRNA delivery efficacy. Cholesterol is essential for efficient liver-targeted delivery but dispensable for extrahepatic delivery. Phospholipid is not directly associated with organ tropism, whereas phospholipid-free LNPs significantly alleviate inflammation. PEG-lipid influences particle size, with PEG-lipid-free LNPs exhibiting preferential spleen tropism. This study comprehensively demonstrates the important roles of each lipid component in determining the in vivo fate of LNPs, guiding the rational design of next-generation LNP-based mRNA delivery systems.
Kexin Su, Zichuan Wang, Lu Shi et al.· ACS Nano· 0 citations
ABSTRACT Despite the growing importance of lipid nanoparticles (LNPs) in mRNA therapeutics, current ionizable lipids, which comprise an ionizable head group, a linker, and hydrophobic tails that collectively govern the delivery behavior, exhibit dose‐limiting toxicity and suboptimal efficiency. Hydrophobic tail chemistry has been implicated in these limitations; however, a systematic structure–activity relationship (SAR) analysis is lacking. In this study, the authors synthesized a 56‐member library of tail‐modified ionizable lipids with biodegradable disulfide linkages and variations in chain length and branching. Systematic SAR evaluation revealed that hydrophobic tail architecture critically determines endosomal escape, biocompatibility, and mRNA translation efficiency. Optimized LNPs delivered mRNA with potent efficacy and markedly reduced cytotoxicity, lower cytokine induction, and balanced Th1/Th2 immune activation in a nonhuman primate model. The findings establish the first comprehensive SAR framework linking tail chemistry to ionizable lipid function, offering molecular design principles for next‐generation LNPs and enabling the development of safer and more effective mRNA‐based vaccines and therapeutics.
Seo-Hyeon Bae, Jisun Lee, J. Ahn et al.· Advancement of science· 0 citations
Lipid nanoparticles (LNPs) are effective carriers for messenger ribonucleic acid (mRNA) delivery in vaccines; however, their reliance on extreme cold-chain storage limits global manufacturing and distribution. Conventional LNPs are formed by rapidly mixing four lipids with mRNA through electrostatic interactions between cationic ionizable lipids and negatively charged nucleic acids, facilitating nucleation and precipitation of mRNA-loaded LNPs. However, this binding also accelerates mRNA degradation, requiring stringent cold storage which limits widespread vaccine deployment. To overcome this limitation, we introduce a post-loading strategy in which empty LNPs (eLNPs) are first fabricated and RNA is subsequently loaded at a later stage. Using scalable confined impinging jet (CIJ) mixers, we optimized pH, buffer composition, lipid concentration, and ethanol content to produce colloidally stable eLNPs. Controlled adjustment of ethanol content and pH enabled efficient incorporation of four distinct RNA payloads while maintaining loaded LNP diameters below 100 nm. Post-loaded LNPs demonstrated mRNA delivery efficiencies in HeLa cells comparable to those of conventionally co-precipitated LNPs. Consistent size distributions and zeta potentials further confirmed comparable surface properties. Structural characterization by x-ray and neutron scattering revealed similar internal architectures for post-loaded and co-precipitated LNPs without compromising RNA loading efficiency. Together, these results demonstrate equivalent cellular delivery performance between the two formulations. This post-loading approach enables decentralized assembly of mRNA LNPs at the point of administration, with both eLNPs and mRNA stored under mild refrigeration, thereby improving vaccine accessibility. Moreover, eLNPs function as modular laboratory reagents, facilitating the translation of mRNA research toward clinical applications.
N. Bizmark, David F. Amelemah, Satya K. Nayagam et al.· bioRxiv· 0 citations
This Perspective revisits cationic polymer-lipid nanoparticles not as historical precursors to ionizable lipid LNPs, but as a macromolecular materials space that can clarify and extend current RNA delivery design.
Intramuscular mRNA lipid nanoparticles (LNPs) often exhibit undesirable liver accumulation, compromising safety and efficacy. While optimization efforts focus on ionizable and helper lipids, the role of trace PEG-lipids remains underexplored. To address this, we constructed a 45-member PEG-lipid library via Ugi/Passerini reactions, keeping the PEG chain constant at 2 kDa while systematically diversifying the hydrophobic tail and linker structures. High-throughput screening identified Pr-182-BNT (P1B LNP) as a lead candidate. P1B LNPs enable highly efficient and selective mRNA delivery to skeletal muscle while drastically minimizing hepatic off-targeting. This precise tropism originates from enhanced muscle cell uptake and optimized membrane interactions, driven by the lipid's unique branched, asymmetric tail. In Ai9 reporter mice, P1B LNPs drive potent muscle-specific gene editing and reduce off-target recombination. As an RSV mRNA vaccine, they elicit robust antigen-specific IgG titers and expands polyfunctional CD8+IFN-γ+ T cells, indicating a Th1-skewed response. By re-engineering only the trace PEG-lipid, this work overcomes a key targeting limitation of classical LNPs and establishes a translatable platform for safer, more effective mRNA vaccines and therapeutics.
mRNA-based nucleic acid therapeutics have gained significant attention and undergone extensive clinical investigation since the FDA's approval of Spikevax and Comirnaty for the treatment of SARS-CoV-2. Lipid nanoparticles (LNPs) are currently the most advanced delivery system for delivering mRNA in vivo. However, concerns persist regarding the tolerability of their repeated use due to lipid accumulation. Over time, lipids have been optimized through the introduction of enzymatically degradable ester linkers. Yet, this modification alone does not fully address lipid clearance from the body. To mitigate these concerns, we have designed a library of novel ionizable lipids that incorporates two nitrogen atoms within the lipid backbone. We hypothesize that the LNPs with these lipids can be potentially administered at lower dosages while maintaining the same payload delivery and translation efficiency. In this study, we identified five lead LNPs that exhibited remarkable transfection efficiency without cytotoxic effects in vitro. We further optimized the LNP formulations by incorporating various cholesterol analogs to fine-tune translation performance. Upon analyzing the endocytic and intracellular pathways of the lead LNPs, we found that multiple pathways were involved in delivering the payload to the cell. This set of novel lipids holds great promise for mRNA delivery and warrants further investigation.
Vishnu Sriram, V. Phatangare, Joo-Youp Lee· Biomaterials Science· 0 citations
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