A hybrid polymeric system for pulmonary mRNA delivery: Advancing mucosal vaccine development
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
TL;DR
A new hybrid polymer system improves mRNA vaccine delivery to the lungs by overcoming mucus barriers and enhancing immune activation.
Contribution
A hybrid PLGA/PBAE system coordinates endosomal escape and mRNA release for efficient pulmonary mRNA delivery.
Findings
The PLGA/PBAE system enables mucus penetration and effective mRNA transfection in airway models.
The system retains transfection efficiency after nebulization and promotes immune activation in dendritic cells.
Abstract
Effective pulmonary messenger RNA (mRNA) vaccination requires delivery systems capable of overcoming the airway barrier and efficiently transfecting pulmonary antigen-presenting cells. Here, we developed a hybrid polymeric system incorporating poly(lactic-co-glycolic) acid (PLGA) and poly(β-amino esters) (PBAEs) to enhance pulmonary mRNA delivery. The components acted through a spatiotemporally coordinated cascade: early PLGA hydrolysis acidified endosomes, boosting PBAE protonation and tightening mRNA condensation for protection; increased buffering, driven by accelerated protonation, strengthened proton-sponge-mediated escape; and weakened electrostatic interactions in the cytosol enabled rapid mRNA release and translation in dendritic cells, supporting immune activation. These findings highlight the need to balance endosomal escape with timely mRNA release for functional expression.…
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TopicsRNA Interference and Gene Delivery · Advanced Drug Delivery Systems · Immunotherapy and Immune Responses
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.
Results and discussion
Characterization of mRNA-loaded nanoparticles
PBAEs have gained increasing attention for nucleic acid delivery due to their tunable structure, biodegradability, and high transfection efficiency.30 Based on our previous findings, amphiphilic PBAEs using bisphenol A glycerolate as the polymeric backbone and polycationic spermine (SP) and lipophilic tetradecylamine (TDA) side chains have demonstrated effectiveness in RNA delivery.21 The synthesis of PBAEs followed our previously established protocol, with detailed structural information provided in Figure S1. The ratios of SP and TDA in our synthetic polymers varied from 40% to 60% and were all included in this study. Through electrostatic interactions with the phosphate backbone, PBAEs enriched with amino groups effectively condensed mRNA to form polyplexes. However, the prepared polyplexes displayed poor dispersity, as evidenced by polydispersity index (PDI) values consistently exceeding 0.3 (Figure 1A) and the presence of multiple peaks in the particle size and concentration distribution plot (Figure 1B). To overcome this limitation, PLGA was incorporated into the delivery system. Hybrid PLGA/PBAE nanoparticles were prepared using a double-emulsion solvent evaporation method, yielding more homogeneous particles with PDIs lower than 0.1 (Figure 1A), and only a single narrow peak was eventually observed from the results of nanoparticle tracking analysis (NTA) (Figure 1B). The hydrodynamic diameters of mRNA-loaded PLGA/PBAE nanoparticles, as measured by dynamic laser scattering (DLS), ranged from 215 to 230 nm, which were approximately 2-fold larger than those of the polyplexes. However, their mode sizes measured by NTA were all lower than 135 nm, which can be attributed to the reduced contribution of large particles to overall scattering and the lesser impact of the hydration layer in this method.31 Cryo-transmission electron microscopy (TEM) images of mRNA-loaded PLGA/PBAE(SP0.5/TDA0.5) also demonstrated well-dispersed nanoparticles, but with even smaller geometric sizes in the range of 65–80 nm (Figure 1C). Similar variations in PLGA particle diameters, as determined by different techniques, have been reported in previous studies.32^,^33 Regardless of the technique used, the measured particle size ranged from 50 to 250 nm, which is considered rather immunogenic due to its resemblance in size to most viruses in the real world.34^,^35 Notably, compared with the cryo-TEM image of PBAE polyplexes (Figure S2), we observed a distinct core-shell structure in Figure 1C, which is consistent with the typical morphological features of PLGA nanoparticles. This feature may facilitate an optimal distribution of amphiphilic PBAEs, with the lipophilic TDA moiety embedded in the hydrophobic core, whereas the cationic SP extends toward the shell. The solid structure of PLGA also ensured the formation of stable nanoparticles.Figure 1. Characterization of PBAE/mRNA polyplexes and mRNA-loaded PLGA/PBAE nanoparticles(A) Hydrodynamic diameter and polydispersity index (PDI) of nanoparticles measured by dynamic light scattering (DLS), represented as bars and individual dots, respectively.(B) Particle size distribution and concentration measured by nanoparticle tracking analysis (NTA).(C) Cryo-TEM image of mRNA-loaded PLGA/PBAE(SP0.5/TDA0.5) nanoparticles. Scale bar, 50 nm.(D) Zeta potential at different pH values determined by phase analysis light scanning.(E) mRNA release from different formulations at pH 5.4 and pH 7.4 in the presence of serially diluted Triton X and heparin, analyzed using the SYBR Gold assay. EC_50_ values were obtained through non-linear fitting analysis of the released mRNA to added interfering substances at pH 7.4. Data are presented as mean ± SD, n = 3.
As the weight ratio of polymer to RNA was maintained at 60:1 across all formulations, PBAEs with a higher ratio of SP resulted in an increased zeta potential due to more protonable amines. Compared with PBAE/mRNA polyplexes, the incorporation of PLGA led to a significant decrease in zeta potential (Figure 1D). Particularly in pH-neutral HEPES buffer, all mRNA-loaded PLGA/PBAE formulations exhibited zeta potentials lower than 6 mV. Importantly, mRNA encapsulation was not affected by this electropositivity shielding and remained above 95% for all tested formulations, as determined by SYBR Gold assay and agarose gel electrophoresis (Figure S3). To assess mRNA integrity under enzymatic stress, we analyzed formulations prepared with PBAE(SP0.5/TDA0.5) and observed efficient protection from RNase degradation relative to naked mRNA processed identically (Figure S4). However, due to electropositivity shielding by the integration of PLGA, mRNA release from PLGA/PBAE nanoparticles under Triton X-100 interference and heparin competition was accelerated compared with polyplexes formulated with the corresponding amount of PBAEs (Figure 1E). This phenomenon was not obvious in acidic HEPES buffer (pH 5.4) due to stronger electrostatic interactions mediated by more protonated amines, which is an advantage for nanoparticles trapped in the acidic endosome, because the mRNA was tightly encapsulated and therefore, protected. However, in pH 7.4 HEPES buffer, we noticed visible mRNA release in two PLGA/PBAE formulations already after adding 0.01% heparin and Triton X. The lower the SP ratio in the polymer used, the faster the mRNA release observed. After performing non-linear fitting of released mRNA to the added interferents, EC_50_ values indicated that achieving the same level of mRNA release required higher concentrations of Triton X and heparin as the SP ratio of the polymers increased, whether in PBAE/mRNA polyplexes or mRNA-loaded PLGA/PBAE nanoparticles. This suggested that mRNA binding strength was primarily driven by electrostatic interactions from protonated SP amines. Reducing the SP ratio in the polymers used lowered cationic charge density (fewer protonable amines), which led to accelerated mRNA release. Although the hydrophobic monomer TDA increased as SP decreased, hydrophobic interactions contributed minimally to mRNA condensation compared with electrostatics. Moreover, the higher EC_50_ values corresponded to slower mRNA release in the tested polyplexes, which was consistent with their stronger electropositivity compared with PLGA/PBAE nanoparticles, as measured by zeta potential analysis.
