Next-Generation mRNA-LNPs: Strategies to Reduce Anti-PEG Antibody Responses

Lipid nanoparticles (LNPs) have recently emerged as one of the most advanced technologies for the delivery of vaccines and gene therapies. A typical LNP includes an ionizable or cationic lipid, cholesterol, at least one phospholipid, and a polyethylene glycol (PEG) modified lipid.

PEG lipids induce a "stealth effect" on LNPs by hindering protein adsorption and opsonization. This enhances LNP circulation stability, reduces macrophage phagocytosis, and prolongs blood residency. However, PEG also introduces several challenges, particularly its potential to limit intracellular delivery and elevate immunogenicity. Emerging strategies are now offering potential ways to overcome these limitations.

Role and Limitations of PEG Lipids in LNPs

PEG lipids consist of a hydrophobic lipid anchor and a hydrophilic PEG chain that extends into the surrounding aqueous environment through a chemical bond. PEG is a flexible polymer made of repeating ethylene oxide units with modifiable end groups. By forming hydrogen bonds with water molecules, PEG creates a hydration layer on the particle surface and generates steric hindrance. Together, these effects give LNPs their “stealth” properties, helping them evade immune detection and prolong circulation time after initial dosing.

However, PEGylation also introduces challenges. The dense PEG coating can act as a physical barrier, reducing interactions between the nanoparticle and biological membranes. This can limit cellular uptake and impair the release of the payload into the cytoplasm. This trade-off between prolonged circulation and reduced intracellular delivery is often referred to as the “PEG dilemma.”

In addition, PEG immunogenicity is an increasing concern. Early animal studies revealed accelerated blood clearance, where an initial dose of PEGylated nanoparticles can trigger faster elimination of subsequent doses. This effect is linked to the formation of anti-PEG antibodies, which have also been detected in humans. These antibodies can reduce therapeutic efficacy and pose potential safety risks.

Strategies to Overcome Anti-PEG Antibodies

next-generation-mRNA-LNPs

Figure 1. Structures of a typical PEGylated mRNA-LNP and next-generation mRNA-LNPs designed to minimize anti-PEG antibody responses. Source: references [1]

Optimizing Route of Administration and Dosage

Route of administration and dosing frequency impact anti-PEG antibody generation risk. Intravenous injection at low doses can trigger a strong immune response, whereas intramuscular injection can create a drug depot, making it more suitable for repeated dosing. Particle size and PEG chain length also influence immunogenicity: higher molecular weight PEGs (>2000 Da) and certain particle sizes are more likely to induce antibody production.

Competition with high-molecular-mass PEG

Co-administration of high–molecular weight PEG (≥30 kDa) can transiently occupy B-cell receptors and block the binding of anti-PEG antibodies, thereby reducing clearance and hypersensitivity reactions. However, it is important to note that high doses of PEG may lead to organ accumulation or complement activation.

PEG Structural Engineering Strategies

PEG structural engineering offers an effective way to address immune challenges driven by anti-PEG antibodies. Three main optimization strategies have been proposed:

● Replacing linear PEG with branched or Y-shaped PEG can significantly reduce anti-PEG antibody production.
● Combining PEG chains of different molecular weights (for example, carboxy-PEG2000 with methoxy-PEG550) to build a dense brush-like layer that lowers immune recognition.
● Designing cleavable PEG (acid- or enzyme-responsive) to maintain circulation time while improving therapeutic efficacy.

Immunomodulation strategies

Immunomodulation approaches include using tolerogenic nanoparticles to suppress the formation of new antibodies, targeting complement pathways to reduce nanoparticle clearance, and applying CD22-modified nanoparticles to selectively deplete B cells that produce anti-PEG antibodies. Hydroxyl-terminated PEG (HO-PEG) lipids, which exhibit lower immunogenicity, are a key focus in LNP development, and their potential has been validated across multiple preclinical and clinical formulations from Moderna.

Preclinical case study: Moderna’s mRNA therapies

Moderna recently reported in Nature Communications on three mRNA therapies targeting inherited metabolic disorders (mRNA-3927, mRNA-3705, and mRNA-3210). All three use the same LNP formulation, built from the cationic lipid SM-86 and the HO-PEG lipid OL-56. In mouse models, these LNPs showed favorable pharmacokinetic and pharmacodynamic profiles, including increased expression of the target proteins and reduced levels of disease-related metabolites. Based on PK/PD modeling, the projected first-in-human doses are well below the corresponding NOAEL values, indicating a promising safety margin. [4]

