Immunogenicity of Lipid Nanoparticles in RNA Therapeutics

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Lipid Nanoparticles in RNA Therapeutics

Compared to other biological agents and small molecule drugs, RNA therapy holds significant advantages, including higher safety, lower cost, and extensive target flexibility, rapidly driving advancements in the nucleic acid field. These advancements owe much to breakthroughs in drug delivery, with lipid nanoparticles (LNPs) standing out as one of the most advanced systems in clinical practice. Currently validated LNP formulations in large populations comprise four types of lipids: ionizable cationic lipids, phospholipids, cholesterol or cholesterol derivatives, and polyethylene glycol (PEG)-lipids. Each lipid plays a crucial role in enabling the normal functionality of RNA-LNP therapeutics. Research also suggests that LNP formulations may induce pro-inflammatory immune responses, potentially affecting therapeutic outcomes. Understanding these immune reactions and the mechanisms behind other potential immune responses to LNP formulations, such as anti-inflammatory or tolerogenic responses, is crucial for the development of LNP-based RNA therapies, such as mRNA-LNP therapeutics.

Simplistic illustration of LNP and its individual components.Simplistic illustration of LNP and its individual components. (Albertsen, C.H.; et al, 2022)

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The Role of Lipid Components in LNPs

Ionizable cationic lipids form complexes with negatively charged RNA molecules, enhancing the biocompatibility of LNPs due to their pH sensitivity. Under normal physiological pH conditions, ionizable cationic lipids are uncharged, reducing their interaction with the anionic membranes of non-target cells. Once LNPs enter the endosome, ionizable cationic lipids undergo protonation, destabilizing the endosomal membrane and promoting RNA molecule escape. Phospholipids play a role in stabilizing the LNP structure during particle formation and may also contribute to endosomal escape. Cholesterol or its derivatives stabilize particles by modulating membrane integrity and rigidity, influencing delivery efficiency and particle biodistribution. PEG-lipids extend the circulation time of LNPs, providing steric hindrance, reducing their size, and preventing aggregation. Ionizable cationic lipids are crucial components of LNPs, while phospholipids and cholesterol and its derivatives are referred to as auxiliary lipids. Auxiliary lipids stabilize lipid nanoparticles, enhance transfection efficiency, and prolong particle circulation time. However, despite the widespread use of LNPs in large populations, the interaction of auxiliary lipids with the human immune system remains largely understudied.

Mechanisms of Immune Response Triggered by Lipid Components in LNPs

Cationic Lipids

The cationic lipids in lipid nanoparticles (LNPs) play a crucial and diverse role in immune responses, involving multiple levels of regulation and influence. Research indicates that using empty LNPs prepared with specific cationic lipids as vaccine adjuvants can effectively induce humoral responses, thereby activating follicular helper T cell (Tfh) responses. The initiation of this response primarily occurs through the release of pro-inflammatory cytokines such as IL-1β and IL-6, as well as the activation of cellular pathways including STING and TLR-4. Meanwhile, cationic lipids also play a crucial role in modulating local immune responses, such as triggering robust inflammatory responses following intradermal injection. Additionally, studies have found that cationic lipids in LNPs exhibit superior performance compared to other types of vaccine adjuvants, thereby providing broader prospects for their application in immunotherapy. A deeper understanding of the mechanisms by which cationic lipids trigger immune responses in LNPs will provide valuable guidance for designing more effective vaccine and immunotherapy strategies in the future, thereby driving progress and development in the field of medicine.


Phospholipids, also known as glycerophospholipids, are essential components of lipid nanoparticles (LNPs). They can be categorized into five classes based on their polar head groups: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS). Among these, dipalmitoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE) are the most commonly utilized phospholipids in LNP formulations. In mammalian cells, PC and PE are the predominant constituents of phospholipids, constituting approximately 40-50% and 25% of total phospholipids, respectively. To maintain cellular homeostasis, phospholipids undergo hydrolysis mediated by phospholipases, yielding various metabolites including oxidized phospholipids and lyso-phospholipids, which possess immunomodulatory properties. For instance, oxidized phospholipids can act as "damage-associated molecular patterns" (DAMPs), regulating inflammatory responses. Furthermore, these metabolites can modulate cellular processes such as phagocytosis, inflammasome activation, and dendritic cell migration by regulating cytokine secretion. Phosphatidylserine (PS) also exhibits immunomodulatory effects, serving as an "eat me" signal on apoptotic cell surfaces, facilitating their engulfment by phagocytes. Additionally, viruses and other pathogens can mimic apoptotic cells by presenting PS on their surfaces, promoting cellular entry and immune evasion.

Studies have shown that LNP formulations containing PS (PS-LNPs) can enhance transfection efficiency and augment protein expression of delivered RNA. Following injection into mice, PS-LNPs achieve effective protein expression in lymph nodes and spleen, with greater distribution observed in cervical lymph nodes, targeting facilitated by monocytes and macrophages. Moreover, PS forms complexes with recombinant factor VIII (FVIII), reducing antibody levels in hemophilia mice. Nanoparticles containing PS or lyso-PS can convert immunogens into tolerogens by inducing tolerogenic dendritic cell generation and promoting regulatory T cell expansion. Thus, phospholipids exhibit immunomodulatory effects in LNP formulations, offering potential value for their application in immunotherapy.

Cholesterol and Its Derivatives

Cholesterol and its derivatives serve as important components in lipid nanoparticle (LNP) formulations, with recent studies indicating their significant role in modulating immune cell activity, including activation, differentiation, and function. Accumulation of intracellular cholesterol can activate inflammasomes, while oxidized forms of cholesterol play crucial regulatory roles in immune responses. However, there is currently limited research on the immune responses triggered by cholesterol in LNP compositions, with most physiological effects studied in the context of diseases like atherosclerosis and cardiovascular diseases.

PEG Lipids

Polyethylene glycol (PEG) lipids are widely utilized in lipid nanoparticles (LNPs) and other nanocarriers to enhance their stability and plasma half-life. However, both pre-existing and newly generated PEG antibodies can potentially induce hypersensitivity reactions and accelerate blood clearance by binding to the surface of nanoparticles, leading to activation of the complement system.

Outlook on the Immunogenicity Research of Lipid Nanoparticles

Currently, each component of clinically approved LNP formulations may contribute to overall immunogenicity. However, the detailed mechanisms and interactions behind this remain largely unexplored, particularly regarding the specific lipid structures (such as the number and length of lipid tails, and the relationship between linear headgroups and heterocycles) and their overall impact on the immunogenicity of LNP formulations. Repeated studies on the mechanisms by which each lipid component triggers immune responses are necessary to continually improve the safety of mRNA-LNP therapy and better optimize the formulation of LNP delivery systems for the screening of next-generation agents.


  1. Albertsen, C.H.; et al. The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv Drug Deliv Rev. 2022, 188: 114416.

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