Formulation screening based on mRNA transfection in different cell types
Successful transfection of mRNA into cells, particularly APCs, is a critical prerequisite for the functionality of mRNA vaccines.22 To efficiently screen formulations composed of PBAEs with varying ratios of SP and TDA, we directly assessed their mRNA transfection across multiple cell types, including DC2.4 cells, THP-1 cells, and 16HBE14o- cells. DC2.4, a murine DC line, and THP-1, a human leukemia monocytic cell line, are both classified as professional APCs and can present antigenic signals to activate T cells following mRNA transfection, thereby eliciting immune responses. 16HBE14o- cells, a human bronchial epithelial cell line, represent the first contact with the formulation upon pulmonary delivery, which is also one of the interests in this study. Notably, transfection of non-APCs can also contribute to humoral immune responses by facilitating antigen production and secretion. Extracellularly secreted soluble antigens can be captured by professional APCs and presented via major histocompatibility complex class II (MHC class II) molecules to helper T cells, which are essential for humoral immunity. Additionally, these antigens can be directly recognized by B cells through their B cell receptors, thereby enhancing antibody production.1
To investigate the versatility of carrier systems in delivering mRNA of varying lengths, we included both enhanced green fluorescent protein (EGFP) mRNA (908 nt) and firefly luciferase (Fluc) mRNA (1,816 nt) in this assessment. Among the tested formulations, PLGA/PBAE(SP0.5/TDA0.5) consistently demonstrated efficient transfection of both EGFP mRNA (Figures 2A, 2B, and S5) and Fluc mRNA (Figure 2C) across all three cell types. The underlying mechanism behind this observation remains complex; however, based on the obtained results, we propose that appropriate mRNA release plays a crucial role. As previously discovered in Figure 1E, PBAE/mRNA polyplexes demonstrated slower mRNA release compared with hybrid PLGA/PBAE formulations, and in most cases, exhibited poor transfection efficiencies. However, an exception to the generally better transfection efficacy of PLGA/PBAE formulations was observed when DC2.4 and 16HBE14o- cells were transfected with polyplexes formulated with PBAE(SP0.4/TDA0.6). The low GFP (Figures 2A and 2B) and Fluc (Figure 2C) expression after transfection with PLGA/PBAE(SP0.4/TDA0.6) could be attributed to the particularly rapid mRNA release from this formulation (Figure 1E), which, to some extent, reflects its instability and may adversely affect transfection efficiency. In contrast, PLGA/PBAE(SP0.6/TDA0.4) upon addition of equal amounts of interferents (Triton X and heparin) showed more restricted release of mRNA (Figure 1E), which may prevent efficient delivery of mRNA into the cytoplasm and hinder mRNA translation (Figures 2B and 2C). Overall, a balanced SP-to-TDA ratio (50:50) in PBAE polymers ensured formulation stability, whereas PLGA integration facilitated a suitable mRNA release profile conducive to efficient transfection and protein expression. Given its versatility and high efficiency in delivering different mRNAs across various cell types, the PLGA/PBAE(SP0.5/TDA0.5) formulation was ultimately selected for further testing.Figure 2. Formulation screening via mRNA transfection in different cell lines(A) Fluorescent images of DC2.4 and 16HBE14o- cells transfected with EGFP mRNA-loaded formulations. Scale bar, 150 μm(B) Histogram of EGFP fluorescence intensity in transfected DC2.4, 16HBE14o-, and THP-1 cells.(C) Luciferase activity of DC2.4, 16HBE14o-, and THP-1 cells after transfection with Fluc mRNA-loaded formulations, represented as relative light unit (RLU) per mg of protein. Data are presented as mean ± SD, n = 3. ^✽✽✽^p < 0.001, ^✽✽✽✽^p < 0.0001; ns, not significant; one-way ANOVA.
In vitro performance in DCs and assessment of the impact of cell type
Based on our in vitro transfection findings (Figure 2), the PLGA/PBAE(SP0.5/TDA0.5) formulation emerged as a promising candidate for mRNA transfection. To better understand the factors contributing to its effectiveness, particularly in the most important APCs, DCs, we further investigated its performance in DC2.4 cells in comparison with PBAE(SP0.5/TDA0.5)/mRNA polyplexes. From this point forward, to streamline notation in data figures, PBAE and PLGA/PBAE will refer specifically to mRNA/PBAE(SP0.5/TDA0.5) polyplexes and mRNA-loaded PLGA/PBAE(SP0.5/TDA0.5) nanoparticles, respectively.
To ensure tolerability, the concentrations of mRNA-loaded formulations added to the medium did not exceed 1 μg/mL of mRNA, according to our cytotoxicity assay (Figure S7A). Due to stronger electropositivity, mRNA/PBAE polyplexes mediated higher internalization in DC2.4 cells (Figure S8A), and only half of the uptake was inhibited by 4°C incubation (Figure S8B). In contrast, mRNA-loaded PLGA/PBAE nanoparticles demonstrated lower uptake and were internalized by the same pathways, including scavenger receptor-mediated36 and clathrin-mediated endocytosis,37 which were mainly energy dependent. Notably, dextran sulfate, a specific inhibitor of scavenger receptor-mediated endocytosis, significantly reduced the cellular uptake of PLGA/PBAE nanoparticles to merely 5% of the original level and further affected mRNA transfection in DC2.4 cells (Figure S8C). However, regardless of whether endocytosis occurs via scavenger receptors or clathrin-mediated pathways, internalized formulations are initially enclosed within endocytic vesicles, where the pH gradually decreases, leading to their transformation into early endosomes.38 If the formulations fail to escape within a short period, the progressively acidifying environment during endosomal maturation would subject them to harsh degradation by enhanced enzymatic activity. As shown in the confocal laser scanning microscopy (CLSM) images, an orange signal, resulting from the overlap of red fluorescence (labeled mRNA) and green fluorescence (LysoTracker), was observed in most of the DC2.4 cells after 2 h of treatment with both formulations (Figure 3A). This indicated a successful internalization but confirmed that mRNA was mainly trapped in the endosomes within the first 2 h. After 8 h, we noticed a significant separation of red and green fluorescence in DC2.4 cells treated with PLGA/PBAE nanoparticles, and the corresponding Pearson correlation coefficient (PCC) value dropped from 0.7 (2 h) to 0.2222 (8 h). The PCC value, a commonly used indicator in CLSM image analysis to assess the degree of colocalization between two variables, ranges from −1 to 1.39 When the PCC value is close to 1, it indicates a high degree of spatial colocalization of the two fluorescence signals, whereas minimal or no overlap is reflected as the value is close to 0 or negative. In contrast to PLGA/PBAE nanoparticles, the overlapped orange signal remained visible in DC2.4 cells treated with mRNA/PBAE polyplexes, suggesting a sustained colocalization of the labeled mRNA and endosomes, which was further supported by the corresponding PCC value, which only slightly decreased from 0.5543 (2 h) to 0.4577 (8 h). Interestingly, stronger LysoTracker Green signals (green fluorescence) were observed in PLGA/PBAE-treated cells at both time points, despite identical acquisition settings to controls. Considering LysoTracker Green is an acidotropic probe whose signal primarily reflects the presence of acidic compartments rather than precise luminal pH, we employed LysoSensor Green, a pH-dependent probe with greater sensitivity to acidity changes. The obtained readouts (Figure 3B) showed more pronounced differences in green fluorescence between PBAE and PLGA/PBAE formulations, consistent with the LysoTracker results. This effect can be attributed to the hydrolysis of PLGA,40 whereby lactic acid and glycolic acid generated in endosomes enhanced LysoSensor fluorescence.Figure 3In vitro performances in DC2.4 cells(A) Confocal laser scanning microscopy (CLSM) images of intracellular tracking in DC2.4 cells after 2 and 8 h incubation with PBAE or PLGA/PBAE formulations. DAPI stains nuclei (blue), LysoTracker Green labels endosomes (green), and Cy5-labeled mRNA is shown in red. Scale bar, 15 μm.(B) CLSM images of DC 2.4 cells treated for 2 and 8 h with PBAE or PLGA/PBAE formulations. LysoSensor Green is shown in green and nuclei in blue. Scale bar, 20 μm.(C) Fluorescence resonance energy transfer (FRET) assessment after loading Cy3/Cy5 dual-labeled mRNA into nanoparticles, including initial spectra and FRET ratios (Ex 500 nm, Em 570 and 67 nm). Data are presented as mean ± SD, n = 3.(D) Intracellular real-time FRET ratio monitoring in DC2.4 cells following treatment.
