Lipid nanoparticles (LNPs) have emerged as a critical delivery platform for mRNA vaccines, particularly following their successful deployment in COVID-19 vaccines that generated over $50 billion in sales and vaccinated more than two billion people by the end of 2021. However, recent research reveals that these delivery systems possess complex immunological properties that can produce both beneficial therapeutic effects and concerning adverse reactions.
LNP-Induced Immune Activation Mechanisms
LNPs consist of four key lipid components: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. The ionizable lipid component, featuring a tertiary amine structure, enables RNA encapsulation and facilitates cytoplasmic transport. Research demonstrates that mRNA-LNPs activate multiple pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), melanoma differentiation-associated protein 5 (MDA5), and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3).
Studies using RNA-LPX (mRNA vaccine-encapsulated liposomes) revealed that IL-1β secretion requires both NLRP3 inflammasome activation and TLR7/8 signaling. When human monocytes were treated with the NLRP3 inhibitor MCC950, IL-1β secretion significantly declined, confirming the necessity of caspase activity for cytokine production. The research also showed that ionizable lipid composition affects immunogenicity, with SM-102 LNPs producing significantly higher IL-1β secretion compared to MC3 LNPs.
MDA5 Pathway Critical for CD8+ T-Cell Response
Investigation of the Pfizer-BioNTech COVID-19 vaccine BNT162b2 using knockout mouse models revealed that MDA5 sensing plays a crucial role in spike-specific CD8+ T-cell responses. While knockout mice lacking TLR3, TLR7, TLR2, TLR4, TLR5, ASC, NLRP3, cGAS, or STING showed no reduction in neutralizing antibodies or T-cell responses, MDA5-deficient mice demonstrated significantly reduced antigen-specific CD8+ T cells and decreased blood IFN-α levels.
The study found that serum IFN-γ levels increased six hours after the second Pfizer vaccine dose, and interferon-stimulating gene (ISG) expression was diminished when IFN-γ receptors were blocked, confirming the importance of type 1 interferon signaling in vaccine-induced immunity.
PEG-Related Adverse Reactions
A significant concern with LNP-based vaccines involves PEG lipids, which can trigger two distinct types of adverse reactions. The first is IgE-mediated anaphylaxis, occurring within 30 minutes to four hours of vaccination and presenting symptoms including urticaria, angioedema, and potentially life-threatening reactions. Research indicates that 72% of the population possesses preexisting IgG and IgM antibodies against PEG due to exposure through everyday products like toothpaste and cosmetics.
The second type is complement activation-related pseudoallergy (CARPA), primarily caused by anti-PEG IgM antibodies. These antibodies bind to PEGylated liposomes, activating the classical complement pathway and producing anaphylatoxins C3a, C4a, and C5a. This process leads to the accelerated blood clearance (ABC) phenomenon, where LNPs are rapidly removed from circulation by Kupffer cells.
Strategies for Controlling LNP Immunogenicity
Size and Charge Modification
Research by Harashima's team demonstrated that LNP size significantly affects lymph node targeting. LNPs with 30 nm diameter showed superior lymph node accumulation compared to 100 nm and 200 nm particles. Surface charge also influences targeting, with neutrally charged particles effectively reaching draining lymph nodes and T-cell zones.
PEG Lipid Optimization
To address PEG-related adverse effects, researchers are exploring several approaches. Shortening the hydrophobic alkyl chain allows easier PEG lipid separation from LNP surfaces. Studies comparing DMG-PEG (14 carbon chains) with DSG-PEG (18 carbon chains) showed that longer chains produced higher anti-PEG IgM levels and increased complement activation.
Alternative polymers are being investigated as PEG replacements. Polysarcosine (PSar) demonstrated longer circulation times with reduced immunogenicity compared to PEGylated liposomes, preventing the ABC phenomenon while showing lower IgM and IgG antibody levels.
Adjuvant Integration
Researchers are developing ionizable lipids that function as built-in adjuvants. Libraries of ionizable lipid-like molecules enable targeted immune activation while limiting systemic cytokine expression. STING agonist-derived amino lipids (SALs) have shown particular promise, with SAL12-LNPs generating stronger neutralizing antibodies than standard COVID-19 vaccine formulations.
Administration Route Optimization
Different injection routes produce varying immune responses. Intravenous administration generates robust CD8+ T-cell responses suitable for cancer immunotherapy by mobilizing splenic antigen-presenting cell pools. Intramuscular injection, used in COVID-19 vaccines, provides balanced immune activation with manageable side effects. Subcutaneous and intradermal routes offer alternative approaches with distinct immunological profiles.
Studies comparing administration routes found that intravenous delivery of mRNA-lipoplexes produced superior T-cell responses compared to subcutaneous or intradermal routes. However, the optimal route depends on the therapeutic application and desired immune outcome.
Clinical Implications and Future Directions
The complex immunological properties of LNPs present both opportunities and challenges for therapeutic development. While immune activation can enhance vaccine efficacy and provide adjuvant effects, excessive or inappropriate immune responses may cause adverse reactions or autoimmune complications.
Current research focuses on developing next-generation LNPs with improved safety profiles through rational design of lipid components, surface modifications, and targeted delivery strategies. The goal is to harness beneficial immune activation while minimizing adverse effects, enabling broader therapeutic applications beyond vaccines to include cancer immunotherapy, gene therapy, and treatment of genetic disorders.
Understanding and controlling LNP immunogenicity represents a critical advancement for the expanding field of nucleic acid therapeutics, with ongoing clinical trials suggesting continued growth in RNA-LNP-based medicines through 2036.