PEG Replacement Strategies

Endosomal Escape and PCB Lipid Strategy

A major hurdle for intracellular delivery is overcoming endosomal entrapment. Conventional PEG-LNPs can interfere with close membrane contact and limit escape from endosomes. To address this, a team at Cornell University developed poly(carboxybetaine) (PCB) lipids as PEG substitutes. PCB is zwitterionic, defined as having both positive and negative charges while retaining a net neutral charge. This design offers two major advantages: first, the PCB headgroup can engage in electrostatic and dipole–dipole interactions with the endosomal membrane, strengthening LNP–membrane association and enhancing endosomal escape; second, the highly hydrophilic microenvironment around the PCB headgroup can help orient polar groups of the LNP toward the membrane interface, increasing the likelihood of membrane fusion. In addition, PCB coatings exhibit extremely low protein adsorption, which is critical for prolonging circulation time, reducing immunogenicity, and mitigating the accelerated blood clearance effect often observed with PEG-containing LNPs, thereby enabling repeated dosing without loss of efficacy. [5]

In vitro studies with immortalized and primary cells show that PCB-containing LNPs consistently achieve higher mRNA transfection efficiency than PEG-containing LNPs across multiple formulations. Primary cell engineering and in vivo immunization studies in mice further demonstrate superior therapeutic efficacy and a favorable immunotoxicity profile for PCB-LNPs. Overall, replacing PEG-lipids with zwitterionic PCB-lipids provides a promising strategy to build mRNA delivery platforms that combine potent endosomal escape, safety, and compatibility with repeated administration, supporting their potential for clinical translation.

Brush-like Polymer Optimization Strategy

Daniel J. Siegwart and colleagues at the University of Texas Southwestern Medical Center engineered brush-shaped polymer–lipid (BPL) conjugates as substitutes for conventional PEG-lipids, aiming to reduce anti-PEG antibody binding to improve protein production consistency in repeated dosing. [6]

Structurally, BPLs adopt a brush-like architecture in which multiple ethylene glycol side chains branch from a single polymer backbone, in contrast to the linear configuration of standard PEG-lipids. At appropriate surface densities on LNPs, these BPLs can adopt a “mushroom regime” conformation: the short backbones splay laterally, creating a dense steric barrier that effectively limits the approach and binding of anti-PEG antibodies. By tuning key parameters—including side-chain length, degree of polymerization, alkyl anchor length, and surface regimes—BPL-containing LNPs can finely modulate anti-PEG antibody binding affinity and control blood-circulation pharmacokinetics.The optimized BPL formulations retain the pharmacokinetic advantages of PEG while reducing the risk of immune-mediated clearance, making them a promising option for repeated dosing in clinical applications.

Together, these two studies offer distinct yet complementary strategies to overcome the limitations of PEGylation. However, it remains unclear whether these materials will introduce new effects outside controlled preclinical settings. Further testing in more clinically relevant models, along with long-term safety studies, is needed to fully assess the therapeutic potential of PEG alternatives and their feasibility in future clinical formulations.

Biopharma PEG is a dynamic science company dedicated to PEG derivatives. We are capable of supplying small to large quantities of a rich selection of PEG derivatives with GMP & non-GMP standards for your LNP-mRNA R&D. We can produce and provide Cholesterol (Plant-Derived), DSPE, classic PEG lipids, such as mPEG-DMG, and mPEG-DSPE for your LNPs R&D.

References:
[1] Xiong, S., & Liu, C. (2025). Breaking the PEG barrier to boost mRNA-LNP therapeutics. Nature Reviews Materials, 10(11), 799-800. https://doi.org/10.1038/s41578-025-00852-9
[2] Gao W, Zhang L. Making way for PEG alternatives. Nat Mater. 2025 Nov;24(11):1682-1683. doi: 10.1038/s41563-025-02381-w. PMID: 41094068.
[3] Chen, Y., Su, Y., & Roffler, S. R. (2025). Polyethylene glycol immunogenicity in nanomedicine. Nature Reviews Bioengineering, 3(9), 742-760. https://doi.org/10.1038/s44222-025-00321-6
[4] Baek R, Coughlan K, Jiang L, Liang M, Ci L, Singh H, Zhang H, Kaushal N, Rajlic IL, Van L, Dimen R, Cavedon A, Yin L, Rice L, Frassetto A, Guey L, Finn P, Martini PGV. Characterizing the mechanism of action for mRNA therapeutics for the treatment of propionic acidemia, methylmalonic acidemia, and phenylketonuria. Nat Commun. 2024 May 7;15(1):3804. doi: 10.1038/s41467-024-47460-9. PMID: 38714648; PMCID: PMC11076592.
[5] Luozhong, S., Liu, P., Li, R. et al. Poly(carboxybetaine) lipids enhance mRNA therapeutics efficacy and reduce their immunogenicity. Nat. Mater. (2025). https://doi.org/10.1038/s41563-025-02240-8
[6] Xiao, Y., Lian, X., Sun, Y. et al. High-density brush-shaped polymer lipids reduce anti-PEG antibody binding for repeated administration of mRNA therapeutics. Nat. Mater. (2025). https://doi.org/10.1038/s41563-024-02116-3