The increased endosomal acidity was expected to enhance protonation of amino groups within the SP moiety of the co-loaded PBAE polymers, thereby tightening electrostatic condensation of mRNA. As shown in Figures 3C and 3D, the fluorescence resonance energy transfer (FRET) ratios of PBAE and PLGA/PBAE formulations were comparable after encapsulation of Cy3/Cy5 dual-labeled mRNA in both cell-free and cellular settings. However, upon incubation with DC2.4 cells for 1–4 h, the FRET ratio increased in the PLGA/PBAE group and remained higher than that of PBAE polyplexes, indicating stronger mRNA condensation and, accordingly, better protection from endosomal nucleases. In parallel, enhanced protonation of the SP moiety increased intraluminal buffering and mediated a proton-sponge-like effect,41^,^42 together with lipophilic TDA, which also facilitated membrane disruption, ultimately promoting endosomal escape (as previously observed in DC2.4 cells). Subsequently, the FRET ratio decreased more rapidly at later time points for PLGA/PBAE, indicating mRNA decondensation and release upon entry into the higher-pH cytosol, where electrostatic interactions were weakened. The released mRNA was then available for translation, whereas a larger fraction of carrier-bound mRNA in PBAE polyplexes was less likely to function in time.
Overall, these findings indicated that PLGA integration provided benefits beyond improved dispersibility and biocompatibility. PLGA and PBAE polymers acted cooperatively in a spatiotemporally synergistic manner. Early after uptake, PLGA hydrolysis increased endosomal acidity, which enhanced PBAE protonation to tighten mRNA condensation, thereby protecting the cargo from nuclease degradation. As protonation accelerated, the buffering capacity of the PBAE drove continued water influx, leading to elevated membrane tension, which facilitated endosomal disruption and escape. After escape, exposure to the higher-pH cytosol weakened electrostatic interactions and facilitated mRNA release from the carrier, which was contrary to the conventional view of PLGA as a slow-released matrix, ultimately yielding more effective mRNA transfection.
Although the above mechanistic assays were performed in DC2.4 cells, we additionally evaluated endosomal escape in two other cell models, namely HeLa-Gal8-mRuby3 and Calu-3, to assess the potential impact of cell type. In HeLa-Gal8-mRuby3 cells, PBAE polyplexes showed significantly higher cellular uptake than PLGA/PBAE; however, both formulations induced comparable levels of Gal8 recruitment, indicating similar extents of endosomal or lysosomal disruption (Figure S9). In Calu-3 epithelial cells, no pronounced differences were detected between the two formulations with respect to endosomal escape (Figure S10), and their mRNA transfection efficiencies were comparable (Figure S6). This lack of advantage in non-APCs underscored that the benefits of PLGA incorporation were tightly linked to APC-specific uptake and intracellular trafficking rather than imparting a universal increase across all cell types.
Antigen presentation and BMDC maturation after transfection with mRNA vaccines
To achieve efficient immune activation, DCs, the unique APCs capable of activating naive T cells,43 should be efficiently transfected and then present antigenic signals to T cells. Additionally, DCs need to provide the second co-stimulatory signal, such as B7 and CD40 molecules, to fully activate T cells. In this context, both transfection and maturation of DCs are equally important. BMDCs are commonly used for this evaluation because of their immature status and similarity to in vivo conditions.44^,^45 In our study, BMDCs were generated by stimulating bone marrow cells isolated from C57BL/6 mice with granulocyte-macrophage colony-stimulating factor (GM-CSF), following well-established protocols (Figure 4A).46Figure 4Ex vivo performances in BMDCs(A) Schematic illustration of the BMDC culture process, in which bone marrow cells isolated from C57BL/6J mice are stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF).(B) Fluc mRNA transfection efficiency in BMDCs after 24 and 48 h of incubation.(C) MHC class I antigen presentation in BMDCs post-transfection with formulations containing OVA mRNA.(D) Surface expression levels of MHC class II, co-stimulatory molecules CD40, CD80, and CD86 in BMDCs post-transfection with formulations containing OVA mRNA for 24 h. Data are presented as mean ± SD, n = 3. All significance indicators represent comparisons with the mRNA group at 24 h post-treatment. ^✽^p < 0.05, ^✽✽^p < 0.01, ^✽✽✽✽^p < 0.0001; one-way ANOVA.
First, as shown in Figure S11, both PBAE polyplexes and PLGA/PBAE nanoparticles were efficiently internalized by BMDCs. We later used Fluc mRNA to assess transfection in BMDCs. As shown in Figure 4B, after 24 h of incubation, the relative light units (RLUs) per mg of protein reached 120,000 with PBAE and 380,000 with PLGA/PBAE, corresponding to 3- and 10-fold increase over free mRNA, respectively. Overall, mRNA transfection was more challenging in primary DCs compared with DC2.4 cells (Figure 2C), in line with prior studies.44 Although Lipofectamine 2000 outperformed the polymeric formulations in this assay, its well-documented cytotoxicity, also evident in our study (Figure S7B), limited its translational utility. Therefore, Lipofectamine 2000 was included here as a positive control rather than a practical delivery option. To link expression with antigen presentation, we next delivered mRNA encoding the model antigen OVA and quantified H-2Kb/SIINFEKL complexes on BMDCs. As a result, we found that mRNA transfection and MHC class I antigen presentation showed a positive association. After 24 h, 6.3% of BMDCs in the free mRNA group were detected H-2Kb/SIINFEKL-positive, increasing to an average of 8.9% with PBAE and 10.8% with PLGA/PBAE, the latter being comparable to Lipofectamine 2000 (averaged 11.2%) (Figure 4C). This result is comparable to that reported by Huang et al., where the PBS control group showed 8.73% positivity and the approved Spikevax mRNA-LNP reached 18.5%.45 By 48 h, both luciferase expression and H-2Kb/SIINFEKL levels declined across all formulations, consistent with prior reports in which expression peaks around 24 h and decreases thereafter.47 From a delivery standpoint, this early 24-h peak followed by a decline remains compatible with pulmonary administration, where airway-resident APCs and local T cells can engage antigen rapidly at the deposition site.
In parallel, we assessed MHC class II presentation after 24 h of OVA mRNA transfection. In contrast to the MHC class I trend, Lipofectamine 2000 (averaged 8.1%) showed levels similar to free mRNA, whereas PBAE exhibited the highest MHC class II-positive population (averaged 15.9%), and PLGA/PBAE (averaged 14.9%) was slightly lower yet remained significantly above free mRNA (Figure 4D). In addition to MHC class II expression, which correlates with BMDC maturation status,46 we quantified the precentages of CD40^+^ and CD80^+^CD86^+^ cell populations (gating strategy in Figure S12) and observed the same trend. A plausible explanation is that our in-house synthesized PBAE, as a cationic polymer, potentially provided an adjuvant-like effect that promoted DC maturation, consistent with frequent reports for cationic materials.48 In addition, the lipophilic moiety TDA might further enhance membrane interactions contributing to this response. Upon PLGA integration, partial charge neutralization and improved biocompatibility appeared to attenuate the maturation signals, resulting in frequencies slightly lower than PBAE but still clearly above free mRNA. Although a definitive mechanism remains uncertain, the low maturation readout observed with Lipofectamine 2000 is concordant with published data on Lipofectamine MessengerMAX in monocytes, which reported restricted CD80 upregulation under similar conditions.49
Coculture of transfected BMDCs and CD8+ T cells harvested from OT-1 mice
Motivated by the above findings, we further investigated whether mRNA-transfected BMDCs could activate T cells. For this purpose, we used C57BL/6-Tg (TcraTcrb)1100Mjb/J (OT-1) mice, which carry transgenic inserts for the mouse Tcra-V2 and Tcrb-V5 genes. This genetic engineering enables CD8^+^ T cells from OT-1 mice to specifically recognize OVA residues 257–264 (SIINFEKL) when presented in the context of H-2Kb.50 Therefore, upon successful transfection of BMDCs with mRNA encoding for OVA, the BMDCs can present the H-2Kb/SIINFEKL complex on their surface. Once CD8^+^ T cells from OT-1 mice interact with transfected BMDCs, they are antigen-specifically activated, as evidenced by proliferation and cytokine secretion (Figure 5A).Figure 5. Coculture of transfected BMDCs and CD8^+^ T cells from OT-1 mice(A) Isolation of CD8^+^ T cells from OT-1 mouse splenocytes using magnetic-activated cell sorting (MACS).(B) Percentages of CD8^+^ T cells before and after isolation.(C) Percentages of IFN-γ-secreting CD8^+^ T cells after 6 h of coculture with transfected BMDCs.(D) IFN-γ concentrations in the cell culture medium after 3 days of co-culture.(E) CD8^+^ T cell proliferation assessed using carboxyfluorescein succinimidyl ester (CFSE) staining, expressed as the percentage of divided cell subsets.(F) Schematic diagram illustrating antigen-specific recognition between DCs and CD8^+^ T cells from OT-1 mice.(G) Percentages of IFN-γ-secreting CD8^+^ T cells.(H) IFN-γ concentrations in the cell culture medium.(I) CD8^+^ T cell proliferation after coculture with BMDCs pretreated with empty PLGA/PBAE nanoparticles, nanoparticles loaded with OVA mRNA, or SARS-CoV-2 spike protein mRNA. Data are presented as mean ± SD, n = 3. ^✽^p < 0.05, ^✽✽^p < 0.01, ^✽✽✽^p < 0.001, ^✽✽✽✽^p < 0.0001; ns, not significant; one-way ANOVA.
