A hybrid polymeric system for pulmonary mRNA delivery: Advancing mucosal vaccine development

The bigger picture

Respiratory viruses, such as coronaviruses, influenza, and respiratory syncytial virus (RSV), continue to challenge global health, highlighting the need for vaccines that can elicit mucosal immunity directly in the airways. Pulmonary mRNA vaccination offers this promise, yet its development has been constrained by the difficulty of transporting mRNA across airway mucus and enabling efficient transfection in lung antigen-presenting cells through clinically relevant aerosol delivery.

In this study, we present a hybrid polymeric delivery system that leverages the complementary properties of poly(lactic-co-glycolic) acid (PLGA) and poly(β-amino esters) (PBAEs) to address these challenges. Designed to act through a coordinated sequence of mRNA protection, intracellular transport, and timely cytosolic release, the hybrid nanoparticles enable efficient mRNA transfection in antigen-presenting cells and support productive immune activation. Importantly, the hybrid nanoparticles can penetrate airway mucus, function in physiologically relevant human lung models, and withstand the mechanical stresses of vibrating-mesh nebulization, features essential for translating pulmonary mRNA delivery toward practical use. Such hybrid systems may help accelerate the development of next-generation inhalable vaccines and broaden the therapeutic reach of mRNA technologies.

Introduction

Messenger RNA (mRNA)-based vaccines have demonstrated rapid development and high efficacy in combating the COVID-19 pandemic, establishing themselves as a leading strategy for addressing future viral outbreaks.1,2 Their design flexibility allows for quick adaptation to emerging variants or novel pathogens through updates to the encoded antigen sequences. However, many viruses, including SARS-CoV-2, primarily infect hosts via the respiratory tract.3 Conventional mRNA vaccines administered via intramuscular injection, a non-respiratory route, have been reported to elicit suboptimal mucosal immunity compared with natural infection, potentially limiting viral clearance at the initial entry site and leaving individuals susceptible to acute infection.4,5,6,7 In this context, pulmonary mRNA vaccines hold great promise, as they can elicit both strong mucosal and systemic immune responses, providing direct protection in the respiratory tract.8,9

Achieving this goal relies on an optimized vehicle, as not only is single-stranded mRNA highly susceptible to RNase degradation and requires protection by suitable carriers,10 but physiological barriers in pulmonary delivery, such as mucus and tight junctions between respiratory epithelial cells, also need to be overcome before transfection can occur.11,12 Although lipid nanoparticles (LNPs) have been transformative for intramuscular mRNA vaccines (Comirnaty, Spikevax, and mRESVIA), their performance is often constrained when shifted to local pulmonary administration.1 Lipid-based carriers encounter marked difficulties in penetrating airway mucus due to strong interactions with its periodic hydrophobic domains13,14,15 and may provoke inflammatory responses in the respiratory tract,16 thereby limiting their effectiveness for pulmonary delivery. Increasing PEG-lipid density can improve mucus permeability and attenuate inflammation, but typically at the cost of transfection efficiency.14,15 In parallel, anti-PEG antibodies have been increasingly reported, particularly in individuals who have received repeated mRNA-LNP vaccinations, raising concerns about the long-term feasibility of PEGylated systems.17,18 To address these challenges, poly(β-amino esters) (PBAEs) have emerged as a promising alternative for effective mRNA delivery. Featuring biodegradable ester bonds and tunable backbones and monomers, PBAEs ensure both safe and efficient transfection in a streamlined, PEG-free manner.19 Notably, previous studies by Patel et al. and Rotolo et al. have demonstrated that PBAEs enable efficient mRNA transfection in the lungs following local inhalation, underscoring their suitability for pulmonary applications.19,20 Consistent with these reports, our in-house synthesized PBAEs used here have previously achieved efficient pulmonary small interfering RNA (siRNA) delivery,21 supporting their capacity to overcome airway barriers encountered post-administration.

For mRNA vaccines, transfecting antigen-presenting cells (APCs), particularly dendritic cells (DCs), is crucial for immune activation, as they play a key role in capturing, processing, and presenting antigens to activate T cells for efficient adaptive immune responses.2,22 However, APCs are intrinsically more difficult to transfect than non-APCs due to harsher endosomal/lysosomal processing that rapidly degrades internalized cargo before translation can occur.23,24 Consistent with this, we observed significantly lower transfection performance in APCs than in non-APCs when using our in-house synthesized PBAE polymers. This places higher demands on the chemical design of PBAEs for improving transfection in APCs, and the synthesis, along with the downstream screening work, becomes rather complicated when navigating the vast library of potential backbones and monomers.25,26 To address this challenge, we found that simple integration of poly(lactic-co-glycolic acid) (PLGA), a widely used biodegradable polymer in Food and Drug Administration (FDA)-approved drugs,27 into our formulations markedly improved mRNA delivery to APCs. Although previous studies have shown that integrating PLGA with protonable polymers, such as polyethyleneimine (PEI)28 and poly-L-lysine (PLL)29 can enhance nucleic acid delivery, the underlying mechanisms have remained unclear. Here, to our knowledge, we provide the first integrated and visual mechanistic framework showing how PLGA coordinates mRNA protection, endosomal escape, and controlled cytosolic release in APCs, thereby overcoming barriers that limit PBAE performance.