To begin, CD8^+^ T cells were isolated from OT-1 mouse splenocytes using magnetic-activated cell sorting (MACS) via negative selection. This purification process increased the percentage of CD8^+^ T cells from 15.8% in the initial samples to 70.7% in the isolated ones (Figure 5B). Transfected BMDCs were then cocultured with the isolated CD8^+^ T cells for 6 h, and intracellular interferon (IFN)-γ staining was performed to assess T cell activation. In both blank and free mRNA-treated samples, IFN-γ^+^ CD8^+^ T cells remained at low levels (<0.08%), whereas the PBAE and PLGA/PBAE groups showed significant increases to 0.14% and 0.65%, respectively (Figure 5C). These findings were further supported by IFN-γ concentrations measured in the culture medium after 3 days of incubation. The highest IFN-γ was observed in the Lipofectamine 2000-treated group (2,946 pg/mL), followed by the PLGA/PBAE (458.9 pg/mL) and PBAE (95.6 pg/mL) groups (Figure 5D). Additionally, CD8^+^ T cell proliferation was assessed using carboxyfluorescein succinimidyl ester (CFSE) staining, where a reduction of CFSE fluorescence intensity and divided peaks in histogram representation from flow cytometry indicated cell division. As shown in Figure 5E, the CFSE signal in the free mRNA-treated group appeared as a single, strong peak on the right side of the fluorescence spectrum. In contrast, the PBAE group displayed multiple peaks on the left, accounting for 40.7% of the fluorescence distribution. The PLGA/PBAE and Lipofectamine 2000-treated groups demonstrated even higher proliferation, reaching 74.4% and 89.1%, respectively, which were comparable with the results reported by Zeng et al., who studied mRNA-LNPs for vaccination.51 Taken together, these data indicate that the extent of mRNA transfection and antigen-specific MHC class I presentation is the primary influencer of CD8^+^ T cell activation under our conditions. Co-stimulatory signals remain important, but their influence appeared to be secondary to this readout.
To confirm the antigen-specificity of the experiment, we replaced OVA mRNA with SARS-CoV-2 spike protein mRNA (Figure 5F). PLGA/PBAE loaded with OVA mRNA successfully induced a high level of IFN-γ secretion, whereas spike protein mRNA loading resulted in minimal secretion, comparable to the blank control (Figures 5G and 5H). Similarly, CFSE staining revealed that CD8^+^ T cell division was only 0.98% in this irrelevant antigen group (Figure 5I). As an additional control, empty nanoparticles without mRNA loading were tested and confirmed to have no impact on antigen-specific immune activation in this OT-1 mouse model (Figure 5I). These findings confirmed that PLGA/PBAE nanocarriers effectively delivered mRNA into DCs, enhanced antigen presentation, and promoted T cell activation in an antigen-specific manner, highlighting their potential as a promising delivery platform for mRNA vaccine development.
mRNA uptake and transfection in ALI-cultured cells
For pulmonary vaccines, the mucus layer on the respiratory tract surface serves as a significant biological barrier, limiting the deep permeability of formulations and preventing direct contact with the tracheal epithelium.12 The ALI culture model, in which the apical side of airway epithelial cells, for example, Calu-3 cells, a lung epithelial cell line, is exposed to air while the basolateral side remains submerged in medium, allows for the differentiation of epithelial cells into a pseudostratified epithelium (Figure 6A).52 This pseudostratified epithelium derived from Calu-3 cells closely mimics in vivo mucosal epithelium, including key features such as mucus production and tight junction formation. Therefore, we used ALI-cultured Calu-3 cells to evaluate the ability of mRNA-loaded nanoparticles to penetrate the mucus barrier. As shown in Figure 6B, CLSM images labeled mucus (green), cell nuclei (blue), and mRNA (red). The widespread green signal overlaying the blue one confirmed successful mucus production on the surface of the cells. For cells treated with free mRNA, the red signal representing labeled mRNA was nearly undetectable. In contrast, red fluorescence was clearly visible beneath the mucus in PBAE-treated cells, overlapping with the nuclei (Figures 6B and S13). Correspondingly, transepithelial electrical resistance (TEER) measurements showed a significant decrease from 663 to 247 Ω∗cm^2^ in PBAE-treated cells after 24 h of incubation (Figure 6C), confirming that the polyplexes altered tight junction integrity after mucus permeabilization. In the PLGA/PBAE group, the red signal was less obvious compared with polyplexes treatment but remained significantly higher than in Lipofectamine 2000-treated cells. This observation aligns with previous findings that lipid-based systems face more challenges in penetrating the mucus barrier.53 However, higher mRNA uptake in ALI-cultured Calu-3 cells did not correlate directly with stronger mRNA transfection efficiency in our study. In contrast, luciferase expression was detected at the highest in the PLGA/PBAE-treated cells, exhibiting a 7-fold increase compared with free mRNA, and significantly higher than both PBAE and Lipofectamine 2000 (Figure 6D). Unlike the previous results observed in submerged cells, the underperformance of Lipofectamine 2000 in the ALI-cultured model confirmed that the mucus barrier adversely affected cellular uptake and subsequent transfection efficiency.Figure 6mRNA uptake and transfection in ALI culture of Calu-3 cells and in hPCLSs(A) Schematic diagram of ALI culture of Calu-3 cells used to evaluate mucus penetration and mRNA transfection efficiency.(B) Mucus penetration of different formulations in ALI-cultured Calu-3 cells after 24 h of transfection. AF647-labeled mRNA is shown in red, nuclei in blue, and mucus layer in green. Scale bar, 100 and 50 μm.(C) TEER measurements of Calu-3 cells before and after transfection with different formulations.(D) Luciferase activity in ALI-cultured Calu-3 cells transfected with different formulations containing Fluc mRNA.(E) Schematic diagram of the preparation process for hPCLSs.(F) CLSM image of hPCLS treated with PLGA/PBAE(SP0.5/TDA0.5) nanoparticles loaded with AF647-labeled mRNA. Nuclei are shown in blue and labeled mRNA in red. Scale bar, 75 μm.(G) Luciferase activity in cell lysates of transfected hPCLS. Data are presented as mean ± SD, n = 3. ^✽^p < 0.05, ^✽✽^p < 0.01, ^✽✽✽^p < 0.001, ^✽✽✽✽^p < 0.0001; one-way ANOVA.
mRNA uptake and transfection in hPCLS
To further assess whether mRNA transfection remains effective in deeper lung regions, we used human precision-cut lung slices (hPCLSs), which preserve native human lung architecture, including airways, alveoli, and blood vessels, in a natural 3D organization. hPCLS also retain multiple lung-resident cell types, such as type I and II alveolar cells, bronchial epithelial cells, endothelial cells, and immune cells, providing a physiologically relevant model for investigating biological responses.54^,^55 The preparation of hPCLS is illustrated in Figure 6E.
After incubating mRNA-loaded PLGA/PBAE nanoparticles with hPCLS for 24 h, CLSM imaging revealed red fluorescence (mRNA) widely distributed throughout the bifurcated lung structure and overlapping with blue (cell nuclei), suggesting efficient cellular uptake (Figure 6F). Subsequent quantification of luciferase activity confirmed that PLGA/PBAE nanoparticles achieved significantly higher transfection efficiency than free mRNA and PBAE polyplexes, reaching an average of RLU 5392 (Figure 6G). In this assay, Lipofectamine 2000 again outperformed due to the absence of the mucus barrier in the agarose-filled lung slices. However, its translational application is limited by its higher toxicity, which has been widely reported56 and was also observed in our cytotoxicity assay (Figure S4).
Nebulization
To explore practical dosing strategies for pulmonary delivery, we aerosolized liquid formulations using a vibrating-mesh nebulizer (Aerogen Pro), which generates fine droplets by driving the liquid through a perforated membrane, enabling efficient lung deposition.57 Laser diffraction showed that, after nebulization of PLGA/PBAE nanoparticles, 50% of droplets fell within 1–4.46 ± 0.08 μm, and nearly 60% were <5 μm, a respirable range suitable for airway deposition (Figure 7A). Importantly, PLGA/PBAE particle size remained stable post-nebulization, whereas SM102-LNP formulated at the component ratios of mRNA-1273 (Moderna) exhibited an average size increase of 78.17 ± 2.89 nm (Figure 7B). Consistent with these trends, nebulized SM102-LNP exhibited a 26.4% decrease in encapsulation efficiency and 44.1% mRNA loss relative to non-nebulized LNP (Figures 7C and 7D), compared with a 36.2% loss for nebulized PLGA/PBAE. Although SYBR Gold and RiboGreen assays both indicated mRNA loss after nebulization, corresponding changes were not readily apparent in the Bioanalyzer traces (Figure 7E). The decrease in mRNA content was reflected functionally by reduced transfection following nebulization, whereas PLGA/PBAE retained significantly higher activity than SM102-LNP under identical conditions (Figure 7F). Together, these results indicated that PLGA/PBAE is more compatible with standard vibrating-mesh nebulization and maintains favorable aerosol, physicochemical, and biological properties for pulmonary delivery. It is worth noting that the formulations nebulized here were unoptimized. In theory, the performance could be improved by choosing alternative nebulizers, adjusting operation parameters, and supplementing excipients to better preserve mRNA and nanoparticle stability during and after aerosolization. These strategies will be investigated in future work.Figure 7. Nebulization of PLGA/PBAE and SM102-LNP using an Aerogen Pro vibrating-mesh nebulizer(A) Laser diffraction analysis of post-nebulization droplet sizes from the PLGA/PBAE formulation.(B) Hydrodynamic diameters of PLGA/PBAE and SM-102 LNP pre- and post-nebulization.(C) Encapsulation efficiency of PLGA/PBAE and SM-102 LNP pre- and post-nebulization.(D) mRNA loss in the samples after nebulization.(E) Bioanalyzer assay showing mRNA integrity in pre- and post-nebulized samples.(F) DC2.4 transfection with EGFP mRNA using pre- and post-nebulized formulations. Data are presented as mean ± SD, n = 3. ^✽✽✽^p < 0.001, Student’s t test.
Methods
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-A^b^ 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).
Synthesis of PBAE
PBAEs polymers were synthesized in our lab according to a previously described protocol.21 In brief, Tri-Boc-SP (0.5 equiv), TDA (0.5 equiv), and bisphenol A diglycidyl ether diacrylate (1.2 equiv) were mixed and stirred at 90°C for 48 h. The obtained product was dissolved in a mixture of trifluoroacetic acid (TFA) and dichloromethane (DCM) (TFA:DCM 1:20 v/v) and stirred at room temperature for 2 h to cleave the Boc groups. The deprotected polymer was purified by precipitation in diethyl ether three times.
Preparation of hybrid PLGA/PBAE nanoparticles
The hybrid PLGA/PBAE nanoparticles were prepared using a double-emulsion solvent evaporation method. In brief, PLGA dissolved in DCM and PBAE dissolved in DEPC-treated water were mixed in equal amounts and emulsified using the MS72 probe of an ultrasonic homogenizer (BANDELIN electronic, Berlin, Germany). Next, the same volume of a 0.5% (w/v) polyvinyl alcohol (PVA) aqueous solution was added to the first emulsion and ultrasonicated to prepare the double emulsion. The obtained emulsion was slowly added dropwise into DEPC-treated water and stirred at 1,200 rpm for 4 h to allow the DCM to evaporate and the particles to harden. The formulations were then ultrafiltrated using a 15 mL Amicon Ultra Centrifugal Filter (molecular weight cut off 30 kDa, Millipore) for 50 min at 500 × g in a Megafuge 16 centrifuge (Thermo Fisher Scientific, Darmstadt, Germany) to obtain a final formulation containing PBAE polymers at a concentration of 1 mg/mL.
Preparation of mRNA-loaded formulations
To prepare mRNA-loaded formulations, the concentrated hybrid PLGA/PBAE nanoparticles and PBAE polymers (1 mg/mL in RNase-free distilled water) were individually mixed with the desired amount of mRNA (diluted in 10 mM HEPES buffer, pH 5.4) via pipetting at a weight/weight (w/w) ratio of 60:1 and incubated at room temperature for 1 h.
Nanoparticle size, PDI, and surface charge
The hydrodynamic diameter and PDI of the nanoparticles were measured by DLS using a Zetasizer Advance Ultra (Malvern Instruments, Malvern, UK). The zeta potential of mRNA-loaded nanoparticles, diluted in either 10 mM pH 5.4 HEPES or 10 mM pH 7.4 HEPES buffer, was determined by phase analysis light scattering (PALS) using the same device. The mode size and concentration of mRNA-loaded nanoparticles were measured by NTA. Specifically, PBAE/mRNA polyplexes were diluted 1:50, and mRNA-loaded PLGA/PBAE nanoparticles were diluted 1:500 using the formulation buffer, then injected into the device via the syringe pump of NanoSight NS300 (Malvern Instruments, Malvern, UK) according to the manufacturer’s instructions.
mRNA encapsulation
mRNA encapsulation was quantified using a SYBR Gold assay. Briefly, 15 μL of prepared nanoparticles was added in 384-well plates, then 5 μL of a 4× SYBR Gold solution was added to each well and incubated in the dark for 10 min. Fluorescence intensity was measured using a plate reader (Tecan, Männedorf, Switzerland) with an excitation (Ex) wavelength of 492 nm and an emission (Em) wavelength of 555 nm. Free mRNA was included as a 100% reference for calculating unencapsulated mRNA. Additionally, agarose gel electrophoresis was performed to visualize mRNA encapsulation. A mixture of 10 μL of prepared nanoparticles and 10 μL of 2× RNA loading dye was loaded into the wells of a 1% agarose gel supplemented with Roti@GelStain. The same amount of free mRNA was loaded as a control. The agarose gel was electrophoresed at a constant voltage of 150 V for 20 min in Tris-borate-EDTA (TBE) buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA), and mRNA bands were visualized using a UV transilluminator (BIO-RAD, Hercules, CA, USA).
mRNA release assay
The SYBR Gold assay was conducted to evaluate the release of mRNA from nanocarriers under different conditions. First, mRNA-loaded formulations at a w/w ratio of 60 were prepared as described before. Different formulations containing 55 ng of mRNA were incubated with 20 μL of serial dilutions of Triton X and heparin in a 384-well plate at 37°C for 30 min. The 100% release control consisted of 10 μL of 2% Triton X and 10 μL of heparin solution (2,000 IU/mL). The buffer used for preparing serial dilutions was 10 mM HEPES, adjusted to pH 5.4 and pH 7.4, respectively. Then, 8.25 μL of a 4× SYBR Gold solution was added to each well and incubated for 10 min. The results were measured as described previously.
RNase protection assay
mRNA-loaded formulations at a w/w ratio of 60 were prepared as previously described. A total of 18 μL of the respective formulations containing 200 ng of mRNA was incubated with 2 μL of pure fetal bovine serum (FBS) to reach a final concentration of 10% FBS for 30 min at 37°C. The same amount of free mRNA was included as controls, either incubated with FBS or left untreated. Subsequently, the mRNA was purified using a RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions and eluted in 10 μL RNase-free water. The recovered samples were analyzed on an Agilent Bioanalyzer using the RNA 6000 Pico kit to assess mRNA integrity.
Cell culture
DC2.4 murine DCs and THP-1 cells were both cultured in RPMI-1640 medium (Sigma-Aldrich, Taufkirchen, Germany) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific, Darmstadt, Germany), 1% (v/v) penicillin-streptomycin (P/S) (Thermo Fisher Scientific, Darmstadt, Germany), and 0.05 mM 2-mercaptoethanol. 16HBE14o- cells and Calu-3 cells were cultured in Eagle’s minimum essential medium (EMEM) supplemented with 10% FBS and 1% P/S. HeLa-Gal8-mRuby3 cells were kindly provided by Professor Ernst Wagner (Ludwig-Maximilians-Universität, Munich, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)—low glucose with 10% FBS and 1% P/S. All cells were cultured in a humidified atmosphere containing 5% CO_2_ at 37°C.