The aim of this study was to develop an optimal carrier system for efficient mRNA transfection and activation of immune cells upon pulmonary administration. To achieve this, we engineered a hybrid PLGA/PBAE system. Mechanistically, PLGA hydrolysis during early endocytosis promotes tighter mRNA condensation, protecting the cargo from endosomal nucleases. In parallel, the generation of lactic and glycolic acids increases intraluminal buffering and strengthens a proton-sponge-like effect, facilitating endosomal escape. Once in the cytosol, where the pH is increased, electrostatic interactions weaken, and mRNA is more readily released from the carrier. Together, this cascade yields superior APC transfection with mRNA-loaded PLGA/PBAE nanoparticles compared with mRNA/PBAE polyplexes. We then evaluated the immunological consequences of delivery. The PLGA/PBAE system facilitated antigen presentation and maturation of bone marrow-derived dendritic cells (BMDCs), prompting further investigation into its immune activation potential using an OT-1 mouse model. Additionally, we assessed mucus penetration in an air-liquid interface (ALI) airway epithelium model and transfection efficiency in ex vivo human precision-cut lung slices (hPCLSs). Importantly, after nebulization with an Aerogen Pro device, the PLGA/PBAE formulation preserved more of its pre-nebulized transfection activity than the SM102-LNP control, underscoring its enhanced tolerance to aerosolization stress. Collectively, these findings highlight the potential of PLGA/PBAE nanocarriers for enhanced pulmonary mRNA vaccine delivery.

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Materials

Resomer RG 502 H, poly(D, L-lactide-co-glycolide), RNase A, Cell Counting Kit-8, nystatin, chlorpromazine hydrochloride, and dextran sulfate sodium salt from Leuconostoc spp. were purchased from Sigma-Aldrich (Merck, Darmstadt, Germany). Diethylpyrocarbonate (DEPC)-treated water and Roti@GelStain were bought from CalRoth (Karlsruhe, Germany). EGFP mRNA, Fluc mRNA, and Alexa Fluor 647-labeled EGFP mRNA were obtained from RiboPro (Oss, Netherlands). OVA mRNA was purchased from GenScript (Piscataway, NJ, USA). SARS-CoV-2 spike protein mRNA was provided by Daiichi Sankyo Europe (Munich, Germany). Invitrogen SYBR Gold Nucleic Acid Gel Stain (10,000× concentrate in DMSO), 2× RNA loading dye, Lipofectamine 2000, LysoTracker Green DND-26, and mouse GM-CSF recombinant protein, PeproTech were bought from Thermo Fisher (Waltham, MA, USA). Aminoallyl-UTP-Cy3 and aminoallyl-UTP-Cy5 were purchased from Jena Bioscience (Dortmund, Germany). HiScribe T7 ARCA mRNA Kit (E2060S) was purchased from New England Biolabs (Ipswich, MA, USA). Zombie Violet Fixable Viability Kit, monensin solution (1,000×), APC anti-mouse I-Ab antibody, fluorescein isothiocyanate (FITC) anti-mouse CD40 antibody, FITC anti-mouse CD80 antibody, APC anti-mouse CD86 antibody, PE anti-mouse CD11c antibody, Brilliant Violet 605 anti-mouse CD11c antibody, PE anti-mouse H-2Kb bound to SIINFEKL antibody, APC anti-mouse IFN-γ antibody, ELISA MAX Standard Set Mouse IFN-γ kit, and CFSE Cell Division Tracker Kit were obtained from Bioligand (San Diego, CA, USA). FITC anti-mouse CD8 antibody and CD8a+ T cell isolation kits (mouse) were purchased from Miltenyi (Bergisch Gladbach, Germany).

Min Jiang, Felix Sieber-Schäfer, Simone P. Carneiro, Dana Matzek, Anny Nguyen, Diana Leidy Porras-Gonzalez, Arun Kumar Verma, Miriam Kolog-Gulko, David C. Jürgens, Gerald Burgstaller, Bastian Popper, Xun Sun, Olivia M. Merkel, A hybrid polymeric system for pulmonary mRNA delivery: Advancing mucosal vaccine development, Cell Biomaterials, 2026, 100311, ISSN 3050-5623, https://doi.org/10.1016/j.celbio.2025.100311.

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