Formulation screening via mRNA transfection in different cell lines
For EGFP mRNA transfection, DC2.4 cells, THP-1 cells, 16HBE14o- cells, and Calu-3 cells were seeded in 96-well plates at densities of 10,000, 10,000, 20,000, and 20,000 cells per well, respectively. Nanoparticles containing 200 ng of EGFP mRNA were added to each well and incubated for 24 h. The same amount of free mRNA was used as a control. After incubation, the cells were trypsinized and collected by centrifugation at 500 × g for 5 min (Eppendorf Centrifuge 5418/5418R). Mean fluorescence intensity (MFI) of mRNA-treated cells was evaluated by flow cytometry (Attune NxT, Thermo Fisher Scientific, Darmstadt, Germany). To observe EGFP expression following the treatment with EGFP mRNA, DC2.4 cells and 16HBE14o- cells cultured in 96-well plates were observed using an EVOS M5000 microscope (Thermo Fisher Scientific, Darmstadt, Germany) with a 20× objective. Fluorescent images were captured in the GFP channel.
For Fluc mRNA transfection, DC2.4 cells, THP-1 cells, and 16HBE14o- cells were seeded as described before and incubated with different formulations containing 200 ng of Fluc mRNA for 24 h. After incubation, different subsequent treatments were performed based on the cell types. For DC2.4 cells and 16HBE14o- cells, the medium in each well was directly aspirated, and the cells were gently rinsed twice with PBS. For THP-1 cells, which grow in suspension, the cells were collected in 1.5 mL Eppendorf tubes by centrifuging at 500 × g for 5 min and then washed once with PBS. Afterward, 100 μL of 0.5× cell culture lysis buffer (Promega) was added to each well of the plates or to each Eppendorf tube. The cells were lysed for 30 min at room temperature. Next, 35 μL of cell lysate was transferred to each well of a 96-well white plate to measure luciferase activity. After adding 100 μL of luciferase assay reagent (LAR) buffer (20 mM glycylglycine, 1 mM MgCl_2_, 0.1 mM EDTA, 3.3 mM dithiothreitol [DTT], 0.55 mM adenosine triphosphate [ATP], 0.27 mM coenzyme A; pH 8.5) supplemented with 5% (v/v) of a mixture of 10 mM luciferin and 29 mM glycylglycine to each well, luminescence counts were measured for 10 s using the Tecan plate reader. The protein amount in each sample was measured using a bicinchoninic acid assay (BCA) kit. Transfection efficiency was presented as RLU/mg by dividing the RLU by the protein amount (mg).
Cell viability
DC2.4 cells seeded in 96-well plates at a density of 10,000 cells per well were used for cytotoxicity assays. Formulations were prepared as described before and added to each well with different amounts of mRNA ranging from 100 to 400 ng. The total volume of the formulations and cell culture medium was 200 μL. After incubation for 24 h, 5 μL of Cell Counting Kit-8 was added to each well and incubated for 3–4 h at 37°C. Absorbance at 450 nm was measured using a Tecan plate reader, and the cell viability was calculated by dividing the absorbance of treated groups by that of the untreated group.
DC2.4 cell uptake and endocytic pathways
DC2.4 cells were seeded at a density of 10,000 cells per well in 96-well plates and incubated for 2 h with formulations containing 200 ng of mRNA, 20% of which was substituted with Alexa Fluor 647-labeled mRNA. Free mRNA was used as a negative control. Lipofectamine 2000 was also included, using 0.2 μL of the reagent to deliver 200 ng of mRNA, prepared following the manufacturer’s instructions. After incubation, the cells were collected and analyzed using flow cytometry (Attune NxT, Thermo Fisher Scientific).
To investigate the uptake mechanisms, the cells were pretreated with various inhibitors, including 5 μg/mL chlorpromazine, 100 μg/mL dextran sulfate, and 20 μg/mL nystatin for 1 h. One plate of seeded cells was incubated for 1 h at 4°C. Afterward, the cells were incubated with the formulations containing the same amount of Alexa Fluor 647-labeled mRNA, either with the inhibitors still present in the medium or by keeping the plate at 4°C for an additional 2 h. Untreated cells served as a blank control, and cells treated with formulations but without inhibitors served as a non-inhibition control. After incubation, the cells were harvested and measured with an Attune NxT flow cytometer.
Endosomal escape
DC2.4 cells were seeded in an 8-well ibiTreat chamber slide (Ibidi, Gräfelfing, Germany) at a density of 10,000 cells per well. The cells were treated with formulations containing 200 ng mRNA with 20% Cy5-labeled negative control mRNA for 2 and 8 h. Cy5-labeled mRNA was generated in-house by in vitro transcription (IVT) without poly(A) tailing. After treatment, the medium in each well was aspirated, and the cells were rinsed twice with PBS. Each well was filled with 200 μL of medium containing LysoTracker Green at a concentration of 75 nM and incubated at 37°C for 1 h. DC2.4 cells were then washed three times with PBS and fixed with a 4% paraformaldehyde (PFA) solution for 15 min at room temperature. After washing with PBS, the cells were stained with a 5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution for 5–10 min. The cells were washed again with PBS and observed using a SP8 inverted CLSM (Leica Camera, Wetzlar, Germany) with a 63× oil-immersion objective. The obtained images were processed using the Fuji plug-in of ImageJ for colocalization analysis by PCC.
Calu-3 cells were seeded in an 8-well ibiTreat chamber slide (Ibidi) at a density of 20,000 cells per well and then treated with formulations containing 200 ng mRNA with 20% Alexa Fluor 647-labeled mRNA for 2 and 8 h. After treatment, the medium in each well was aspirated, and the cells were rinsed twice with PBS. Each well was filled with 200 μL of medium containing LysoSensor Green DND-189 at a concentration of 1 μM and incubated at 37°C for 1 h. Then, the cells were stained with Hoechst 33342 (1 μg/mL) for 8 min at room temperature. After washing three times with PBS, the samples were imaged using an SP8 inverted CLSM using a 63× oil-immersion objective.
Endosomal/lysosomal acidity
DC2.4 cells were seeded in 8-well ibiTreat chamber slides (Ibidi, Gräfelfing, Germany) at 10,000 cells per well. Cells were treated with formulations containing 200 ng OVA mRNA for 2, 4, and 8 h. After treatment, the medium was aspirated, and cells were rinsed twice with PBS. Each well was then incubated with 200 μL of medium containing LysoSensor Green (1 μM) at 37°C for 30 min. Cells were washed three times with PBS and counterstained with Hoechst 33342 (1 μg/mL, 8 min), followed by a final PBS wash. Imaging was performed on a Leica SP8 inverted CLSM using a 63× oil-immersion objective.
Endosomal escape in HeLa-Gal8-mRuby3 cells
HeLa-Gal8-mRuby3 cells were seeded in an 8-well ibiTreat chamber (Ibidi) at a density of 10,000 cells per well and then treated with formulations containing 200 ng of mRNA, of which 20% was replaced by Alexa Fluor 674-labeled mRNA for 1, 4, and 8 h. Then, the cells were fixed with 4% PFA for 15 min at room temperature and subsequently stained with 5 μg/mL of DAPI for 5–10 min. Finally, the cells were observed using the SP8 inverted CLSM (Leica Camera) with a 63× oil objective. The fluorescent dots of Gal8-mRuby3 and Alexa Fluor 647 were analyzed using the Fuji plug-in of ImageJ.
FRET assessment
Cy3/Cy5 dual-labeled mRNA was generated in-house by IVT using the HiScribe T7 ARCA mRNA Kit (New England Biolabs) with aminoallyl-UTP-Cy3 and aminoallyl-UTP-Cy5 added to the reaction. The dual-labeled mRNA was purified with an RNeasy Mini Kit (QIAGEN) and quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific). mRNA-loaded nanoparticles were prepared as previously described using 100% dual-labeled mRNA. For spectral characterization, nanoparticles containing 200 ng labeled mRNA were diluted in 200 μL PBS, and a fluorescence Em scan was acquired with Ex wavelength at 500 nm and Em wavelength from 545 to 745 nm on the Tecan plate reader. For in vitro FRET measurements, 10,000 DC2.4 cells per well were seeded in a black, clear-bottom 96-well plate (μCLEAR, Greiner Bio-One) and treated with either 200 ng free dual-labeled mRNA or the corresponding mRNA-loaded nanoparticles. Fluorescence was recorded in real time on a Spark Cyto plate reader (Tecan) with Ex 500 nm and dual Em at 570 nm (Cy3) and 670 nm (Cy5) at 1 h intervals. The FRET ratio was calculated as I_Cy5/I_Cy3.
Animals
All experiments involving animal organs or tissues were conducted according to the German law of animal protection and were approved by the Government of Upper Bavaria, Munich, Germany (5.1-231 5682/LMU/BMC/Core Facility Animal Models [CAM]). C57BL/6J mice were obtained from the CAM at the Ludwig-Maximilians-Universität München (LMU) Biomedical Center (BMC). C57BL/6-Tg (TcraTcrb)1100Mjb/J (OT-1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained through in-house breeding at CAM. All mice were housed under specified pathogen-free conditions with room temperature maintained at 22°C ± 2°C and relative humidity (RH) set at 50% ± 5%. The light cycle was adjusted to a 12 h light/12 h dark period. All mice had free access to water and standard chow (irradiated, 10 mm pellet; 1314P, Altromin, Laage, Germany).58
BMDC culture
C57BL/6J mice aged 6–8 weeks were euthanized to harvest their hind legs. After removing muscles on the leg, the obtained femur and tibia were immersed in 70% ethanol for 5 min and then placed in normal RPMI-1640 medium. All subsequent treatments were performed under the laminar flow hood. One side of the femur or tibia was incised and placed face down into a 0.5 mL tube with the tube bottom pre-punctured by a G18 needle. The smaller tube containing bones was then inserted into a 1.5 mL Eppendorf tube and centrifuged at 10,000 × g for 15 s. The cell pellet was collected and resuspended at a density of 1 × 10^7^ cells per dish in a 100 mm Petri dish with 10 mL RPMI-1640 medium supplemented with 0.05 mM 2-mercaptoethanol and 20 ng/mL mouse GM-CSF. After 3 days, 10 mL of the medium containing 20 ng/mL mouse GM-CSF was added to each dish. On day 5, the loosely adherent cells were collected, resuspended, and kept in 50% FBS for 30 min at 37°C. Subsequently, the cells were centrifuged at 200 × g for 10 min and stained with PE anti-mouse CD11c antibody to evaluate the purity of DCs using an Attune NxT flow cytometer. BMDCs were considered ready for the subsequent experiments if the purity reached 80%.
mRNA uptake and transfection in BMDCs
BMDCs were seeded at a density of 120,000 cells per well in 96-well plates and treated with formulations containing a total of 200 ng mRNA, including 20% Alexa Fluor 647-labeled mRNA, for 1, 4, and 8 h. After incubation, BMDCs were collected to measure both the percentage of Alexa Fluor 647-positive cells and the MFI by flow cytometry (Attune NxT). Additionally, cells treated with formulations but not detached from the plates were stained with 5 μg/mL DAPI and imaged using an EVOS M5000 microscope (Thermo Fisher Scientific) with a 20× objective.
For Fluc mRNA transfection in BMDCs, 120,000 cells were seeded per well in 96-well plates and then incubated with formulations containing 200 ng of Fluc mRNA for 24 and 48 h, respectively. The cells in each well were collected by centrifuging at 500 × g for 5 min in a 1.5 mL Eppendorf tube and then resuspended in 100 μL of 0.5× cell culture lysis buffer. After 30 min of incubation at room temperature, the luciferase activity in 35 μL of cell lysate was measured as previously described. The transfection results are presented as RLU/mg.
Antigen presentation and BMDC maturation
BMDCs were seeded at a density of 300,000 cells per well in 24-well plates and incubated with formulations containing 500 ng of OVA mRNA for 24 and 48 h, respectively. Cells treated with the same amount of free OVA mRNA served as a negative control. After incubation, the cells were stained with Zombie Violet at room temperature for 20 min and divided equally. Half of the cells were used for investigating antigen presentation, with a subsequent staining of PE anti-mouse H-2Kb/SIINFEKL antibody for 30 min. The percentage of PE-positive cells was analyzed using an Attune NxT flow cytometer. The other half were used for BMDC maturation analysis, after staining with BV605 anti-mouse CD11c, APC anti-mouse MHC class II, FITC anti-mouse CD40 or FITC anti-mouse CD80, APC anti-mouse CD86 antibodies for 30 min at 4°C. The expression of the co-stimulatory molecules on the surface of BMDCs was determined by flow cytometry (Attune NxT).
Coculture of transfected BMDCs and CD8+ T cells
OT-1 mice aged 16 weeks were sacrificed, and their spleens were harvested for further processing. The isolation of CD8^+^ T cells was performed using a mouse CD8a^+^ T cell isolation kit purchased from Miltenyi. First, the spleens were ground through 70 μm cell meshes to obtain single-cell suspensions. The cells were counted and then resuspended in MACS buffer containing Biotin-Antibody Cocktail and incubated for 5 min. Afterward, the cells were incubated with Anti-Biotin MicroBeads for another 10 min at 4°C. In the end, a total of 1 × 10^8^ cells were applied on one LS column for further separation. The isolated cells were stained with FITC anti-mouse CD8a antibody, and later were measured using an Attune NxT flow cytometer to check the percentages of CD8^+^ T cells.
BMDCs were seeded at a density of 600,000 cells per well in 12-well plates and then transfected with different formulations containing 1 μg OVA mRNA for 24 h. BMDCs treated with empty PLGA/PBAE nanoparticles and PLGA/PBAE loaded with SARS-CoV-2 spike protein mRNA were applied as a negative control and to test an irrelevant antigen control, respectively. After 24 h of transfection, 200,000 BMDCs were seeded in 12-well plates, and 1 × 10^6^ isolated CD8^+^ T cells from OT-1 mice were subsequently added to each well.
After a coculture period of 1 h, diluted monensin solution was added into each well to inhibit cytokine exocytosis.59 The cells were cocultured for another 5 h, and then incubated with Zombie Violet for live/dead staining, followed by staining with FITC anti-mouse CD8a and APC anti-mouse IFN-γ antibodies for 30 min at 4°C. Finally, the cells were analyzed by Attune NxT flow cytometer. In parallel, another set of cells was continuously cocultured for 72 h without the addition of monensin. After incubation, the cell culture medium from each well was collected to measure the IFN-γ concentration using the ELISA MAX Standard Set Mouse IFN-γ kit according to the manufacturer’s instructions.
To investigate T cell proliferation, CD8^+^ T cells isolated from OT-1 mice were first stained with CFSE at a concentration of 4 μM in cell culture medium, and then cocultured with the transfected BMDCs as previously described. After 72 h, the cells were collected and stained with Zombie Violet, and then with APC-Cy7 anti-mouse CD8 antibody. Finally, the percentages of the divided subset of CD8^+^ T cells were analyzed using an Attune NxT flow cytometer.
mRNA uptake and transfection in ALI Cells
Calu-3 cells were seeded at a density of 250,000 cells per well on the apical side of Transwell polyester cell culture inserts (6.5 mm, 0.4 μm pore size). The basolateral compartment was filled with 700 μL of normal EMEM medium. After 3 days, the medium in the apical chamber was removed, and the medium in the basolateral chamber was replaced with 300 μL PneumaCult ALI medium (Stemcell Technologies, Vancouver, Canada) to obtain an ALI condition. The ALI medium was changed every 3 days. After 7 days of culture, TEER was measured using an EVOM epithelial volt/Ω meter (World Precision Instruments, Sarasota, USA). When the TEER values reached 300 Ω∗cm^2^, the ALI cells were considered ready for the subsequent experiments.
ALI-cultured Calu-3 cells were treated with different formulations containing 500 ng of mRNA (20% Alexa Fluor 647-labeled mRNA) and incubated for 24 h. After incubation, the liquid in the apical chamber was gently aspirated. A 100 μL mixture of Hoechst 33342 (concentration 50 μg/mL) and AF488-labeled wheat germ agglutinin was added to the apical side. The Transwell inserts were placed in the basolateral chamber filled with 400 μL of Hoechst 33342 solution at the same concentration. After incubation at 37°C for 20 min, the ALI monolayers were gently rinsed with PBS from both apical and basolateral sides three times. The membrane was cut and mounted on glass slides using FluorSave reagent and immediately observed using an SP8 inverted CLSM (Leica) with a 40× oil-immersion objective. Images were exported from the Leica Image Analysis Suite and processed with the Fuji plug-in of ImageJ.
For Fluc mRNA transfection in ALI cells, the same treatments with formulations containing 500 ng of Fluc mRNA were performed on the apical side. After 24 h of incubation, the Transwells were washed twice with 300 μL of PBS, and then 200 μL of 0.5× cell culture lysis buffer (Promega) was added to each well. The cell lysates were obtained after 1 h of incubation at room temperature followed by thorough pipetting. Luciferase activity in 35 μL of cell lysate was measured as previously described. The transfection efficiency is presented as RLU/mg.
mRNA uptake and transfection in hPCLS
Human lung tissues were obtained from the CPC-M bioArchive at the Comprehensive Pneumology Center (CPC), the University Hospital Großhadern of the Ludwig-Maximilian University (Munich, Germany), and the Asklepios Biobank of Lung Diseases (Gauting, Germany). The use of human lung tissues in this study was approved by the local ethics committee of the Ludwig-Maximilian University, Munich, Germany (Project 19-630), and all participants provided written informed consent to participate. We gratefully acknowledge the provision of human lung tissues and clinical data from the CPC-M bioArchive and its partners. We thank the patients and their families for their support. hPCLSs were prepared from tumor-free tissue according to a previous description.60 The lung tissue was inflated with a 3% agarose solution and solidified at 4°C. Then, lung slices with a thickness of 500 μm were cut from tissue blocks using a vibration microtome (HyraxV50) (Karl Zeiss AG, Oberkochen, Germany). hPCLS were cultured in DMEM F-12 medium supplemented with 0.1% FBS. Prior to experiments, the hPCLS were processed into small punches with a diameter of 4 mm.
hPCLS were submerged in the culture medium containing mRNA-loaded formulations (with 20% Alexa Fluor 647-labeled mRNA) at a final concentration of 1 μg/mL total mRNA for 24 h. The hPCLS were then fixed with a 4% PFA solution for 30 min. After being washed three times with PBS, the slices were stained with a 5 μg/mL DAPI solution for another 30 min. Subsequently, the hPCLS were mounted on glass slides using FluorSave reagent and observed using an SP8 inverted CLSM (Leica) with a 20× objective. The acquired images were processed with the Fiji plug-in of ImageJ.
For Fluc mRNA transfection, hPCLS were treated with different formulations containing 1 μg of Fluc mRNA for 24 h. Once the incubation time was completed, the slices were rinsed with PBS twice and then cut into small pieces. The hPCLS fragments were transferred into 1.5 mL Eppendorf tubes and submerged in 200 μL of 0.5× cell culture lysis buffer (Promega). After three freeze-thaw cycles, the tissue lysate was used for measuring the luciferase activity as previously described.
Nebulization
mRNA-loaded PLGA/PBAE nanoparticles were prepared as previously described. SM-102 LNPs were formulated at a molar ratio of SM-102 (ionizable lipid):cholesterol:1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):PEG-2000-DMG = 50:38.5:10:1.5 (N/P 6). Freshly prepared suspensions were immediately nebulized using an Aerogen Pro device, and the obtained aerosols were collected into pre-cooled 15 mL Falcon tubes for downstream analyses.
Aerosol characterization by laser diffraction
Aerosol droplet size distribution was measured by laser diffraction. EGFP mRNA-loaded PLGA/PBAE nanoparticles were nebulized with the Aerogen Pro directly into a HELOS laser diffractor (Sympatec, Clausthal-Zellerfeld, Germany) equipped with an R2 lens and INHALER module. Aerosol was delivered through a punched silicone mouthpiece using an Aerogen Pro T-piece positioned 50–100 mm upstream of the laser beam. The setup was enclosed in a Plexiglas box to maintain RH > 70% during acquisition. Aerosol was extracted at 13.9 L/min to prevent re-entry into the beam path. Each measurement comprised three runs with 5 s per run and a signal integration time of 200 ms. Experiments were performed in triplicate (n = 3).
Encapsulation efficiency and mRNA loss post-nebulization
mRNA encapsulation was quantified for both pre- and post-nebulized PLGA/PBAE samples using the SYBR Gold assay, whereas mRNA loss was calculated exclusively for post-nebulized samples relative to their pre-nebulization inputs. Free mRNA was included as a 100% reference. PLGA/PBAE samples containing a theoretical 55 ng of mRNA were incubated either with 10 μL of 2% Triton X and 10 μL of heparin solution (2,000 IU/mL) in a 384-well plate at 37°C for 30 min to achieve complete mRNA release, or with 20 μL of buffer to quantify unencapsulated mRNA. Subsequently, 8.25 μL of a 4× SYBR Gold solution was added to each well and incubated for 10 min in the dark before fluorescence measurement, as previously described.
For LNP formulations, mRNA encapsulation was quantified using the Quant-iT RiboGreen RNA Assay. Briefly, 5 μL of each LNP sample was added into a 384-well plate and incubated with 10 μL of 2% Triton X or 10 μL of 1× Tris-EDTA buffer as the complete release control and unencapsulated control, respectively. After 1 h incubation at 37°C, 5 μL of 100-fold diluted RiboGreen reagent was added to each well and incubated for 10 min in the dark. Fluorescence was measured at an Ex wavelength of 480 nm and an Em wavelength of 525 nm.
mRNA integrity post-nebulization
Aerosols generated from nebulized PLGA/PBAE and SM-102 LNP formulations were collected in cooled 15 mL tubes. mRNA from pre- and post-nebulization samples was purified using an RNeasy Mini Kit (QIAGEN), and the corresponding integrity was assessed using an Agilent Bioanalyzer using the RNA 6000 Pico kit.
mRNA transfection in DC2.4 cells post-nebulization
DC2.4 cells were seeded at 10,000 cells per well in 96-well plates and treated for 24 h with pre- and post-nebulized PLGA/PBAE and SM-102 LNP formulations. After incubation, GFP expression was quantified by flow cytometry (Attune NxT, Thermo Fisher Scientific).
Statistical analysis
All data were expressed as means ± SD. All statistical analyses were performed using one-way analysis of variance (ANOVA) in GraphPad Prism or Student’s t test when specifically stated. Levels of significant differences were expressed as follows: ns, not significant; ^✽^p < 0.05; ^✽✽^p < 0.01; ^✽✽✽^p < 0.001; ^✽✽✽✽^p < 0.0001.
Resource availability
Lead contact
Request for further information and requests should be directed to and will be fulfilled by the lead contact, Olivia M. Merkel ([email protected]).
Materials availability
This study did not generate new unique reagents.
Data and code availability
Any additional information or data reported in this paper will be shared by the lead contact upon request.
Acknowledgments
This project was partially funded by the Bavarian Research Foundation (AZ-1449-20C) and ERC-2022-COG-101088587. M.J. acknowledges financial support from the China Scholarship Council. We gratefully acknowledge Prof. Roland Beckmann and Dr. Otto Berninghausen for the cryo-TEM imaging.
Author contributions
Conceptualization, M.J., F.S.-S., and O.M.M.; methodology, M.J., F.S.-S., S.P.C., D.M., and A.N.; investigation, F.S.-S. contributed to polymer synthesis; D.M. and B.P. contributed to animal tissue harvesting; D.L.P.-G., A.K.V., and G.B. provided hPCLS; M.K.-G. provided SARS-CoV-2 spike protein mRNA; writing – original draft, M.J.; writing – review and editing, M.J., D.C.J., S.P.C., X.S., and O.M.M.; funding acquisition, resources, and supervision, O.M.M.
Declaration of interests
O.M.M. and D.C.J. are co-founders of RNhale GmbH. O.M.M. is a Scientific Advisory Board Member of Coriolis Pharma, AMW, and Corden Pharma as well as a consultant for PARI Pharma, AbbVie Deutschland, and Boehringer-Ingelheim International. M.K.-G. is an employee of Daiichi Sankyo Europe and may own stock.
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